Mutant wnt7 proteins with c-terminal amino acid addition

Abstract

The current invention relates to mutant Wnt7 proteins, comprising at their C-terminal end an amino acid addition having a length of 1 to 8 amino acids. The invention also relates to a nucleic acid encoding the mutant Wnt7 protein, a nucleic acid expression cassette, a vector, a pharmaceutical composition; and their uses in medicine and therapy.

Description

[0001] MUTANT WNT7 PROTEINS WITH C-TERMINAL AMINO ACID ADDITION

[0002] FIELD OF THE INVENTION

[0003] The present invention is directed to mutant Wnt7 proteins comprising at their C- terminal end an amino acid addition, having a length of 1 to 8 amino acids. The invention also relates to nucleic acids encoding such mutant proteins and to their use in medicine.

[0004] BACKGROUND

[0005] Endothelial Wnt / 0-catenin signaling acts as a master regulator of Blood-Brain Barrier (BBB) physiology in response to neural-derived Wnt7a / b ligands. Wnt / 0-catenin signaling initiates the BBB differentiation cascade at the earliest steps of central nervous system (CNS) vascular invasion, and then maintains BBB function in adults. Recent evidence suggests that inhibition of Wnt signaling by conditional deletion of 0-catenin signaling in cerebral endothelial cells (ECs) causes BBB breakdown and accelerates disease progression in stroke, glioblastoma, and multiple sclerosis murine models. Conversely, recombining a constitutively active form of 0-catenin in the CNS endothelium is protective in models of brain cancer and stroke.

[0006] Wnt7a / b being the endogenous ligands controlling p-catenin-dependent BBB maturation, they are in principle, legitimate therapeutic agents to repair the dysfunctional BBB. However, activation of Wnt signaling via Wnt7 ligands has pleiotropic outcomes across a range of tissues and organs, in both health and disease. More so, the structural modalities of Wnt / Frizzled (FZD) interactions disqualify natural Wnt ligands as safe therapeutics. On the other hand, it is known that Wnt7 ligands activate Wnt signaling via two distinct types of membrane receptor complexes. The first one is the Frizzled (FZD) family receptors / LRP5 / 6 complex which has broad tissue distribution, binds non-discriminately to Wnt7a / b and leads to systemic activation of Wnt signaling, the second one is the GPR124 / RECK / FZ / LRP5 / 6 complex which is enriched at cerebral endothelial cells where GPR124 and / or RECK are expressed.

[0007] Recently, proof-of-concept mutagenesis has revealed that Wnt7a ligands can be engineered into highly-specific GPR124 / RECK agonists to target Wnt signaling pathway in cells expressing GPR124 and RECK such as in cerebral endothelial cells ("on-target") and not inducing Wnt signaling in the absence of GPR124 and / or RECK ("off-target"). Results suggest a promising use of engineered Wnt7 ligands as therapeutics, especially for CNS related disorders.

[0008] W02019180204 and Martin et al., 2022 describe Wnt7 mutants which activate GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling with impaired activation of Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124, the latter which would lead to "off-target" activity. To be used in therapy, unwanted "off- target" activity should be as low as possible, especially when targeting a pathway such as Wnt signaling which has broad functions across the developmental stages and which deregulation is known to be implicated in many human diseases, ranging from cancers to skeletal disorders. Although the previously discovered Wnt7 variants showed low "off-target" activity, there is still a need for additional GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling specific agonists with low off- target effect.

[0009] The present invention aims to resolve at least some of the problems and disadvantages mentioned above. The invention thereto aims to provide further novel Wnt7a or Wnt7b-derived GPR124 / RECK co-receptor complex specific agonists.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention relates to a mutant Wnt7 protein according to claim 1. In particular, the mutant Wnt7 protein comprises at its C-terminal end an amino acid addition, having a length of 1 to 8 amino acids. Said mutant is preferably an agonist of a G-protein coupled receptor GPR124 / RECK / Frizzled / lipoprotein receptor-related protein (LRP) complex. Said mutant is not able to or has an impaired capacity to bind to Frizzled and / or lipoprotein receptor-related protein (LRP) in the absence of RECK and / or GPR124; and / or said mutant is not able to or has an impaired capacity to activate Frizzled and / or lipoprotein receptor-related protein (LRP) in the absence of RECK and / or GPR124.

[0012] It was shown that such mutant Wnt7 proteins are particularly efficient agonists of GPR124 / RECK specific signaling, with no or only limited activation of canonical Wnt signaling via Frizzled / LPR in the absence of GPR124 and / or RECK. Wnt proteins exert pleiotropic functions by regulating cell proliferation, differentiation, migration, apoptosis, polarity and genetic stability. Dysregulated Wnt signaling levels are associated with a large spectrum of human pathologies. Intervening in Wnt signaling pathways generally leads to multiple effects, of which many were unwanted ("off- target").

[0013] As the GPR124 / RECK co-receptor complex is enriched at the level of the blood brain barrier endothelial cells, said mutant Wnt proteins of the present invention can selectively (re-)activate Wnt signaling in cerebral endothelial cells ("on-target"), without activating unwanted "off-target" Wnt-related pathways.

[0014] Preferred embodiments of the Wnt7 mutant protein of claim 1 are shown in any of the claims 2 to 10.

[0015] In next aspects, the invention relates to a nucleic acid encoding the mutant Wnt7 protein according to claim 11, to nucleic acid expression cassette according to claim 12, to a vector according to claim 13, and to a pharmaceutical composition according to claim 14.

[0016] In a further aspect, the invention relates to the mutant Wnt7 protein, the nucleic acid, the nucleic acid cassette, the vector or the pharmaceutical composition for use as a medicament according to claim 15, for use in the prevention or treatment of a neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction according to claim 18, or for use in the treatment of retinopathy according to claim 19.

[0017] In another aspect, the invention relates to the nucleic acid, the nucleic acid expression cassette, or the vector, for use in gene therapy according to claim 16.

[0018] In a final aspect, the invention relates to the nucleic acid, or the vector, for use in RIMA therapy according to claim 17.

[0019] DESCRIPTION OF FIGURES

[0020] 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. Throughout the drawings, corresponding reference numerals indicate like or corresponding parts and features. Figure 1 shows the effect of the addition of 1 to 8 amino acids to the C-terminal end of wildtype or mutant (K190A) mouse Wnt7a, on "off-target" activation of the canonical Wnt pathway without GPR124 / RECK (off-target). The resulting constructs (Fig. 1A) were transfected in HEK293 STF cells together with Renilla luciferase, Lrp5 and Fz5 receptor (Off-target) and tested in a dual luciferase assay. Due to the C- terminal additions, off-target activity is significantly decreased and even reaches the detection limit (Fig. IB).

[0021] Figure 2 shows the effects of the addition of each of 20 natural amino acids to the C-terminal end of mutant (K190A) mouse Wnt7a on "on-target" activation and "off- target" activation of the canonical Wnt pathway either with GPR124 / RECK (on target) or without GPR124 / RECK (off-target). For many amino acid additions, the on-target activity remains high, while the off-target activity is significantly decreased and even reaches the detection limit.

[0022] Figure 3 shows the effect of one alanine residue addition to the C-terminus of various mutant Wnt7 proteins on "on-target" activation and "off-target" activation of the canonical Wnt pathway either with GPR124 / RECK (on target) or without GPR124 / RECK (off-target). The on-target activity remains high, while the off-target activity is significantly decreased and even reaches the detection limit.

[0023] Figure 4 shows the effect of various amino acid additions of variable lengths (2-4- 5-6-8 amino acids) fused to the C-terminus of mutant (K190A) mouse Wnt7a on "on-target" and "off-target" signaling activation of the canonical Wnt pathway either with GPR124 / RECK (on target) or without GPR124 / RECK (off-target). The on-target activity remains high, while the off-target activity is significantly decreased and even reaches the detection limit.

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention concerns further Wnt7-derived proteins that are selective agonists of the Gprl24-and Reck-dependent Wnt / 0-catenin pathway. The invention thereto relates to mutant Wnt7 proteins that are agonists of a G-protein coupled receptor GPR124 / RECK / Frizzled / lipoprotein receptor-related protein (LRP) complex, and are not able to or have an impaired capacity to bind to or to activate Frizzled and / or lipoprotein receptor-related protein (LRP) in the absence of RECK and / or GPR124. The invention in particular concerns mutant Wnt7 proteins, having at their C-terminal end an amino acid addition, having a length of 1 to 8 amino acids, preferably having a length of one amino acid, wherein said amino acid is chosen from an alanine and a glycine. The present invention further concerns the nucleic acids and vectors encoding said mutant Wnt7 proteins and pharmaceutical compositions comprising such mutant proteins, nucleic acid and / or vector, and their uses in medicine.

[0026] Definitions

[0027] 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 present invention.

[0028] 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 synthesized. 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.

[0029] The term "variant" or "mutant" 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 (FEMS 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).

[0030] 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 (BI2seq) algorithm, available as a web application or as a standalone executable program (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 BI2seq already conveniently includes the percentage of identities.

[0031] 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. 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.

[0032] 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.

[0033] 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. 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.

[0034] The term "biologically active" is interchangeable with terms such as "functionally active" or "functional", denoting that the proteins disclosed herein at least partly retain 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.

[0035] Preferably, a functionally active protein 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 protein.

[0036] In certain embodiments, a functionally active protein 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 protein. By means of an example, where the activity of a given 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 protein. "Wnt7" in the present context refers to both "Wnt7a" and / or "Wnt7b", which are part of the Wnt-family of proteins. The terms "Wnt7" and "WNT7" are used interchangeably throughout this specification.

[0037] A "GPR124 / RECK / FZD / LRP receptor complex" "GPR124 / RECK / FZD / LRP co-receptor complex", "GPR124 / RECK / FZD / LRP complex" or "GPR124 / RECK / FZD / LRP protein 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 protein, at least one RECK protein, at least one FZD protein and at least one LRP protein. A GPR124 / RECK / FZD / LRP receptor complex, when located at the plasma membrane of a cell, is capable of activating Wnt / 0- catenin signaling in said cell in response to extracellularly provided Wnt7 ligand.

[0038] A "FZD / LRP receptor complex", "FZD / LRP co-receptor complex", "FZD / LRP complex" or "FZD / LRP protein 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 protein and at least one LRP protein. A FZD / LRP receptor complex, when located at the plasma membrane of a cell, is capable of activating Wnt / 0-catenin signaling in said cell in response to extracellularly provided Wnt ligand, such as, but not limited to Wnt7 ligand.

[0039] "LRP" or "lipoprotein receptor-related protein" 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. In certain particularly preferred embodiments, the terms denote LRP5, LRP6, or LRP5 and LRP6 (LRP5 / 6).

[0040] 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.

[0041] The term "amino acid addition" as used throughout this specification means an entity of one to eight amino acids which can be added to C-terminal end of a protein or (poly)peptide, preferably via peptide bonds. The term may be used to refer to only one, two, three, four, five, six, seven, or eight amino acids. If longer than one amino acid, the amino acids of the amino acid addition are preferably joined together by peptide bonds.

[0042] The term "gene therapy" and / or "RIMA 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. The RNA molecule may be any type of RNA molecule. For instance, the RNA molecule may be cytoplasmic RNA, a nuclear RNA, an mRNA, antisense RNA or a noncoding RNA, preferably an mRNA.

[0043] 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 cells are inter alia unicellular organisms, such as bacteria (e.g., E. coli, Salmonella typhimurium, Serratia marcescens, or Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris), (cultured) plant cells (e.g., from Arabidopsis thaliana or Nicotiana tabacum) and (cultured) animal cells (e.g., vertebrate animal cells, mammalian cells, primate cells, human cells or insect cells). Contemplated as host organisms are inter alia multi-cellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrate animals, still more preferably mammals, yet more preferably primates; particularly contemplated are such animals and animal categories which are non-human.

[0044] 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 neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction. 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.

[0045] The term "therapeutically active / 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 active / 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. In present context, the "therapeutically active amount" is used to refer to both the "therapeutically active amount" and "prophylactically active amount" as described above, unless the distinction is clear from the context. Methods are known in the art for determining therapeutically and / or prophylactically effective doses of a compound, a protein, a nucleic acid encoding the compound / protein, a nucleic acid expression cassette, or a pharmaceutical composition, as taught herein. The term "therapeutically effective dose" as used herein refers to an amount of a compound, a protein, a nucleic acid encoding the compound / protein, a nucleic acid expression cassette, or a pharmaceutical composition, as taught herein, that when administered brings about a positive therapeutic response with respect to treatment of a patient having a specific disease or disorder.

[0046] Description

[0047] Compounds Previously the inventors characterized 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 the herein characterized Wnt decoding module, selectivity is conferred by RECK, which mediates Wnt7-specific binding in a Frizzled-independent manner and is part of Wnt7 ligand-specific RECK / GPR124 / Frizzled / lipoprotein receptor-related protein (LRP) signalosomes.

[0048] The inventors further demonstrated the design of agonists capable of activating Wnt signaling selectively in cells expressing RECK and GPR124 i.e. for instance cerebral endothelial cells. They further showed that such agonists are useful as therapeutics, such as particularly for the treatment of neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction. Hence, the invention allowed to provide inter alia agonists capable of stimulating Wnt / 0-catenin signaling in cerebral endothelial cells ("on-target activity") with only low cross-reactivity with other Frizzled pathways ("unwanted" or "off-target activity"), and useful as therapeutics, particularly for neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction.

[0049] The inventors now surprisingly discovered further Wnt7 related proteins, in particular the mutant Wnt7 proteins of the invention, that display highly specific "on-target" Gprl24 / Reck activity, wherein the "off-target" activity is preferably even lower than previously shown. Such mutant proteins are agonists of a GPR124 / RECK co-receptor complex, and are preferably not able to or have an impaired capacity to bind to or activate Frizzled and / or LRP in the absence of RECK and / or GPR124. Said mutants are thus selective agonists of Gprl24-and Reck-dependent Wnt / p-catenin pathway. In particular, the mutants of the present invention comprise at their C-terminal end an amino acid addition, having a length of 1 to 8 amino acids.

[0050] Herein disclosed is a surprising discovery that said mutant Wnt7 proteins with additional amino acids display highly specific "on-target" GPR124 / RECK activity, wherein the "off-target" activity is even lower than earlier described mutant variants of Wnt7.

[0051] 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.

[0052] Accordingly, in a first aspect, the invention provides a mutant Wnt7 protein, wherein said mutant comprises at its C-terminal end an amino acid addition, having a length of 1 to 8 amino acids.

[0053] In an embodiment, said mutant Wnt7 protein is an agonist, preferably a selective agonist, of the Gprl24-and Reck-dependent Wnt / p-catenin pathway.

[0054] In an embodiment, the term "selective agonist of the Gprl24-and Reck-dependent Wnt / p-catenin pathway" is to be understood as an agonist of said pathway that is able to activate said pathway with high selectivity and specificity (on-target activity), with no or only minimal activation of other Wnt signaling pathways (off-target activity). In embodiments, the Wnt7 mutants disclosed herein as an agonist of Gprl24-and Reck-dependent Wnt / p-catenin pathway show less than 20% off-target activity, for example, showed less than 15%, 10%, 5% or 1% off-target activity, preferably less than 10% off-target activity as measured in cell culture Super TOP- Flash assays (as known it the art and discussed in the example section below).

[0055] In an embodiment, said mutant Wnt7 protein is a modulator of the Gprl24-and Reck-dependent Wnt / 0-catenin pathway, comprising at its C-terminal end an amino acid addition, having a length of 1 to 8 amino acids.

[0056] In embodiments, the mutant Wnt7 protein as disclosed herein is an agonist of the GPR124 / RECK complex, preferably a specific GPR124 / RECK complex agonist, wherein said protein is capable of binding to the GPR124 / RECK complex, the GPR124 protein and / or the RECK protein. In embodiments, said mutant is not able to or has an impaired capacity bind to or activate Frizzled and / or lipoprotein receptor-related protein (LRP) in the absence of RECK and / or GPR124.

[0057] In the present specification, 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 "Wnt" and in particular to "Wnt7", 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.

[0058] In certain embodiments, one or more and preferably all of Wnt7, 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.

[0059] A skilled person can appreciate that any sequences represented in sequence databases or in the present specification may be precursors of the respective peptides, polypeptides, proteins, or nucleic acids and may include parts that are processed away from mature molecules; or may represent sequences that are of mature molecules while also the precursor sequences are considered part of the present concept.

[0060] 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.

[0061] 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, palmitoylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, ubiquitination, oxidation of methionine to methionine sulphoxide or methionine sulphone, signal peptide removal, N-terminal Met removal, conversion of proenzymes or pre-hormones into active forms, and the like. A broader definition is given above.

[0062] In certain embodiments, the mutant Wnt7 protein 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 mouse or human origin. In another embodiment, the mutant Wnt7 protein is of synthetic origin. In certain embodiments, the amino acid addition has a length of 8 amino acids, or a length of 7 amino acids, or a length of 6 amino acids, or a length of 5 amino acids, or a length of 4 amino acids, or a length of 3 amino acids, preferably a length of 2 amino acids, most preferably a length of 1 amino acid.

[0063] The length of the amino acid addition is optimized as to provide the highest possible on-target activity combined with the lowest possible off-target activity of the mutant Wnt7 protein. In addition, amino acid additions with a length of maximum 8 amino acids were shown to provide the required activity.

[0064] In another or further embodiment, the amino acid addition has a length of at least 1 and maximum 8 amino acids, preferably at least 2 and maximum 8 amino acids, preferably at least 3 and maximum 8 amino acids, preferably at least 4 and maximum 8 amino acids, preferably at least 5 and maximum 8 amino acids, preferably at least 6 and maximum 8 amino acids, preferably at least 7 and maximum 8 amino acids.

[0065] In another or further embodiment, the amino acid addition has a length of at least 1 and maximum 7 amino acids, preferably at least 2 and maximum 7 amino acids, preferably at least 3 and maximum 7 amino acids, preferably at least 4 and maximum 7 amino acids, preferably at least 5 and maximum 7 amino acids, preferably at least 6 and maximum 7 amino acids.

[0066] In another or further embodiment, the amino acid addition has a length of at least 1 and maximum 6 amino acids, preferably at least 2 and maximum 6 amino acids, preferably at least 3 and maximum 6 amino acids, preferably at least 4 and maximum 6 amino acids, preferably at least 5 and maximum 6 amino acids.

[0067] In another or further embodiment, the amino acid addition has a length of at least 1 and maximum 5 amino acids, preferably at least 2 and maximum 5 amino acids, preferably at least 3 and maximum 5 amino acids, preferably at least 4 and maximum 5 amino acids.

[0068] In another or further embodiment, the amino acid addition has a length of at least 1 and maximum 4 amino acids, preferably at least 2 and maximum 4 amino acids, preferably at least 3 and maximum 4 amino acids. In another or further embodiment, the amino acid addition has a length of maximum

[0069] 2 amino acids, preferably of one amino acid.

[0070] In an embodiment, said amino acid addition comprises amino acids selected from the 22 alpha-amino acids known to build proteins (so-called proteinogenic amino acids). As a non-limiting example, the amino acid addition can be formed of repetition of amino acids such as repetitions of Alanine (A), or of repetitions of Glycine-Serine (GS) di-peptides.

[0071] In an embodiment, the amount of negatively charged amino acids in the addition is kept to a minimum (such as one or two). In an embodiment, the addition does not comprise any negatively charged amino acids.

[0072] In a preferred embodiment, the amino acid addition has a length of one amino acid and said one amino acid is preferably alanine or glycine.

[0073] In an embodiment, the amino acid sequence of the mutant Wnt7 protein comprises one or more substitutions, deletions, truncations, or insertions, such as 5, 4, 3, 2 or preferably 1 substitution(s), deletion(s), truncation(s), or insertion(s) compared to a sequence according to SEQ ID NO: 1 or SEQ ID NO:2, and has at its C-terminus an amino acid addition, having a length of 1 to 8 amino acids, or a length as described above in any of the embodiments.

[0074] SEQ ID NO: 1 and SEQ ID NO:2 correspond to the mature amino acid sequences of human or mouse Wnt7a and Wnt7b protein sequences respectively. The mutant Wnt7 protein of the present invention is therefore preferably a mutant Wnt7a or Wnt7b protein, having at its C-terminus an amino acid addition, having a length of 1 to 8 amino acids.

[0075] The amino acid sequence corresponding to SEQ ID NO: 1 (mature human or mouse Wnt7a) is:

[0076] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFG KELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSA DIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWT TLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEE DPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTC SERTEMYTCK The amino acid sequence corresponding to SEQ ID NO:2 (mature human or mouse

[0077] Wnt7b) is:

[0078] LGANIICNKIPGLAPRQRAICQSRPDAIIVIGEGAQMGINECQYQFRFGRWNCSALGEKTVFG QELRVGSREAAFTYAITAAGVAHAVTAACSQGNLSNCGCDREKQGYYNQAEGWKWGGCSA DVRYGIDFSRRFVDAREIKKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCW TTLPKFREVGHLLKEKYNAAVQVEVVRASRLRQPTFLRIKQLRSYQKPMETDLVYIEKSPNYCE EDAATGSVGTQGRLCNRTSPGADGCDTMCCGRGYNTHQYTKVWQCNCKFHWCCFVKCNT CSERTEVFTCK

[0079] In a further embodiment, the amino acid sequence of said mutant Wnt7 protein comprises one or more amino acid substitutions, such as 5, 4, 3 or 2 amino acid substitutions, preferably one amino acid substitution, compared to a sequence according to SEQ ID NO: 1 or SEQ ID NO:2, and having at its C-terminus an amino acid addition, having a length of 1 to 8 amino acids.

[0080] In an embodiment, the amino acids of the C-terminal amino acid addition are chosen from alanine, glycine, proline, serine, or a combination thereof, most preferably the amino acid addition has a length of one amino acid, wherein said amino acid is chosen from an alanine and a glycine.

[0081] As indicated, the term "amino acid addition" as used throughout this specification means an entity of one to eight amino acids which can be added to C-terminal end of a protein or (poly)peptide, preferably via peptide bonds. The term may be used to refer to only one, two, three, four, five, six, seven, or eight amino acids. If longer than one amino acid, the amino acids of the amino acid addition are preferably joined together by peptide bonds.

[0082] Therefore, where this mutant Wnt7 protein comprises one or more amino acid substitutions, such as 5, 4, 3 or 2 amino acid substitutions, preferably one amino acid substitution, compared to a sequence according to SEQ ID NO: 1 or SEQ ID NO:2, has at its C-terminus an amino acid addition, having a length of 1 to 8 amino acids, or a length as described in any of the embodiments, said mutant Wnt7 protein has a sequence length similar to SEQ ID NO: 1 or 2 plus 1 to 8 additional amino acids of the amino acid addition.

[0083] SEQ ID NO: 1 and 2 each have a length of 348 amino acids, and therefore, in an embodiment, the mutant Wnt7 protein preferably has a length of more than 348 amino acids, preferably 349 amino acids to 356 amino acids, more preferably 349 amino acids to 355 amino acids, more preferably 349 amino acids to 354 amino acids, more preferably 349 amino acids to 353 amino acids, more preferably 354 amino acids to 352 amino acids, more preferably 349 amino acids to 351 amino acids, more preferably 349 amino acids to 350 amino acids, most preferably 349 amino acids.

[0084] In embodiments, the protein can bind to RECK protein.

[0085] In embodiments, the mutant Wnt7 protein may bind RECK protein, optionally to the cysteine knot 4 (CK4) region, to the CK5 region, or to the CK4 and CK5 regions of RECK protein.

[0086] In embodiments, the mutant Wnt7 protein disclosed herein can concurrently bind to Frizzled (FZD), LRP and RECK. The mutant Wnt7 protein can bind to the cysteine- rich domain (CRD) of the Frizzled protein. The mutant Wnt7 protein can bind to the LRP protein, for example to the extracellular domain of the LRP protein, to the DKK- binding site and / or the Wnt-binding site of the LRP protein. In more particular embodiments, the mutant Wnt7 protein disclosed herein is capable of binding to the DKKl-binding site of the LRP5 and / or LRP6 protein.

[0087] In particular embodiments the mutant Wnt7 protein as disclosed herein is capable of binding to 0-propeller-EGF-like domains 1 and 2 (P1E1P2E2) and / or p-propeller- EGF-like domains 3 and 4(P3E3P4E4) of the LRP protein.

[0088] In particular embodiments, the mutant Wnt7 protein as disclosed herein is capable of concurrently binding to Frizzled and / or LRP proteins in addition to the GPR124 and / or the RECK proteins.

[0089] According to embodiments, the mutant Wnt7 protein disclosed herein is not able to bind FZD and / or LPR if GPR124 and / or RECK is absent.

[0090] In embodiments, the absence of GPR124 and / or RECK proteins may denote to a cell expressing FZD and / or LRP proteins (FZD / LRP positive) but having no or low expression of GPR124 and / or RECK proteins (GRP124 / RECK negative).

[0091] In embodiments, GRP124 / RECK negative can mean the no or low expression of GPR124 and / or RECK in a cell, no or low presence of GPR124 and / or RECK proteins in a cell and / or no or low presence of GPR124 and / or RECK proteins at a cell surface. In embodiments, GRP124 / RECK positive can mean the expression of GPR124 and / or RECK in a cell, presence of GPR124 and / or RECK proteins in a cell and / or presence of GPR124 and / or RECK proteins at a cell surface.

[0092] In embodiments, FZD / LRP positive can mean the expression of FZD and / or LRP in a cell, presence of FZD and / or LRP protein in a cell, and / or presence of FZD and / or LRP proteins at a cell surface.

[0093] In particular embodiments, cells expressing GPR124, RECK, FZD and LRP proteins at its plasma membrane is a cell naturally expressing all GPR124, RECK, FZD and LRP proteins at the cell surface, such as a cerebral endothelial cell.

[0094] In some embodiments "the presence of RECK and GPR124" may refer to the presence of the RECK and GPR124 proteins at or close to the cell membrane, preferably in close proximity with the Frizzled and LRP proteins. The close mutual proximity can facilitate the formation of the GPR124 / RECK / Frizzled / LRP receptor complex under conditions conducive thereto, such as a when a mutant Wnt7 protein as taught herein is externally supplied to a cell. In embodiments, when used in gene or RNA therapy, a mutant Wnt7 protein as taught herein could be expressed from the target cell population. On the other hand, the phrase "the absence of RECK and / or GPR124" may refer to lack or non-occurrence of the RECK and / or GPR124 proteins at the cell membrane. The 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 protein at the cell membrane, but may for example refer to an amount of RECK and / or GPR124 protein 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.

[0095] In some embodiments, the mutant Wnt7 protein as disclosed here should first bind to RECK and / or GPR124 to be able to bind FZD and / or LRP proteins.

[0096] In embodiments, said mutant Wnt7 protein is capable of activating GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling, wherein said mutant Wnt7 protein does not activate Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124. In particular embodiments, the capability of the mutant Wnt7 protein 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 mutant Wnt7 protein to activate Wnt signaling in cells positive for GPR124, RECK, FZD and LRP, but not in cells positive for FZ and LRP and negative for GPR124 and / or RECK, wherein the cells positive GPR124, RECK, FZ and LRP and the cells positive for FZD and LRP and negative for GPR124 and / or RECK are otherwise substantially identical.

[0097] In particular embodiments, the capability of the Wnt7 protein 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 said mutant Wnt7 protein to activate canonical Wnt signaling in the presence of RECK and GPR124, but not in the absence of RECK and / or GPR124.

[0098] The capability of activating Wnt signaling refers to the ability of the protein 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 an FZD / LRP complex.

[0099] 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. 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, n-myc, LEF1, or c-jun. 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.

[0100] In particular embodiments, the mutant Wnt7 protein as disclosed herein may be considered capable of activating (canonical) Wnt signaling if the compound 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 / 0- catenin signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.

[0101] In particular embodiments, the mutant Wnt7 protein as disclosed herein may be considered to not activate (canonical) Wnt signaling if the compound enhances Wnt / 0-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 protein 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 / 0-catenin signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.

[0102] In particular embodiments, the mutant Wnt7 protein as disclosed herein may be considered to activate the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling (denoted "on-target activity"), but not activate Frizzled / LRP-mediated Wnt in the absence of RECK and / or GPR124 (denoted "off-target activity"), if the on-target signaling activity is induced at least 30% and off-target activity is induced less than 20% by said mutant Wnt7 protein. In embodiments, said mutant Wnt7 protein can activate GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99% and all ranges and subranges therein, preferably more than 70%.

[0103] In embodiments, said mutant Wnt7 protein can activate Frizzled / LRP-mediated Wnt in the absence of RECK and / or GPR124 less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1% and all ranges and subranges therein, preferable less than 10%. 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 mutant Wnt7 protein may be normalized to activity 1 and activity 2 induced by wildtype Wnt7a, respectively, the latter for example set to represent 100% activity.

[0104] In an embodiment, the mutant Wnt7 protein of the present invention acts by Wnt7- specific RECK / GPR124 / Frizzled / LRP-mediated signaling, in which said mutant Wnt7 (Wnt7a or Wnt7b) binds specifically to RECK in a FZD-independent manner and GPR124, a RECK binding partner, bridges RECK-bound Wnt7 to the FZD / LRP complex via intracellular DVL scaffolds, thereby assembling Wnt7-ligand specific RECK / GPR124 / FZD / LRP signalosomes and activating canonical Wnt signaling.

[0105] 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.

[0106] Activation of the Wnt signaling pathway may occur by promoting the close association or mutual proximity of the Frizzled and LRP proteins at the cell membrane, thereby forming membrane-associated hetero-oligomers comprising the Frizzled and LRP proteins (ligand-driven formation of the Frizzled-LRP heterooligomer).

[0107] In some tissues expressing GPR124 and / or RECK such as cerebral ECs, a second Wnt7a / b-specific receptor complex is formed to activate Wnt / p-catenin signaling. RECK stabilizes the ligand in a signaling competent lipophilic conformation and delivers it to Frizzled receptors via GPR124. Thereby, RECK and GPR124 synergistically stimulate Wnt7a / b specific responses by assembling higher-order GPR124 / RECK / FZD / LRP5 / 6 complex.

[0108] According to embodiments, the mutant Wnt7 protein of disclosed invention induces the heteromerization of Frizzled and LRP proteins in the presence of RECK and / or GPR124. The Wnt7 protein of disclosed invention preferably first binds to the RECK protein to induce the heteromerization of Frizzled and LRP protein, more preferably first binds to the RECK / GPR124 complex to induce the heteromerization of Frizzled and LRP protein. According to embodiments, the mutant Wnt7 protein of the disclosed invention is not able to induce heteromerization of Frizzled and LRP proteins in the absence of RECK and / or GPR124, more specifically said Wnt7 protein is not able to induce heteromerization of Frizzled and LRP proteins when it is not bound to RECK and / or when it is not bound to GPR124 protein and / or when it is not bound to GPR124 / RECK complex.

[0109] Accordingly, in particular embodiments, the mutant Wnt7 protein as disclosed herein is able to bind to Frizzled and LRP proteins, preferably at a cell membrane, in the presence of RECK and GPR124, but is not able to bind to said proteins in the absence of RECK and / or GPR124.

[0110] In particular embodiments, the mutant Wnt7 protein as disclosed herein is capable of binding to one or more different Frizzled proteins, such as one or more Frizzled proteins selected from the group consisting of Fzd 1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and FzdlO. Preferably, the compound as disclosed herein may be capable of specifically binding to at least Fzd4, and optionally to one or more other Fzd; or may be capable of specifically binding to Fzd4 substantially to the exclusion of other Fzd. Fzd4 is believed to be the dominant Fzd family member in endothelial cells of the central nervous system. More preferably, the compound as disclosed herein is capable of specifically binding to human Fzd4. The compound as disclosed herein may be selective for the one or more preferred Frizzled proteins, for example having specificity for the one or more preferred Frizzled proteins 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 proteins.

[0111] In particular embodiments, the mutant Wnt7 protein as disclosed herein is capable of binding to one or more different LRP proteins involved in Wnt signaling. Preferably, the protein 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 protein 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 protein as disclosed herein may be selective for the one or more preferred LRP proteins, for example having a specificity for the one or more preferred LRP proteins 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 IxlO5- fold, compared to other non-preferred LRP proteins. In particular embodiments, the mutant Wnt7 protein as disclosed herein is capable of concurrently binding to Frizzled and LRP proteins, and in addition to the GPR124 and / or the RECK protein.

[0112] 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, in embodiments, the protein capable of binding to the RECK protein 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 protein, preferably said protein binds to the CK4 and / or CK5 regions of the RECK protein.

[0113] In particular embodiments, the mutant Wnt7 protein of the invention as disclosed herein 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 protein. In preferred embodiments, the protein as disclosed herein has at least 70% of the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling activity of the full-length wild-type Wnt7 protein. In particular embodiments, the mutant Wnt7 protein of the invention as disclosed herein may activate GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling in the presence of RECK and / or GPR124 more than 30% of the activation by full-length wild-type Wnt7 protein. For example, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99% and all ranges and subranges therein.

[0114] In particular embodiments, the mutant Wnt7 protein of the invention as disclosed herein may activate Wnt signaling in the absence of RECK and / or GPR124 less than 20% of the activation by full-length wild-type Wnt7 protein. For example, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1% and all ranges and subranges therein.

[0115] In certain embodiments, in the mutant Wnt7 protein having at its C-terminal end an amino acid addition, the glutamine (Q) residue at the position corresponding to position 17 in SEQ ID NO: 1; the isoleucine (I) residue at the position corresponding to position 20 in SEQ ID NO: 1; the proline (P) residue at the position corresponding to position 25 in SEQ ID NO: 1; the alanine (A) residue at the position corresponding to position 27 in SEQ ID NO: 1; the isoleucine (I) residue at position corresponding to position 28 in SEQ ID NO: 1; the glutamate (E) residue at the position corresponding to position 33 in SEQ ID NO: 1; the methionine (M) residue at the position corresponding to position 37 in SEQ ID NO: 1; the leucine (L) residue at the position corresponding to position 39 in SEQ ID NO: 1; the glutamate (E) residue at the position corresponding to position 41 in SEQ ID NO: 1; the phenylalanine (F) residue at the position corresponding to position 44 in SEQ ID NO: 1; the arginine (R) residue at the position corresponding to position 50 in SEQ ID NO: 1; the asparagine (N) residue at the position corresponding to position 52 in SEQ ID NO: 1; the valine (V) residue at the position corresponding to position 68 in SEQ ID NO: 1; the isoleucine (I) residue at the position corresponding to position 129 in SEQ ID NO: 1; the phenylalanine (F) residue at the position corresponding to position 131 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 133 in SEQ ID NO: 1; the phenylalanine (F) residue at the position corresponding to position 135 in SEQ ID NO: 1; the isoleucine (I) residue at the position corresponding to position 141 in SEQ ID NO: 1; the arginine (R) residue at the position corresponding to position 146 in SEQ ID NO: 1; the arginine (R) residue at the position corresponding to position 158 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 181 in SEQ ID NO: 1; the arginine (R) residue at the position corresponding to position 191 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 198 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 200 in SEQ ID NO: 1; the valine (V) residue at the position corresponding to position 205 in SEQ ID NO: 1; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 1; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 1; the arginine (R) residue at the position corresponding to position 214 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 216 in SEQ ID NO: 1; the proline (P) residue at the position corresponding to position 218 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 222 in SEQ ID NO: 1; the isoleucine (I) residue at the position corresponding to position 223 in SEQ ID NO: 1; the tyrosine (Y) residue at the position corresponding to position 229 in SEQ ID NO: 1; the proline (P) residue at the position corresponding to position 232 in SEQ ID NO: 1; the threonine (T) residue at the position corresponding to position 235 in SEQ ID NO: 1; the glutamate (E) residue at the position corresponding to position 248 in SEQ ID NO: 1; the arginine (R) residue at the position corresponding to position 289 in SEQ ID NO: 1; the tryptophan (W) residue at the position corresponding to position 291 in SEQ ID NO: 1; the threonine (T) residue at the position corresponding to position 307 in SEQ ID NO: 1; and / or the lysine (K) residue at the position corresponding to position 318 in SEQ ID NO: 1, 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

[0116] (Q) residue, more preferably by an alanine (A) residue.

[0117] In certain embodiments, in the mutant Wnt7 protein having at its C-terminal end an amino acid addition, the glutamine (Q) residue at the position corresponding to position 17 in SEQ ID NO: 1; the isoleucine (I) residue at the position corresponding to position 20 in SEQ ID NO: 1; the proline (P) residue at the position corresponding to position 25 in SEQ ID NO: 1; the isoleucine (I) residue at position corresponding to position 28 in SEQ ID NO: 1; the glutamate (E) residue at the position corresponding to position 33 in SEQ ID NO: 1; the methionine (M) residue at the position corresponding to position 37 in SEQ ID NO: 1; the leucine (L) residue at the position corresponding to position 39 in SEQ ID NO: 1; the glutamate (E) residue at the position corresponding to position 41 in SEQ ID NO: 1; the phenylalanine (F) residue at the position corresponding to position 44 in SEQ ID NO: 1; the arginine

[0118] (R) residue at the position corresponding to position 50 in SEQ ID NO: 1; the valine

[0119] (V) residue at the position corresponding to position 68 in SEQ ID NO: 1; the isoleucine (I) residue at the position corresponding to position 129 in SEQ ID NO: 1; the phenylalanine (F) residue at the position corresponding to position 131 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 133 in SEQ ID NO: 1; the phenylalanine (F) residue at the position corresponding to position 135 in SEQ ID NO: 1; the isoleucine (I) residue at the position corresponding to position 141 in SEQ ID NO: 1; the arginine (R) residue at the position corresponding to position 146 in SEQ ID NO: 1; the arginine (R) residue at the position corresponding to position 158 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 181 in SEQ ID NO: 1; the arginine (R) residue at the position corresponding to position 191 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 198 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 200 in SEQ ID NO: 1; the valine (V) residue at the position corresponding to position 205 in SEQ ID NO: 1; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 1; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 1; the arginine (R) residue at the position corresponding to position 214 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 216 in SEQ ID NO: 1; the proline (P) residue at the position corresponding to position 218 in SEQ ID NO: 1; the lysine (K) residue at the position corresponding to position 222 in SEQ ID NO: 1; the isoleucine (I) residue at the position corresponding to position 223 in SEQ ID NO: 1; the tyrosine (Y) residue at the position corresponding to position 229 in SEQ ID NO: 1; the proline (P) residue at the position corresponding to position 232 in SEQ ID NO: 1; the threonine (T) residue at the position corresponding to position 235 in SEQ ID NO: 1; the glutamate (E) residue at the position corresponding to position 248 in SEQ ID NO: 1; the arginine (R) residue at the position corresponding to position 289 in SEQ ID NO: 1; the tryptophan (W) residue at the position corresponding to position 291 in SEQ ID NO: 1; the threonine (T) residue at the position corresponding to position 307 in SEQ ID NO: 1; and / or the lysine (K) residue at the position corresponding to position 318 in SEQ ID NO: 1, is substituted by an alanine (A) residue; and / or the alanine (A) residue at the position corresponding to position 27 in SEQ ID NO: 1 is substituted by an arginine (R) residue; and / or the asparagine (N) residue at the position corresponding to position 52 in SEQ ID NO: 1 is substituted by a glutamine (Q) residue; and / or the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1 is substituted by an alanine (A), a serine (S) or a leucine (L) residue.

[0120] In certain embodiments, the mutant Wnt7 protein comprises two or more (e.g., preferably two, preferably three, more preferably four) of the amino acid substitutions listed above.

[0121] Hence, in particular embodiments, the mutant Wnt7 protein has amino acid sequence having at least at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 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.

[0122] In particular embodiments, the mutant Wnt7a protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 5 to SEQ ID NO: 46, preferably SEQ ID NO: 25, or preferably SEQ ID NO: 13.

[0123] Sequences below are amino acid sequences of mature mutant Wnt7a proteins, wherein one amino acid residue has been substituted by another residue (in bold). hWnt7aQ17A:

[0124] LGASIICNKIPGLAPRARAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 5) hWnt7aI20A:

[0125] LGASIICNKIPGLAPRQRAACQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 6) hWnt7aP25A:

[0126] LGASIICNKIPGLAPRQRAICQSRADAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 7) hWnt7aA27R:

[0127] LGASIICNKIPGLAPRQRAICQSRPDRIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 8) hWnt7aI28A:

[0128] LGASIICNKIPGLAPRQRAICQSRPDAAIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 9) hWnt7aE33A:

[0129] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGAGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 10) hWnt7aM37A:

[0130] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQAGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 11) hWnt7aL39A:

[0131] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGADECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 12) hWnt7aE41A:

[0132] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDACQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 13) hWnt7aF44A:

[0133] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQAQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 14) hWnt7aR50A:

[0134] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGAWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 15) hWnt7aN52Q:

[0135] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWQCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 16) hWnt7aV68A:

[0136] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKAG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 17) hWnt7aI129A:

[0137] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGAGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 18) hWnt7aF131A:

[0138] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGAAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 19) hWnt7aK133A:

[0139] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAAVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 20) hWnt7aF135A:

[0140] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVAVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 21) hWnt7aI141A:

[0141] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REAKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 22) hWnt7aR146A:

[0142] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNAATLM N LH N N EAGRKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 23) hWnt7aR158A:

[0143] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAGAKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 24) hWnt7aK159A:

[0144] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLM N LHN N EAGRAILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 25) hWnt7aK159L:

[0145] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLMNLHNNEAGRLILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 26) hWnt7aK159S:

[0146] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLMNLHNNEAGRSILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 27) hWnt7aK181A:

[0147] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLMNLHNN EAGRKILEENMKLECKCHGVSGSCTTATCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 28) hWnt7aR191A:

[0148] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFAELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 29) hWnt7aK198A:

[0149] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLADKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 30) hWnt7aK200A:

[0150] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDAYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 31) hWnt7aV205A:

[0151] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAAH V EPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYEEDPVTGSVGTQGRACNKTAPQASGCDLMC CGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 32) hWnt7aE208A:

[0152] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV APVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 33) hWnt7aR214A:

[0153] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASANKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 34) hWnt7aK216A:

[0154] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNARPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 35) hWnt7aP218A:

[0155] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRATFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 36) hWnt7aK222A:

[0156] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLAIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 37) hWnt7aI223A:

[0157] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKAKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 38) hWnt7aY229A:

[0158] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSARKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 39) hWnt7aP232A:

[0159] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKAMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 40) hWnt7aT235A:

[0160] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKPMDADLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 41) hWnt7aE248A:

[0161] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCAEDPVTGSVGTQGRACN KTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 42) hWnt7aR289A:

[0162] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYAAVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 43) hWnt7aW291A:

[0163] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVAQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 44) hWnt7aT307A:

[0164] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNACSERTEMYTCK (SEQ ID NO: 45) hWnt7aK318A:

[0165] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCA (SEQ ID NO: 46)

[0166] Table 1 gives an overview of the position of amino acid substitutions in the amino acid sequence of the (mouse) Wnt7a precursor polypeptide and their corresponding position in the amino acid sequence of the mature (mouse) Wnt7a polypeptide:

[0167] Table 1

[0168] In particularly preferred embodiments, in such mutant Wnt7a protein, the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1 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. It will be clear to a person skilled in the art that said K residue at the position corresponding to position 159 in SEQ ID NO: 1, relates to the K residue at position 190 in the Wnt7a precursor polypeptide, as shown in Table 1.

[0169] The invention thus also relates to mutant Wnt7 proteins, wherein the amino acid sequence of said mutant Wnt7 proteins has one amino acid substitution compared to a sequence according to SEQ ID NO: 1 or SEQ ID NO:2, and said mutants comprise at their C-terminal end an amino acid addition, having a length of 1 to 8 amino acids.

[0170] In an embodiment, the C-terminal amino acid addition is as described above in any of the embodiments. In another or further embodiment, the amino acid substitution can be as described above in any of the embodiments.

[0171] In a preferred embodiment, the C-terminal amino acid addition has a length of one amino acid, and preferably is an alanine.

[0172] In a preferred embodiment, as amino acid substitution, the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1 is substituted by an alanine (A), serine (S) or leucine (L) residue, more preferably by an alanine (A) residue.

[0173] In a preferred embodiment, the C-terminal amino acid addition has a length of one amino acid, wherein said amino acid is chosen from an alanine and a glycine, and the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1 is substituted by an alanine (A), serine (S) or leucine (L) residue, most preferably by an alanine (A) residue.

[0174] In a second aspect, the invention relates to a nucleic acid encoding the mutant Wnt7 protein as described in any of the embodiments described above. Said nucleic acid may be inserted into a nucleic acid expression cassette and / or vector, as is well- known in the art.

[0175] Nucleic acid can refer to deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), wherein said RNA is preferably an mRNA.

[0176] Accordingly, a further aspect relates to a nucleic acid expression cassette comprising the nucleic acid encoding the mutant Wnt7 protein as disclosed herein, operably linked to a promoter and / or transcriptional and translational regulatory signals.

[0177] Preferably, the nucleic acid expression cassette may comprise one or more open reading frames (ORF) encoding said one or more proteins, polypeptides or peptides.

[0178] 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.

[0179] In particular embodiments, the nucleic acid expression cassette comprises the nucleic acid encoding the mutant Wnt7 protein as disclosed herein, operably linked to one or more promoters, enhancers, ORFs and / or transcription terminators.

[0180] A further aspect relates to vector comprising the nucleic acid encoding the mutant Wnt7 protein as disclosed herein, or the nucleic acid expression cassette as disclosed herein, such as a viral vector.

[0181] 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 recognized 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. Expression vectors can be autonomous or integrative. A nucleic acid can be 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 extra-chromosomally 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 ARS18 or ARS68.

[0182] 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. 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.

[0183] In other particular embodiments, the vector comprising the nucleic acid as described herein is a viral vector, preferably a viral vector specifically directed towards the central and / or peripheral nervous system (e.g., a brain-specific viral vector). In further particular embodiments, the viral vector is a central nervous system (CNS) neuron-specific adeno-associated virus serotype 9 (AAV9) mutant. In preferred embodiments, the viral vector is a blood brain barrier endothelial cellspecific viral vector. In further preferred embodiments, the viral vector is a blood brain barrier endothelial cell-specific capsid adeno-associated virus serotype 2 (AAV2) mutant.

[0184] In embodiments the mutant Wnt7 protein, the nucleic acid, the nucleic acid expression cassette and / or the vector disclosed herein can be used in a method of activating canonical Wnt signaling in the presence of RECK and GPR124, but not in the absence of RECK and / or GPR124, comprising administering to a mammal, a therapeutically effective amount of at least one of said molecules.

[0185] Pharmaceutical Compositions and Formulations

[0186] Disclosed herein are pharmaceutical compositions comprising the mutant Wnt7 protein, the nucleic acid, the nucleic acid expression cassette, or the vector as described herein in any of the embodiments, with an optional a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0187] In embodiments, the pharmaceutical composition may comprise a protein, such as the mutant Wnt7 protein as disclosed herein. Such protein may be suitably obtained through expression by host cells or host organisms, transformed with an expression construct encoding and configured for expression of said protein in said host cells or host organisms, followed by purification of the protein.

[0188] Hence, a further aspect provides a host cell comprising the nucleic acid, nucleic acid expression cassette or vector as taught herein.

[0189] In certain embodiments, the host cell may be a bacterial cell, a yeast cell, an animal cell, or a mammalian cell. The mutant Wnt7 protein may be isolated from such cells using any suitable method known to the skilled person.

[0190] In embodiments, the pharmaceutical composition as disclosed herein can further comprise a pharmaceutically acceptable carrier.

[0191] 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. 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.

[0192] 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 surfactant-containing vesicles, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to one of ordinary skill in the art.

[0193] In particular embodiments, the mutant Wnt7 protein, the nucleic acid, the nucleic acid expression cassette, or the vector of the pharmaceutical composition is provided in a liposome or lipid nanoparticle.

[0194] 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, intrathecal or intracisternal injection or infusion), transdermal or transmucosal (such as, e.g., oral, sublingual, intranasal) administration, topical administration, rectal, vaginal or intratracheal 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 on the specific needs of a given application.

[0195] 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. For example, for oral or nasal aerosol or inhalation administration, pharmaceutical compositions may be formulated for administration in the form of aerosols or sprays, for example, as solutions, suspensions or emulsions of the compound 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.

[0196] Examples of carriers for administration via mucosal surfaces depend upon the particular route, e.g., oral, sublingual, intranasal, etc. and are generally known in the art.

[0197] For example, for parenteral administration, pharmaceutical compositions may be advantageously formulated as solutions, suspensions or emulsions with suitable solvents, diluents, solubilisers or emulsifiers, etc. as generally known in the art. Injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents. The compound 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.

[0198] 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. Various types of phospholipids are known in the art. 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.

[0199] In preferred embodiments, the pharmaceutical composition comprising the mutant Wnt7 protein or nucleic acid encoding the protein, as taught herein is administered parenterally. More preferably, the pharmaceutical composition as taught herein is administered intravenously, for example by infusion, or intrathecally.

[0200] When rectally administered in the form of suppositories, these formulations may be prepared by mixing the compounds according to the invention with a suitable nonirritating 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.

[0201] In embodiments the pharmaceutical composition can be used in a method of activating canonical Wnt signaling in the presence of RECK and GPR124, but not in the absence of RECK and / or GPR124, comprising administering to a mammal, a therapeutically effective amount of at least one of said mutant Wnt7 proteins, nucleic acids, or vectors.

[0202] 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.

[0203] It will be obvious for a skilled person that the aspects as described in the "molecules"-section above for the determination, analysis and quantification of activation of Wnt signaling are suitable here as well.

[0204] Methods Of Treatment and Use Of Compounds Or Compositions

[0205] Also disclosed herein are methods of treatment and usages of the compounds and / or compositions disclosed herein. More particularly, disclosed herein are proteins, nucleic acids, expression cassettes, vectors and / or pharmaceutical compositions as described herein for use as a human or veterinary medicament.

[0206] In the following paragraphs, the term "compound(s)" is used to refer to either of said mutant Wnt7 proteins, nucleic acids, expression cassettes, vectors as disclosed above in any of the embodiments.

[0207] The compounds and pharmaceutical composition disclosed herein allow to provide agonists or modulators capable of stimulating Wnt / 0-catenin signaling in cerebral endothelial cells substantially without cross-reactivity with other Frizzled pathways, and useful as therapeutics, particularly for neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction. In particular embodiments, said compounds and pharmaceutical compositions, as disclosed herein, are also suitable to be used in the treatment or prevention of a disorder presenting an abnormal Wnt signaling in RECK and / or GPR124 expressing cells, tissues and / or organs.

[0208] In particular embodiments, said neurovascular disorder is selected from the group consisting of ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, brain aneurysms, arteriovenous malformations (AVMs), cavernous malformations, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformations, carotid artery stenosis, Moyamoya disease and intracranial atherosclerosis, and retinal vascular disorders, including but not limited to Norrie disease, familial exudative vitreoretinopathy, osteoporosis-pseudoglioma syndrome, diabetic retinopathy and macular degeneration and combinations thereof.

[0209] In particular embodiments, said CNS disorder comprising neurovascular dysfunction is selected from the group consisting of multiple sclerosis, ischemic stroke, brain cancer, glioblastoma, human monogenic neurological disorders (e.g. SLC2A1), epilepsy, neurodegenerative disorders, dementia, vascular dementia, HIV-1- associated dementia, Alzheimer's disease, Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, infectious brain diseases, traumatic brain injuries, migraine, chronic traumatic encephalopathy, neuroinflammation, neurocovid, and combinations thereof.

[0210] 'Neuroinflammation' can refer to a neurovascular inflammation. Further, neuroinflammation can be triggered by different factors. For example, in non-limiting embodiments neuroinflammation is caused by an injury such as traumatic brain injury and / or spinal cord injury, an infection such as viral or bacterial or fungal infection, exposure to a toxin or due to toxic metabolites, neurodegenerative disease, autoimmune disease, smoking or passive smoking, aging or any combination thereof.

[0211] Neuroinflammation can be caused by an infectious disease. Said infection may be of bacterial, fungal, parasitic or viral origin. In some instances, the infectious disease is a viral infection. In certain instances, the viral infection comprises infection by one or more of Herpesviridae (Herpes Zoster Virus (VZV) infection, Herpes Simplex Virus (HSV-1 or HSV-2), cytomegalovirus (CMV)), Paramoxyviridae, coronaviruses (SARS- CoV-1 or MERS-CoV or SARS-CoV-2, HCoVNL63, HCoV-229E, HCoV-OC43, or HKU1 or a variant thereof), influenza viruses (influenza virus A, group 1 (H1N1), group 2 (H2N2), group 3 (H3N2), group 5 (H5N1, H5N2, H5N8) or group 7 (H7N7, H7N9), influenza B, influenza C), Zika virus, Japanese encephalitis virus, Epstein-Barr virus, Hepatitis B virus, Hepatitis C virus, Human immunodeficiency virus type 1 (HIV-1), Human papilloma virus (HPV), Human T-cell lymphotropic virus type I (HTLV-1) and Kaposi sarcoma herpesvirus (KSHV).

[0212] Neuroinflammation can be associated with lung condition such as, acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS), possibly associated with or arising from ventilator use, viral infection, sepsis, or systemic bacterial infections in a subject in need thereof have been established. Said lung condition can be caused by an infectious disease caused by bacterial, fungal, parasitic or viral infection. Preferably said infection is an infection causing respiratory problems or is a respiratory tract infection. Preferably said infection is a viral infection.

[0213] Neuroinflammation can be associated with a bacterial infection. Such inflammation is mostly caused by blood-borne bacteria that cross the blood-brain barrier (BBB) and finally invade the brain parenchyma. Pathogens such as Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae are the main etiological causes of bacterial meningitis.

[0214] In the present context, prevention and / or treatment of neuroinflammation may include encephalitis. In certain instances, the encephalitis is viral encephalitis, such as COVID-19 associated encephalitis. Neuroinflammation may also be related to inflammation of the optic nerve. Neuroinflammation may also be related to acute disseminated encephalomyelitis, such as COVID-19 associated acute disseminated encephalomyelitis. Neuroinflammation may also be related to inflammation of the vitreous retinal ganglion cells.

[0215] In embodiments said compounds and compositions are suitable particularly suitable to be administered to a patient prone to develop or suffering from such disorder.

[0216] In particular embodiments, the mutant Wnt7a protein, the nucleic acid encoding the mutant Wnt7a protein, or the nucleic acid expression cassette comprising the nucleic acid as taught herein is used in gene therapy, in particular in blood brain barrier endothelial cell-directed gene therapy.

[0217] Accordingly, also provided herein is a method for gene therapy, in particular central and / or peripheral nervous system-directed gene therapy, in a subject in need of said gene therapy comprising: introducing in the subject, in particular in the central and / or peripheral nervous system of the subject, a nucleic acid expression cassette or a vector as described herein; and expressing a therapeutically effective amount of the mutant Wnt7a protein encoded by the nucleic acid as taught herein in the subject, in particular the central and / or peripheral nervous system of the subject.

[0218] In particular embodiments, the mutant Wnt7a protein or the nucleic acid encoding the mutant Wnt7a protein as taught herein is used in mRNA therapy, in particular in blood brain barrier endothelial cell-directed mRNA therapy.

[0219] Accordingly, also provided herein is a method for RNA therapy, preferably mRNA therapy, in particular central and / or peripheral nervous system-directed mRNA therapy, in a subject in need of said mRNA therapy comprising: introducing in the subject, in particular in the central and / or peripheral nervous system of the subject, a nucleic acid encoding the mutant Wnt7a protein as taught therein; and expressing a therapeutically effective amount of the mutant Wnt7a protein encoded by the nucleic acid as taught herein in the subject, in particular the central and / or peripheral nervous system of the subject.

[0220] An advantage of the use of RNAs therapy, it is generally believed that RNAs do not integrate into the genome and therefore do not have the risk of insertional mutagenesis.

[0221] Any other well-known methods of introducing nucleic acids into animal cells may be used herein. At the simplest, the nucleic acid can be directly injected into the target cell I 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 (NA) 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 or providing 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.

[0222] In particular embodiments, the compound is provided in carriers, such as liposomes, lipid nanoparticles, nanostructured lipid carriers, nanoemulsions, polymer nanoparticles, polymer micelle or dendrimers. In a preferred embodiment, said carriers are liposomes or lipid nanoparticles (LNPs).

[0223] In an embodiment, said carriers are lipid-based carriers. Said lipid-based carriers comprise one or more lipids. The one or more lipids can be in solid and / or liquid form. Said lipid-based carriers may be LNPs, lipoplexes, liposomes, phospholipid micelles, solid lipid nanoparticles, nanostructured lipid carriers or nano-emulsions. Lipid-based carriers useful according to the invention include, for example, cationic lipids, liposomes, in particular cationic liposomes, and micelles, and nanoparticles. Cationic lipids may form complexes with negatively charged nucleic acids. Any cationic lipid may be used according to the invention. Liposomes are phospholipid and cholesterol self-assembled bilayer membranes that enclose an aqueous core, where hydrophilic molecules can be incorporated. Hydrophobic compounds can also be incorporated in the lipid bilayer. Liposomes can be classified in (i) small unilamellar vesicles (SUVs); (ii) large unilamellar vesicles (LUVs) and (iii) multilamellar vesicles (MLVs), according to their size and lamellarity. Solid lipid nanoparticles (SLNs) have a spherical shape with an average diameter of 10-1000 nm. They are used as a colloidal NP drug delivery system in which lipid drug carrier solidifies at room temperature as well as at body temperature. Different solid lipids can be exploited to produce SLNs, such as, tripalmitin, cetyl alcohol, cetyl palmitate, glyceryl monostearate, trimyristin, tristearin, stearic acid, etc. SLNs comprise of solid lipid, such as triglycerides, fatty acids, waxes, partial glycerides, and polyethylene glycosylated lipid; emulsifiers, such as polysorbates, poloxamer and lecithin; and water. Nanostructured lipid carriers (NLC), comprise a blend of solid and liquid lipids which results in a partially crystallized lipid system and many have advantages such as enhanced drug loading capacity, drug release modulation flexibility and improved stability.

[0224] In an embodiment, said lipid-based carrier is a lipid nanoparticle. Solid lipid nanoparticles (SLNs, sLNPs), or lipid nanoparticles (LNPs), are nanoparticles composed of lipids that are suited to be used as a drug delivery vehicle for drug compounds, especially polynucleotides such as RIMA or DNA.

[0225] In particular embodiments, 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 compound as taught herein in vivo.

[0226] The dosage or amount of the compound 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 compound described in any of the embodiments above. In order to optimize therapeutic efficacy, the compound as taught herein can be first administered at different dosing regimens. Typically, levels of the compound 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.

[0227] Toxicity and therapeutic efficacy of the compound as described herein or pharmaceutical composition of the invention as described in any of the embodiments, 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.

[0228] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages 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.

[0229] In particular embodiments, the compound 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 compound as taught herein, a pump and infusion means (e.g., a tubing system). For example, the sustained delivery system may be a mini- osmotic pump system implanted in the brain.

[0230] In particular embodiment, the compound as disclosed herein is the main or only active ingredient of the pharmaceutical composition.

[0231] In another embodiment, the pharmaceutical composition as described in any of the embodiments is combined with a second therapy, preferably chosen from surgery, chemotherapy, radiotherapy or immunotherapy. A further aspect relates to the pharmaceutical composition comprising the compound as disclosed herein, the nucleic acid encoding the compound as disclosed herein the nucleic acid expression cassette as disclosed herein or the vector as disclosed herein, for use as a medicament.

[0232] A further aspect relates to the pharmaceutical composition comprising the mutant Wnt7 protein as disclosed herein, the nucleic acid encoding the mutant Wnt7 protein as disclosed herein the nucleic acid expression cassette as disclosed herein or the vector as disclosed herein, for use in the prevention or treatment of a neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction.

[0233] In particular embodiments, said neurovascular disorder is selected from the group consisting of ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, brain aneurysms, arteriovenous malformations (AVMs), cavernous malformations, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformations, carotid artery stenosis, Moyamoya disease, and intracranial atherosclerosis and combinations thereof, retinal vascular disorders such as Norrie disease, familial exudative vitreoretinopathy, osteoporosis-pseudoglioma syndrome, diabetic retinopathy, macular degeneration and combinations thereof.

[0234] In an embodiment, said neurovascular disorder is retinopathy, wherein said retinopathy is a retinal vascular disease. In an embodiment, said retinal vascular disease is caused by inhibition of vascular development. In an embodiment, said retinopathy is caused by excessive angiogenesis. In an embodiment, said retinal vascular disease is selected from the group consisting of: familiar exudative vitreoretionopathy (FEVR), exudative vitreoretinopathy, Norrie disease, Nome disease, diabetic retinopathy (DR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats disease.

[0235] In particular embodiments, said CNS disorder comprising neurovascular dysfunction is selected from the group consisting of multiple sclerosis, ischemic stroke, brain cancer, glioblastoma, human monogenic neurological disorders (e.g. SLC2A1), epilepsy, neurodegenerative disorders, dementia, vascular dementia, HIV-1- associated dementia, Alzheimer's disease, Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, infectious brain diseases, traumatic brain injuries, migraine, chronic traumatic encephalopathy, neuroinflammation, neurocovid, COVID-19, and combinations thereof.

[0236] 'Neuroinflammation' can refer to a neurovascular inflammation as described above.

[0237] A further aspect provides a method of preventing, reducing in progression or treating a neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction, such as but not limited to the disorders listed above, in a subject in need thereof, comprising administering to a subject a therapeutically active amount of pharmaceutical composition comprising a mutant Wnt7 protein wherein said mutant protein is a selective agonist of Gprl24-and Reck-dependent Wnt / 0-catenin pathway, and comprises at its C-terminal end an amino acid addition having a length of minimal 1 to maximum 8 amino acids, or a nucleic acid encoding for such mutant Wnt7 protein, as described in any of the embodiments throughout this specification.

[0238] It will be clear that the current invention equally provides methods for the treatment of a neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction, as well as is directed to the use of a therapeutically active amount of pharmaceutical composition comprising mutant Wnt7 protein wherein said mutant protein is a selective agonist of Gprl24-and Reck-dependent Wnt / p-catenin pathway, and comprises at its C-terminal end an amino acid addition having a length of minimal 1 to maximum 8 amino acids, or a nucleic acid encoding for such mutant Wnt7 protein, as described in any of the embodiments throughout this specification for the manufacture of a medicament for the treatment or reduction of progression of a neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction in a subject. Further embodiments are as described above.

[0239] 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.

[0240] Synthesis The addition of amino acids to the C-terminus of the protein can be done with any method known to the field. For example, the amino acids can be introduced via site- directed mutagenesis to the nucleic acid sequence encoding the protein, via PCR with overhanging primers and / or cloning into vectors. Protein can be made via translation of said vector, or via de novo protein synthesis.

[0241] EXAMPLES

[0242] The present invention will now be further exemplified with reference to the following examples. The present invention is in no way limited to the given examples or to the embodiments presented in the figures.

[0243] Materials and Methods

[0244] Expression plasmid constructs

[0245] Deletion, insertion, and substitution mutants of mouse Wnt7a (UniProtKB #P24383), mouse Wnt7b (UniProtKB #P28047), and human Wnt7a (UniProtKB #000755), and C-terminal modifications (i.e. amino acid addition), were created using the Q5® Site- Directed Mutagenesis Kit (New England Biolabs®). All modified genes were cloned using pCS2+ vector as template. The identity of all constructs was confirmed by Sanger sequencing.

[0246] Super TOP-Flash reporter gene assay

[0247] Dual luciferase assays were performed using Super TOP-Flash HEK293 luciferase reporter cell lines as known in the art.

[0248] For ON-target setting, cells were transiently transfected with Renilla luciferase (0.5 ng), Wnt7 mutants (20 ng), Fzl receptor (5 ng), Lrp5 (2.5 ng), Gprl24 (10 ng), Reck (10 ng) in Lipofectamine™ 2000 Transfection Reagent (Invitrogen).

[0249] For OFF-target setting, cells were only transfected with plasmids encoding Renilla luciferase (0.5 ng), Wnt7 mutants (20 ng), Fz5 receptor (5 ng), and Lrp5 (2.5 ng). The assays were performed 48h after transfection and the activity was measured. The data shown (mean ± standard deviation (SD)) are pooled from at least three independent experiments each consisting of three technical replicates.

[0250] Example 1 : effect of amino acid addition to the C-terminus of Wnt7a-K190A mutant on "off-target" activation of the canonical Wnt pathway without GPR124 / RECK One to eight amino acids (Figure 1A) were added to the C-terminus of either wild type mouse Wnt7a or mouse Wnt7a mutant K190A. The resulting constructs were transfected in HEK293 STF cells together with Renilla luciferase (0.5ng), Lrp5 (2.5ng) and Fz5 receptor (5 ng) constructs (Off-target - no GPR124 / RECK) and tested in a dual luciferase assay. Briefly, after addition of the plasmids, the total amount of DNA was adjusted to 100 ng per well with the empty pCS2 vector. 48h after transfection, dual luciferase assays were performed using the Dual-Luciferase Reporter Assay system (E1960, Promega) according to the manufacturers' instructions.

[0251] The data showed the relative luciferase activity pooled from at least five independent experiments (each conducted in triplicate) normalized to unmodified Wnt7a (without addition) (Figure IB). The data showed that a one to eight amino acid addition to the K190A mutant is sufficient to make this mutant specific for Gprl24 / Reck agonism, while without amino acid addition, there still is some unwanted off-target activity.

[0252] "On-target" activity remained high after the addition of one to eight amino acids at the C-terminus of the mWnt7a-K190A mutant.

[0253] Example 2: effect of one amino acid addition to the C-terminus of Wnt7a-K190A mutant on "on-target" activation and "off-target" activation of the canonical Wnt pathway either with GPR124 / RECK (on target) or without GPR124 / RECK (off-target)

[0254] The effect of the addition of any of 20 amino acids was then tested on agonism property of mWnt7a-K190A mutant. Each of the 20 different amino acids was added at the C-terminus of the mouse Wnt7a-K190A mutant. The resulting constructs were transfected in HEK293 STF cells together with Renilla luciferase, Lrp5 and Fz5 receptor constructs (Off-target) or Fzl receptor, Gprl24 and Reck constructs (On- target) and tested in a dual luciferase assay as described above. The results are shown in Figure 2.

[0255] Interestingly, the addition of only one amino acids to the C-terminal end of the mWnt7a-K190A mutant protein lead to an undetectable off-target activity, such as is desired for use in the clinic, while the mWnt7a-K190A mutant protein without addition still showed some off-target activity.

[0256] Example 3: effect of one amino acid addition to the C-terminus of various mutant Wnt7 proteins on "on-target" activation and "off-target" activation of the canonical Wnt pathway either with GPR124 / RECK (on target) or without GPR124 / RECK (off- ta rqet)

[0257] In Figure 3A, examples are shown of mWnt7a mutants with a single C-terminal alanine addition exhibiting no or undetectable "off-target" activity. In addition, in Figure 3B examples are shown of mWnt7a mutants with a single C-terminal alanine addition where "on-target" activity remains high and is practically unaffected .

[0258] Example 4: in vitro signaling activity of Wnt7a-K190A with various extensions

[0259] The effect of amino acid extensions on "On- and Off-target" signaling activity was determined by fusing 3 sequences: GKPIPNPL, GSGSGSGS, or AAAAAAAA, and variants thereof of variable lengths (2-4-5-6-8 amino acids) to Wnt7a-K190A (Figure 4).

[0260] The results of the in vitro signaling assay showed that the addition of the three sequences lead to high "On-target" signaling activities, and low to undetectable "Off- target" activity.

[0261] These data highlight that the nature of the exact amino acids included in the C- terminal addition is not important for achieving specific Gprl24 / Reck signaling.

[0262] The present invention is in no way limited to the embodiments described in the examples and / or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

Claims

CLAIMS1. A mutant Wnt7 protein, characterized in that, said mutant comprises at its C- terminal end an amino acid addition, having a length of 1 to 8 amino acids.

2. The mutant Wnt7 protein according to claim 1, wherein said mutant is an agonist, preferably a selective agonist, of Gprl24-and Reck-dependent Wnt / p- catenin pathway.

3. The mutant Wnt7 protein according to claim 1 or 2, wherein the amino acid sequence of said mutant Wnt7 protein comprises one or more substitutions, deletions, truncations, or insertions compared to a sequence according to SEQ ID NO: 1 or SEQ ID NO:2.

4. The mutant Wnt7 protein according to claim 3, wherein the amino acid sequence of said mutant Wnt7 protein comprises one or more amino acid substitutions, preferably one amino acid substitution, compared to a sequence according to SEQ ID NO: 1 or SEQ ID NO:2.

5. The mutant Wnt7 protein according to any one of the previous claims, wherein the amino acids of the C-terminal amino acid addition are chosen from alanine, glycine, proline, serine, or a combination thereof.

6. The mutant Wnt7 protein according to any one of the previous claims, wherein the C-terminal amino acid addition has a length of one amino acid, wherein said amino acid is chosen from an alanine or a glycine.

7. The mutant Wnt7 protein according to any one of the previous claims, wherein the amino acid sequence of said mutant Wnt7 protein comprises one or more amino acid substitutions, preferably one amino acid substitution, compared to a sequence according to SEQ ID NO: 1 or SEQ ID NO:2, and wherein the C-terminal amino acid addition has a length of one amino acid, wherein said amino acid is chosen from an alanine and a glycine.

8. The mutant Wnt7 protein according to any one of the previous claims, wherein :- the glutamine (Q) residue at the position corresponding to position 17 in SEQ ID NO: 1 is substituted by an amino acid residue other than glutamine (Q), preferably by an alanine (A) residue; or- the isoleucine (1) residue at the position corresponding to position 20 in SEQ ID NO: 1 is substituted by an amino acid residue other than isoleucine (1), preferably by an alanine (A) residue; or- the proline (P) residue at the position corresponding to position 25 in SEQ ID NO: 1 is substituted by an amino acid residue other than proline (P), preferably by an alanine (A) residue; or- the alanine (A) residue at the position corresponding to position 27 in SEQ ID NO: 1 is substituted by an amino acid residue other than alanine (A), preferably by an arginine (R) residue; or- the isoleucine (I) residue at position corresponding to position 28 in SEQ ID NO: 1 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue; or- the glutamate (E) residue at the position corresponding to position 33 in SEQ ID NO: 1 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue; or the methionine (M) residue at the position corresponding to position 37 in SEQ ID NO: 1 is substituted by one or more amino acid residues other than methionine (M), preferably by an alanine (A) residue; or- the leucine (L) residue at the position corresponding to position 39 in SEQ ID NO: 1 is substituted by an amino acid residue other than leucine (L), preferably by an alanine (A) residue; or- the glutamate (E) residue at the position corresponding to position 41 in SEQ ID NO: 1 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue; or- the phenylalanine (F) residue at the position corresponding to position 44 in SEQ ID NO: 1 is substituted by an amino acid residue other than phenylalanine (F), preferably by an alanine (A) residue; or- the arginine (R) residue at the position corresponding to position 50 in SEQ ID NO: 1 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue; or- the asparagine (N) residue at the position corresponding to position 52 in SEQ ID NO: 1 is substituted by an amino acid residue other than asparagine (N), preferably by a glutamine (Q) residue; or the valine (V) residue at the position corresponding to position 68 in SEQ ID NO: 1 is substituted by an amino acid residue other than valine (V), preferably by an alanine (A) residue; or- the isoleucine (I) residue at the position corresponding to position 129 in SEQ ID NO: 1 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue; or- the phenylalanine (F) residue at the position corresponding to position 131 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than phenylalanine (F), preferably by an alanine (A) residue; or- the lysine (K) residue at the position corresponding to position 133 in SEQ ID NO: 1 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue; or- the phenylalanine (F) residue at the position corresponding to position 135 in SEQ ID NO: 1 is substituted by an amino acid residue other than phenylalanine (F), preferably by an alanine (A) residue; or- the isoleucine (I) residue at the position corresponding to position 141 in SEQ ID NO: 1 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue; or- the arginine (R) residue at the position corresponding to position 146 in SEQ ID NO: 1 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue; or- the arginine (R) residue at the position corresponding to position 158 in SEQ ID NO: 1 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue; or- the lysine (K) residue at the position corresponding to position 159 in SEQID NO: 1 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A), serine (S) or leucine (L) residue; or- the lysine (K) residue at the position corresponding to position 181 in SEQID NO: 1 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue; or- the arginine (R) residue at the position corresponding to position 191 in SEQ ID NO: 1 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue; or the lysine (K) residue at the position corresponding to position 198 in SEQ ID NO: 1 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue; or- the lysine (K) residue at the position corresponding to position 200 in SEQID NO: 1 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue; or- the valine (V) residue at the position corresponding to position 205 in SEQID NO: 1 is substituted by an amino acid residue other than valine (V), preferably by an alanine (A) residue; or- the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 1 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue; or the arginine (R) residue at the position corresponding to position 214 in SEQ ID NO: 1 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue; or- the lysine (K) residue at the position corresponding to position 216 in SEQ ID NO: 1 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue; or- the proline (P) residue at the position corresponding to position 218 in SEQ ID NO: 1 is substituted by an amino acid residue other than proline (P), preferably by an alanine (A) residue; or- the lysine (K) residue at the position corresponding to position 222 in SEQ ID NO: 1 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue; or- the isoleucine (I) residue at the position corresponding to position 223 in SEQ ID NO: 1 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue; or- the tyrosine (Y) residue at the position corresponding to position 229 in SEQ ID NO: 1 is substituted by an amino acid residue other than tyrosine (Y), preferably by an alanine (A) residue; or- the proline (P) residue at the position corresponding to position 232 in SEQ ID NO: 1 is substituted by an amino acid residue other than proline (P), preferably by an alanine (A) residue; or- the threonine (T) residue at the position corresponding to position 235 in SEQ ID NO: 1 is substituted by an amino acid residue other than threonine (T), preferably by an alanine (A) residue; or- the glutamate (E) residue at the position corresponding to position 248 in SEQ ID NO: 1 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue; or- the arginine (R) residue at the position corresponding to position 289 in SEQ ID NO: 1 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue; or- the tryptophan (W) residue at the position corresponding to position 291 in SEQ ID NO: 1 is substituted by an amino acid residue other than tryptophan (W), preferably by an alanine (A) residue; or- the threonine (T) residue at the position corresponding to position 307 in SEQ ID NO: 1 is substituted by an amino acid residue other than threonine (T), preferably by an alanine (A) residue; or- the lysine (K) residue at the position corresponding to position 318 in SEQ ID NO: 1 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue.

9. The mutant Wnt7 protein according to any one of the previous claims, wherein the amino acid addition has a length of one amino acid, wherein said amino acid is chosen from an alanine and a glycine, and wherein the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1 is substituted by an alanine (A), serine (S) or leucine (L) residue, preferably by an alanine (A) residue.

10. The mutant Wnt7 protein according to any one of the previous claims, having a length of between 349 and 356 amino acids, preferably having a length of 349 amino acids.

11. A nucleic acid encoding for the mutant Wnt7 protein according to any one of claims 1 to 10.

12. A nucleic acid expression cassette comprising the nucleic acid according to claim 11, operably linked to a promoter and / or transcriptional and translational regulatory signals.

13. A vector comprising the nucleic acid according to claim 11, or the nucleic acid expression cassette according to claim 12, such as a viral vector.

14. A pharmaceutical composition comprising the mutant Wnt7 protein according to any one of claims 1 to 10, the nucleic acid according to claim 11, the nucleicacid expression cassette according to claim 12, or the vector according to claim 13, and a pharmaceutically acceptable carrier.

15. The mutant Wnt7 protein according to any one of claims 1 to 10, the nucleic acid according to claim 11, the nucleic acid expression cassette according to claim 12, the vector according to claim 13, or the pharmaceutical composition according to claim 14, for use as a medicament.

16. The nucleic acid according to claim 11, the nucleic acid expression cassette according to claim 12, or the vector according to claim 13, for use in gene therapy, preferably for use in blood brain barrier endothelial cell-directed gene therapy.

17. The nucleic acid according to claim 11, or the vector according to claim 13, for use in RIMA therapy, preferably for use in blood brain barrier endothelial cell- directed RNA therapy.

18. The mutant Wnt7 protein according to any one of the claims 1 to 10, the nucleic acid according to claim 11, the nucleic acid expression cassette according to claim 12, or the vector according to claim 13, for use in the prevention or treatment of a neurovascular disorder, a neuroinflammation disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction.

19. The mutant Wnt7 protein according to any one of the claims 1 to 10, the nucleic acid according to claim 11, the nucleic acid expression cassette according to claim 12, or the vector according to claim 13 for use according to claim 18, wherein said neurovascular disorder is selected from the group consisting of retinopathy, ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, brain aneurysms, arteriovenous malformations (AVMs), cavernous malformations, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformations, carotid artery stenosis, Moyamoya disease and intracranial atherosclerosis and combinations thereof, or said CNS disorder is selected from the group consisting of multiple sclerosis, ischemic stroke, brain cancer, epilepsy, human monogenic neurological disorders, neurodegenerative disorders, dementia, vascular dementia, HIV-l-associated dementia, Alzheimer's disease, Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, infectious brain diseases, traumatic brain injuries, migraine, chronic traumatic encephalopathy, neuroinflammation, COVID-19, and combinations thereof.

20. The mutant Wnt7 protein according to any one of the claims 1 to 10, the nucleic acid according to claim 11, the nucleic acid expression cassette according toclaim 12, or the vector according to claim 13, for use in the treatment of glioblastoma.

21. The mutant Wnt7 protein according to any one of the claims 1 to 10, the nucleic acid according to claim 11, the nucleic acid expression cassette according to claim 12, or the vector according to claim 13, for use in the treatment of retinopathy, wherein said retinopathy is a retinal vascular disease or disorder.

22. The mutant Wnt7 protein for use according to claim 21, wherein said retinal vascular disease or disorder is caused by inhibition of vascular development.

23. The mutant Wnt7 protein for use according to claim 21, wherein said retinal vascular disease or disorder is caused by excessive angiogenesis.

24. The mutant Wnt7 protein for use according to claim 21, wherein the retinal vascular disease or disorder is selected from the group consisting of: familiar exudative vitreoretionopathy (FEVR), exudative vitreoretinopathy, Norrie disease, Nome disease, diabetic retinopathy (DR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), osteoporosis- pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats disease.

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