Mutant WNT7 protein with C-terminal amino acid additive

Abstract

The present invention relates to a mutant Wnt7 protein comprising at the C-terminus an amino acid additive having a length of 1 to 8 amino acids. The invention also relates to nucleic acid for coding the mutant Wnt7 protein, a nucleic acid expression cassette, a carrier and a pharmaceutical composition. And their use in medicine and therapy.

Description

Technical Field

[0001] This invention relates to mutant Wnt7 proteins containing amino acid additives at their C-terminus, said amino acid additives being 1 to 8 amino acids in length. The invention also relates to nucleic acids encoding such mutant proteins and their uses in medicine. Background Technology

[0002] Endothelial Wnt / β-catenin signaling functions as a key regulator of blood-brain barrier (BBB) ​​physiology in response to neurogenic Wnt7a / b ligands. Wnt / β-catenin signaling initiates the BBB differentiation cascade in the initial stages of central nervous system (CNS) vascular invasion and then sustains BBB function into adulthood. Recent evidence suggests that conditional knockout of β-catenin signaling in brain vascular endothelial cells (ECs) to inhibit Wnt signaling leads to BBB disruption and accelerates disease progression in mouse models of stroke, glioblastoma, and multiple sclerosis. Conversely, recombinant constitutive active forms of β-catenin in the CNS endothelium exhibit protective effects in brain cancer and stroke models.

[0003] Wnt7a / b is an endogenous ligand that regulates β-catenin-dependent BBB maturation, and therefore theoretically a rational therapeutic agent for repairing dysfunctional BBB. However, Wnt signaling is activated via Wnt7 ligand in both healthy and disease states, exhibiting pleiotropic effects in multiple tissues and organs. Furthermore, the structural pattern of Wnt / Frizzled (FZD) interactions makes natural Wnt ligands unsuitable as safe therapeutic agents. On the other hand, Wnt7 ligands are known to activate Wnt signaling through two different types of membrane receptor complexes. The first is the Frizzled (FZD) family receptor / LRP5 / 6 complex, which has a wide tissue distribution, nonselectively binds to Wnt7a / b, and leads to systemic activation of Wnt signaling; the second is the GPR124 / RECK / FZ / LRP5 / 6 complex, which is enriched in brain endothelial cells expressing GPR124 and / or RECK.

[0004] Recent proof-of-concept mutation experiments have demonstrated that Wnt7a ligands can be engineered into highly specific GPR124 / RECK agonists, thereby targeting the Wnt signaling pathway in cells expressing GPR124 and RECK (“targeting”) while not inducing Wnt signaling in the absence of GPR124 and / or RECK (“off-target”). These results suggest that engineered Wnt7a ligands show promising potential as therapeutic agents, particularly for CNS-related conditions.

[0005] WO2019180204 and Martin et al. (2022) described Wnt7 mutants that activate Wnt signaling mediated by GPR124 / RECK / Frizzled / LRP, but whose activation of Frizzled / LRP-mediated Wnt signaling is weakened in the absence of RECK and / or GPR124, leading to “off-target” activity. For therapeutic applications, undesirable “off-target” activity should be minimized, especially when targeting pathways such as Wnt signaling, which have broad functions across developmental stages and whose dysregulation is known to be associated with a variety of human diseases, from cancer to bone disorders. Despite previously identified Wnt7 variants exhibiting lower “off-target” activity, there is still a need for more GPR124 / RECK / Frizzled / LRP-specific agonists of Wnt signaling with low off-target effects.

[0006] The present invention aims to address at least some of the problems and disadvantages described above. Therefore, the object of the present invention is to provide more novel Wnt7a or Wnt7b-derived GPR124 / RECK co-receptor complex-specific agonists. Summary of the Invention

[0007] The present invention and its embodiments aim to provide a solution to one or more of the aforementioned disadvantages. To this end, the present invention relates to the mutant Wnt7 protein of claim 1. Specifically, the mutant Wnt7 protein includes an amino acid addition of 1 to 8 amino acids in length at its C-terminus. The mutant is preferably an agonist of the G protein-coupled receptor GPR124 / RECK / Frizzled / lipoprotein receptor-associated protein (LRP) complex. In the absence of RECK and / or GPR124, the mutant cannot bind to Frizzled and / or lipoprotein receptor-associated protein (LRP), or its ability to bind to it is weakened; and / or in the absence of RECK and / or GPR124, the mutant cannot activate Frizzled and / or lipoprotein receptor-associated protein (LRP), or its ability to activate it is weakened.

[0008] Studies have shown that these mutant Wnt7 proteins are particularly potent agonists of GPR124 / RECK-specific signaling, activating, or only partially activating, classical 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. Dysregulation of Wnt signaling levels is associated with a variety of human pathological conditions. Intervention in Wnt signaling pathways often produces a variety of effects, many of which are undesirable ("off-target").

[0009] Since the GPR124 / RECK co-receptor complex is enriched at the level of blood-brain barrier endothelial cells, the mutant Wnt protein of the present invention can selectively (re)activate Wnt signaling in brain endothelial cells ("targeting") without activating unwanted "off-target" Wnt-related pathways.

[0010] A preferred embodiment of the Wnt7 mutant protein of claim 1 is shown in any one of claims 2 to 10.

[0011] The present invention relates to the nucleic acid encoding a mutant Wnt7 protein of claim 11, the nucleic acid expression cassette of claim 12, the vector of claim 13, and the pharmaceutical composition of claim 14 in the following aspects.

[0012] In another aspect, the present invention relates to mutant Wnt7 protein, nucleic acid, nucleic acid cassette, carrier or pharmaceutical composition for use in the medicament of claim 15, for use in the prevention or treatment of neurovascular diseases or central nervous system (CNS) diseases including neurovascular dysfunction as claimed in claim 18, or for use in the treatment of retinopathy as claimed in claim 19.

[0013] In another aspect, the present invention relates to the use of nucleic acids, nucleic acid expression cassettes or vectors for the gene therapy of claim 16.

[0014] In a final aspect, the present invention relates to the use of nucleic acids or vectors for the RNA therapy of claim 17. Attached Figure Description

[0015] The following description of specific embodiments of the present invention is merely illustrative and is not intended to limit the teachings, application, or uses of the invention. In all the figures, corresponding reference numerals denote the same or corresponding parts and features.

[0016] Figure 1 This demonstrates the effect of adding 1 to 8 amino acids to the C-terminus of Wnt7a in wild-type or mutant (K190A) mice on off-target activation of the classical Wnt pathway in the absence of GPR124 / RECK (off-target). The resulting constructs ( Figure 1 A) Co-transfected with Renalis luciferase, Lrp5, and Fz5 receptors (off-target) into HEK293 STF cells and tested using a dual-luciferase assay. Due to C-terminal addition, off-target activity was significantly reduced, even reaching the detection limit. Figure 1 B).

[0017] Figure 2This study demonstrates the effects of adding each of the 20 native amino acids to the C-terminus of Wnt7a in mutant (K190A) mice, in the presence or absence of GPR124 / RECK (targeted) or off-target conditions, on the “targeted” and “off-target” activation of the classical Wnt pathway. For many amino acid additions, the targeting activity remained at a high level, while the off-target activity was significantly reduced, even reaching the detection limit.

[0018] Figure 3 This study demonstrates the effect of adding an alanine residue to the C-terminus of various mutant Wnt7 proteins on the "targeted" and "untargeted" activation of the classical Wnt pathway, under conditions of GPR124 / RECK (targeted) or GPR124 / RECK (untargeted) presence. Targeted activity remained at a high level, while off-target activity was significantly reduced, even reaching the detection limit.

[0019] Figure 4 This study demonstrates the effects of fusing multiple amino acid additives of various lengths (2, 4, 5, 6, or 8 amino acids) to the C-terminus of Wnt7a in mutant (K190A) mice with or without GPR124 / RECK (targeting) on ​​the activation of “targeted” and “off-target” signaling in the classical Wnt pathway. Targeting activity remained at a high level, while off-target activity was significantly reduced, even reaching the detection limit. Detailed Implementation

[0020] This invention also relates to Wnt7-derived proteins as selective agonists of the Gpr124 and Reck-dependent Wnt / β-catenin pathway. Therefore, this invention relates to mutant Wnt7 proteins that are agonists of the G protein-coupled receptor GPR124 / RECK / Frizzled / lipoprotein receptor-associated protein (LRP) complex and, in the absence of RECK and / or GPR124, are unable to bind to or activate Frizzled and / or LRP, or have a diminished ability to bind to or activate them.

[0021] This invention particularly relates to a mutant Wnt7 protein with an amino acid additive at its C-terminus, said amino acid additive being 1 to 8 amino acids in length, preferably one amino acid in length, wherein said amino acid is selected from alanine and glycine. This invention further relates to nucleic acids and vectors encoding said mutant Wnt7 protein, and pharmaceutical compositions comprising such mutant proteins, nucleic acids, and / or vectors, and their use in medicine.

[0022] definition

[0023] Unless otherwise defined, all terms used in disclosing this invention, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For further illustration, definitions of terms are included to better understand the teachings of this invention.

[0024] Peptides, polypeptides, or proteins can be naturally occurring, for example, present in or isolated from nature, such as being naturally or endogenously produced or expressed by cells or tissues and optionally isolated therefrom. Peptides, polypeptides, or proteins can be recombinant, i.e., produced by recombinant DNA technology, and / or can be synthesized partially or wholly by chemical or biochemical methods. Without limitation, peptides, polypeptides, or proteins can be recombinantly produced and optionally isolated from suitable host or host cell expression systems (e.g., suitable bacterial, yeast, fungal, plant, or animal host or host cell expression systems), or produced through cell-free translation or cell-free transcription-translation and recombination, or produced by non-biological peptide, polypeptide, or protein synthesis methods.

[0025] The term "variant" or "mutant" of a protein, polypeptide, peptide, or nucleic acid generally refers to a protein, polypeptide, peptide, or nucleic acid whose amino acid or nucleotide sequence is substantially identical (i.e., substantially identical) to the sequence of the stated protein, polypeptide, peptide, or nucleic acid, for example, at least about 80% or at least about 85% identical, preferably at least about 90% identical, for example at least 91% or 92% identical, more preferably at least about 93% identical, for example at least 94% identical, even more preferably at least about 95% identical, for example at least 96% identical, even more preferably at least about 97% identical, for example at least 98% identical, and most preferably at least 99% identical. Preferably, when the complete sequence of the listed protein, polypeptide, peptide, or nucleic acid is queried in sequence alignment, the variant can show the degree of identity (i.e., overall sequence identity) with the listed protein, polypeptide, peptide, or nucleic acid. Sequence identity can be determined by suitable algorithms known in the art for performing sequence alignment and determining sequence identity. Exemplary but non-limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST), originally described by Altschul et al., 1990 (J MolBiol 215: 403-10), such as the "Blast 2 Sequence" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), for example, using publicly available default settings or other suitable settings (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, expected value = 10.0, word length = 28; or for the BLASTP algorithm: matrix = Blosum62). (Henikoff et al., 1992, Proc. Natl. Acad. Sci., 89: 10915-10919), Empty space open penalty = 11, Empty space expansion penalty = 1, Expected value = 10.0, Word length = 3).

[0026] An example procedure for determining the percentage of identity between a specific amino acid sequence and the amino acid sequence of a query peptide will require using the Blast 2 Sequence (Bl2seq) algorithm. Two amino acid sequences will be aligned using suitable algorithm parameters. This algorithm is available as a web application or as a standalone executable (BLAST version 2.2.31+) at the NCBI website (www.ncbi.nlm.nih.gov). Examples of suitable algorithm parameters include: matrix = Blosum62, vacancy opening penalty = 11, vacancy expansion penalty = 1, expectation = 10.0, word length = 3. If the two compared sequences are homologous, the output will present these homologous regions as the aligned sequences. If the two compared sequences are not homologous, the output will not display the aligned sequences. Once aligned, the number of matches is determined by counting the positions of the same amino acid residues in both sequences. The percentage of identity is determined by dividing the number of matches by the length of the query peptide and then multiplying the result by 100. The identity percentage values ​​can, but do not need to, be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 can be rounded to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 can be rounded to 78.2. It should also be noted that the detailed view of each aligned fragment output by Bl2seq conveniently includes the identity percentage.

[0027] Variants of proteins, polypeptides, peptides, or nucleic acids can be homologs (e.g., orthologs or paralogs) of said proteins, polypeptides, peptides, or nucleic acids. As used herein, the term "homology" generally refers to the structural similarity between two macromolecules from the same or different taxa, where said similarity is attributed to a common ancestor.

[0028] Variants of proteins, polypeptides, or peptides may contain one or more amino acid additions, deletions, or substitutions relative to (i.e., compared to) the corresponding protein or polypeptide. For example, variants of proteins, polypeptides, or peptides (substituted variants) may contain up to 70 (e.g., no more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, 50, 60, or 70) conserved amino acid substitutions relative to (i.e., compared to) the corresponding protein or polypeptide; and / or variants of proteins, polypeptides, or peptides (substituted variants) may contain up to 20 (e.g., no more than one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19) non-conserved amino acid substitutions relative to (i.e., compared to) the corresponding protein or polypeptide.

[0029] Conservative amino acid substitution is the substitution of one amino acid by another amino acid with similar characteristics. Conservative amino acid substitutions include substitutions from 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. Nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (i.e., basic) amino acids include arginine, lysine, and histidine. Negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above polar, basic, or acidic groups by another member of the same group can be considered a conservative substitution. Conversely, nonconservative substitution is the substitution of one amino acid by another amino acid with dissimilar characteristics.

[0030] Alternatively or otherwise, variants of proteins, polypeptides, or peptides (deletion variants) may be derived from (i.e., compared to) the corresponding protein or polypeptide by deleting up to 20 amino acid segments (e.g., segments of one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 segments). Each deleted segment may independently consist of one amino acid, two consecutive amino acids, or three consecutive amino acids. Deletions may be discontinuous, or two or more, or all, of the deleted segments may be continuous.

[0031] Variants of nucleic acids can contain one or more nucleotide additions, deletions, or substitutions relative to the corresponding nucleic acid (i.e., compared to it).

[0032] The term “biologically active” is interchangeable with terms such as “functionally active” or “functional”, indicating that the protein disclosed herein retains at least part of the biological activity or intended function of the corresponding or related peptide, polypeptide, or protein. References to the “activity” of a peptide, polypeptide, or protein may generally encompass any one or more aspects of its biological activity, such as, but not limited to, any one or more aspects of its biochemical activity, enzymatic activity, signal transduction activity, interaction activity, ligand activity, and / or structural activity, for example, in cells, tissues, organs, or organisms.

[0033] Preferably, compared with the corresponding protein, the functionally active protein retains 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%), further 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 expected biological activity or function.

[0034] In some embodiments, the functionally active protein may even exhibit higher biological activity or function compared to the corresponding peptide, polypeptide, or protein, for example, it may exhibit 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 expected biological activity or function compared to the corresponding protein. For example, where the activity of a given protein can be easily measured by a quantitative output assay (e.g., an enzyme assay, a signal transduction assay, or a binding assay that produces a quantifiable signal transduction), the signal transduction produced by the functionally active fragment or variant of the peptide, polypeptide, or protein is at least about 20%, at least about 25%, at least 30%, at least about 40%, at least about 50%, at least 60%, more preferably at least about 70%, at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%.

[0035] In the context of this invention, "Wnt7" refers to "Wnt7a" and / or "Wnt7b", which are part of the Wnt protein family. In this specification, the terms "Wnt7" and "WNT7" are used interchangeably.

[0036] The terms "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 refer to protein complexes, particularly membrane-associated protein complexes, and more specifically, plasma membrane-associated protein complexes, comprising at least one GPR124 protein, at least one RECK protein, at least one FZD protein, and at least one LRP protein. When the GPR124 / RECK / FZD / LRP receptor complex is located at the plasma membrane of a cell, it can activate Wnt / β-catenin signaling in the cell in response to an extracellularly provided Wnt7 ligand.

[0037] The terms "FZD / LRP receptor complex," "FZD / LRP co-receptor complex," "FZD / LRP complex," or "FZD / LRP protein complex" broadly refer to protein complexes, particularly membrane-associated protein complexes, and more specifically, plasma membrane-associated protein complexes, which comprise at least one FZD protein and at least one LRP protein. When the FZD / LRP receptor complex is located at the plasma membrane of a cell, it is capable of activating Wnt / β-catenin signaling in the cell in response to extracellularly provided Wnt ligands (e.g., but not limited to Wnt7 ligands).

[0038] The term "LRP" or "lipoprotein receptor-related protein" encompasses any and all lipoprotein receptor-related proteins, also referred to in the art as low-density lipoprotein receptor-related proteins or prolow-density lipoprotein receptor-related proteins. In some particularly preferred embodiments, the term refers to LRP5, LRP6, or LRP5 and LRP6 (LRP5 / 6).

[0039] As used in this specification, the terms “binding,” “interaction,” “specific binding,” or “specific interaction” mean that a reagent binds to or influences one or more desired molecules or analytes, substantially excluding other random or unrelated molecules, and optionally substantially excluding other structurally related molecules. This term does not necessarily require that the reagent bind only to its intended target. For example, if the reagent, under binding conditions, has an affinity for the intended target that is at least about 2 times higher, preferably at least about 5 times higher, more preferably at least about 10 times higher, even more preferably at least about 25 times higher, still more preferably at least about 50 times higher, and even more preferably at least about 100 times higher (e.g., at least about 1000 times higher, at least about 1 x 10⁻⁶) than its affinity for non-target molecules. 4 More than 1x10, or at least about 1x10 5 If the reagent binds to the target specifically (more than twice the amount of the target), then the reagent can be said to bind to the target specifically.

[0040] As used in this specification, the term "amino acid addition" refers to an entity of one to eight amino acids that can be added to the C-terminus of a protein or (poly)peptide via peptide bonds, preferably via peptide bonds. This term can 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 in the amino acid addition are preferably linked together by peptide bonds.

[0041] As used herein, the terms “gene therapy” and / or “RNA therapy” refer to the introduction of exogenous polynucleotides into host cells for therapeutic or preventative purposes, regardless of the method used for such introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (via, for example, viral infection / transfection, or a variety of other protein- or lipid-based gene delivery complexes) as described elsewhere herein. The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide contains a replication origin compatible with the host cell or integrates into a replicon of the host cell, such as an extrachromosomal replicon (e.g., a plasmid) or integrates into a nuclear or mitochondrial chromosome. Various vectors are known in the art capable of mediating gene transfer into mammalian cells. The RNA molecule can be any type of RNA molecule. For example, the RNA molecule can be cytoplasmic RNA, nuclear RNA, mRNA, antisense RNA, or non-coding RNA, preferably mRNA.

[0042] The terms “host cell” and “host organism” may suitably include cells or organisms of prokaryotes (e.g., bacteria) and eukaryotes (e.g., yeast, fungi, protozoa, plants, and animals). Considerable host cells include, but are not limited to, single-celled organisms such as bacteria (e.g., *Escherichia coli*, *Salmonella typhimurium*, *Serratia marcescens*, or *Bacillus subtilis*), yeasts (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 cells, mammalian cells, primate cells, human cells, or insect cells). The host organisms that may be considered include, but are not limited to, multicellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrates, even more preferably mammals, and even more preferably primates; in particular, non-humans such as animals and animal species are considered.

[0043] Unless otherwise stated, the terms "subject" or "patient" are used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, and even more preferably primates, and specifically include human patients as well as non-human mammals and primates. Preferred subjects are human subjects. The terms "subject" or "patient" include subjects who require treatment, and more particularly, subjects who will benefit from treatment of a given condition, especially a neurovascular disease or a central nervous system (CNS) disease involving neurovascular dysfunction. Such subjects may include, but are not limited to: individuals diagnosed with the condition, individuals susceptible to the condition, and / or individuals requiring prevention of the condition.

[0044] As used herein, the term "therapeutic effective amount" refers to the amount of an active compound or agent sought by a surgeon, researcher, veterinarian, physician, or other clinician to elicit a biological or medical response in a subject, which may include, but is not limited to, alleviating symptoms of the treated disease or condition. The term "preventive effective amount" refers to the amount of an active compound or agent sought by a researcher, veterinarian, physician, or other clinician to inhibit or delay the onset of a disease in a subject. In the context of this invention, "therapeutic effective amount" is used to refer to both the aforementioned "therapeutic effective amount" and "preventive effective amount," unless the context clearly distinguishes the two. Methods for determining the therapeutic and / or preventive effective doses of compounds, proteins, nucleic acids encoding such compounds / proteins, nucleic acid expression cassettes, or pharmaceutical compositions as taught herein are known in the art. As used herein, the term "therapeutic effective dose" refers to the amount of a compound, protein, nucleic acid encoding such compound / protein, nucleic acid expression cassette, or pharmaceutical composition as taught herein that elicits a positive therapeutic response when administered to a patient suffering from a specific disease or disorder.

[0045] describe:

[0046] compound

[0047] Previously, the inventors characterized the first "Wnt decoding module" capable of distinguishing Wnt ligands. This module could differentiate between Wnt ligands that were substantially similar in their ability to bind frizzled, thereby helping cells interpret complex Wnt signaling inputs to coordinate tissue development and homeostasis. In the Wnt decoding module characterized herein, selectivity is conferred by RECK, which mediates Wnt7-specific binding in a frizzled-independent manner and is part of the Wnt7 ligand-specific RECK / GPR124 / Frizzled / lipoprotein receptor-associated protein (LRP) signaling pathway.

[0048] The inventors further demonstrated the design of agonists capable of selectively activating Wnt signaling in cells expressing RECK and GPR124 (i.e., brain endothelial cells). They also further demonstrated that such agonists can be used as therapeutic agents, particularly for the treatment of neurovascular diseases or central nervous system (CNS) diseases involving neurovascular dysfunction. Therefore, the present invention allows for the provision of agonists capable, but not limited to, stimulating Wnt / β-catenin signaling in brain endothelial cells (“targeting activity”) and exhibiting only low cross-reactivity with other frizzled pathways (“undesirable” or “off-target activity”), and which can be used as therapeutic agents, particularly for the treatment of neurovascular diseases or central nervous system (CNS) diseases involving neurovascular dysfunction.

[0049] The inventors have now surprisingly discovered more Wnt7-related proteins, particularly the mutant Wnt7 protein of this invention, which exhibits highly specific “targeting” Gpr124 / Reck activity, with “off-target” activity preferably even lower than previously shown. Such mutant proteins are agonists of the GPR124 / RECK co-receptor complex and preferably cannot bind to or activate Frizzled and / or LRP, or have reduced binding or activation capacity, in the absence of RECK and / or GPR124. Therefore, the mutants are selective agonists of the Gpr124 and Reck-dependent Wnt / β-catenin pathway. In particular, the mutants of this invention contain an amino acid addition of 1 to 8 amino acids in length at the C-terminus.

[0050] This article discloses a surprising finding: the mutant Wnt7 protein with the extra amino acid exhibits highly specific “targeting” GPR124 / RECK activity, with “off-target” activity even lower than that of the previously described mutant variants of Wnt7.

[0051] In the following paragraphs, different aspects or embodiments of the invention are defined in more detail. Each of these aspects or embodiments may be combined with any other aspect or embodiment unless expressly stated to the contrary. Specifically, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous.

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

[0053] In one embodiment, the mutant Wnt7 protein is an agonist of the Gpr124 and Reck-dependent Wnt / β-catenin pathway, preferably a selective agonist.

[0054] In one embodiment, the term "selective agonist of the Gpr124 and Reck-dependent Wnt / β-catenin pathway" should be understood as an agonist capable of activating said pathway with high selectivity and specificity (targeting activity) and without activating or only minimally activating other Wnt signaling pathways (off-target activity). In this embodiment, as a Gpr124 and Reck-dependent Wnt / β-catenin pathway agonist, the Wnt7 mutant disclosed herein exhibits less than 20% off-target activity, for example, less than 15%, 10%, 5%, or 1%, preferably less than 10%, as determined by a cell culture Super TOP-Flash assay (known in the art and discussed in the Examples section below).

[0055] In one embodiment, the mutant Wnt7 protein is a regulator of the Gpr124 and Reck-dependent Wnt / β-catenin pathway, and its C-terminus contains an amino acid addition of 1 to 8 amino acids in length.

[0056] In embodiments, the mutant Wnt7 protein disclosed herein is an agonist of the GPR124 / RECK complex, preferably an agonist of a specific GPR124 / RECK complex, wherein the protein is capable of binding the GPR124 / RECK complex, the GPR124 protein, and / or the RECK protein. In embodiments, in the absence of RECK and / or GPR124, the mutant cannot bind or activate Frizzled and / or lipoprotein receptor-associated proteins (LRPs), or its binding or activation ability is weakened.

[0057] In this specification, references to any peptide, polypeptide, protein, or nucleic acid indicate the corresponding peptide, polypeptide, protein, or nucleic acid commonly known under the various names described in the art. More specifically, references to “Wnt,” particularly “Wnt7,” “G protein-coupled receptor 124” (GPR124), “reversion-inducing cysteine-rich protein with Kazal motifs” (RECK), “Frizzled” (FZD), or “lipoprotein receptor-associated protein” (LRP) indicate the corresponding peptide, polypeptide, protein, or nucleic acid as is evident from the context and commonly known under the names described in the art.

[0058] In some embodiments, one or more, preferably all, of Wnt7, GPR124, RECK, FZD and LRP as used herein are of animal origin, preferably of warm-blooded animal origin, more preferably of vertebrate origin, and even more preferably of mammalian origin, including human and non-human mammalian origins, and even more preferably of human origin.

[0059] Those skilled in the art will understand that any sequence represented in a sequence database or this specification may belong to the precursor of the corresponding peptide, polypeptide, protein, or nucleic acid, and may include portions processed from mature molecules; or, these sequences may represent sequences of mature molecules while precursor sequences are also considered part of this concept.

[0060] The term covers peptides, polypeptides, proteins, or nucleic acids when they form part of a living organism, organ, tissue, or cell; when they form part of a biological sample; and when they are at least partially isolated from such sources. The term also covers peptides, polypeptides, proteins, or nucleic acids when they are produced by recombinant or synthetic means.

[0061] Unless otherwise apparent from the context, any peptide, polypeptide, protein, or nucleic acid mentioned herein also encompasses modified forms of said peptide, polypeptide, protein, or nucleic acid, such as those with post-expression modifications, including, for example, phosphorylation, glycosylation, esterification, methylation, cysteine ​​modification, sulfonation, glutathioneization, acetylation, ubiquitination, oxidation of methionine to methionine sulfoxide or methionine sulfone, cleavage of the signal peptide, removal of the N-terminal Met, and conversion of a prozymogen or precursor hormone to its active form. The above provides a broad definition.

[0062] In some embodiments, the mutant Wnt7 protein is of animal origin, preferably warm-blooded animal origin, more preferably vertebrate origin, and even more preferably mammalian origin, including human and non-human mammalian origins, and even more preferably mouse or human origin. In another embodiment, the mutant Wnt7 protein is of synthetic origin.

[0063] In some embodiments, the length of the amino acid additive is 8 amino acids, or 7 amino acids, or 6 amino acids, or 5 amino acids, or 4 amino acids, or 3 amino acids, preferably 2 amino acids, and most preferably 1 amino acid.

[0064] The length of the amino acid additives was optimized to provide the mutant Wnt7 protein with the highest possible targeting activity and the lowest possible off-target activity. Furthermore, additions of up to eight amino acids have been shown to provide the desired activity.

[0065] In another or further embodiment, the length of the amino acid additive is at least 1 and at most 8 amino acids, preferably at least 2 and at most 8 amino acids, preferably at least 3 and at most 8 amino acids, preferably at least 4 and at most 8 amino acids, preferably at least 5 and at most 8 amino acids, preferably at least 6 and at most 8 amino acids, preferably at least 7 and at most 8 amino acids.

[0066] In another or further embodiment, the length of the amino acid additive is at least 1 and at most 7 amino acids, preferably at least 2 and at most 7 amino acids, preferably at least 3 and at most 7 amino acids, preferably at least 4 and at most 7 amino acids, preferably at least 5 and at most 7 amino acids, and preferably at least 6 and at most 7 amino acids.

[0067] In another or further embodiment, the length of the amino acid additive is at least 1 and at most 6 amino acids, preferably at least 2 and at most 6 amino acids, preferably at least 3 and at most 6 amino acids, preferably at least 4 and at most 6 amino acids, and preferably at least 5 and at most 6 amino acids.

[0068] In another or further embodiment, the length of the amino acid additive is at least 1 and at most 5 amino acids, preferably at least 2 and at most 5 amino acids, preferably at least 3 and at most 5 amino acids, and preferably at least 4 and at most 5 amino acids.

[0069] In another or further embodiment, the length of the amino acid additive is at least one and at most four amino acids, preferably at least two and at most four amino acids, and preferably at least three and at most four amino acids.

[0070] In another or further embodiment, the length of the amino acid additive is at most two amino acids, preferably one amino acid.

[0071] In one embodiment, the amino acid additive comprises an amino acid selected from 22 known α-amino acids (i.e., proteogenic amino acids) that constitute proteins. As a non-limiting example, the amino acid additive may be formed from repeating amino acids, such as repeating alanine (A) or repeating glycine-serine (GS) dipeptides.

[0072] In this embodiment, the number of negatively charged amino acids in the additive is kept to a minimum (e.g., one or two). In this embodiment, the additive does not contain any negatively charged amino acids.

[0073] In a preferred embodiment, the length of the amino acid additive is one amino acid, preferably alanine or glycine.

[0074] In one embodiment, the amino acid sequence of the mutant Wnt7 protein, compared to the sequence of SEQ ID NO:1 or SEQ ID NO:2, includes one or more substitutions, deletions, truncations, or insertions, such as 5, 4, 3, or 2, preferably 1 substitution, deletion, truncation, or insertion, and has an amino acid addition at the C-terminus, the length of which is 1 to 8 amino acids, or has the length of any one of the foregoing embodiments.

[0075] SEQ ID NO:1 and SEQ ID NO:2 correspond to the mature amino acid sequences of human or mouse Wnt7a and Wnt7b proteins, respectively. Therefore, the mutant Wnt7 protein of the present invention is preferably a mutant Wnt7a or Wnt7b protein having an amino acid addition of 1 to 8 amino acids at its C-terminus.

[0076] The amino acid sequence corresponding to SEQ ID NO:1 (mature human or mouse Wnt7a) is:

[0077] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRK ILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK

[0078] The amino acid sequence corresponding to SEQ ID NO:2 (mature human or mouse Wnt7b) is as follows:

[0079] LGANIICNKIPGLAPRQRAICQSRPDAIIVIGEGAQMGINECQYQFRFGRWNCSALGEKTVFGQELRVGSREAAFTYAITAAGVAHAVTAACSQGNLSNCGCDREKQGYYNQAEGWKWGGCSADVRYGIDFSRRFVDAREIKKNARRLMNLHNNEAGRK VLEDRMQLECKCHGVSSGSCTTKTCWTTLPKFREVGHLLKEKYNAAVQVEVVRASRLRQPTFLRIKQLRSYQKPMETDLVYIEKSPNYCEEDAATGSVGTQGRLCNRTSPGADGCDTMCCGRGYNTHQYTKVWQCNCKFHWCCFVKCNTCSERTEVFTCK

[0080] In a further embodiment, the amino acid sequence of the mutant Wnt7 protein, compared with the sequence of SEQ ID NO:1 or SEQ ID NO:2, includes one or more amino acid substitutions, such as 5, 4, 3 or 2 amino acid substitutions, preferably one amino acid substitution, and has an amino acid addition of 1 to 8 amino acids in length at the C-terminus.

[0081] In one embodiment, the amino acid in the C-terminal amino acid additive is selected from alanine, glycine, proline, serine, or a combination thereof. Most preferably, the length of the amino acid additive is one amino acid, wherein the amino acid is selected from alanine and glycine.

[0082] As previously stated, the term "amino acid addition" as used in this specification refers to an entity of one to eight amino acids that can be added to the C-terminus of a protein or (poly)peptide, preferably via peptide bonds. This term can 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 in the amino acid addition are preferably linked together by peptide bonds.

[0083] Therefore, when this mutant Wnt7 protein contains one or more amino acid substitutions, such as 5, 4, 3 or 2 amino acid substitutions, preferably one amino acid substitution, compared to the sequence of SEQ ID NO:1 or SEQ ID NO:2, and has an amino acid addition at the C-terminus, the length of the amino acid addition being 1 to 8 amino acids, or having the length of any one of the foregoing embodiments, then the sequence length of the mutant Wnt7 protein is similar to the length of SEQ ID NO:1 or SEQ ID NO:2 plus 1 to 8 additional amino acids of the amino acid addition.

[0084] SEQ ID NO: 1 and 2 are both 348 amino acids in length. Therefore, in one embodiment, the length of the mutant Wnt7 protein is preferably more than 348 amino acids, preferably 349 to 356 amino acids, more preferably 349 to 355 amino acids, more preferably 349 to 354 amino acids, more preferably 349 to 353 amino acids, more preferably 354 to 352 amino acids, more preferably 349 to 351 amino acids, more preferably 349 to 350 amino acids, and most preferably 349 amino acids.

[0085] In this implementation, the protein is able to bind to the RECK protein.

[0086] In an embodiment, the mutant Wnt7 protein can bind to the RECK protein, optionally to the cysteine ​​knot 4 (CK4) region or the CK5 region of the RECK protein, or to both the CK4 and CK5 regions.

[0087] In embodiments, the mutant Wnt7 protein disclosed herein can simultaneously bind to Frizzled (FZD), LRP, and RECK. The mutant Wnt7 protein can bind to the cysteine-rich domain (CRD) of Frizzled proteins. This mutant Wnt7 protein can bind to LRP proteins, for example, to the extracellular domain of LRP proteins, to the DKK binding site of LRP proteins, and / or to the Wnt binding site. In more specific embodiments, the mutant Wnt7 protein disclosed herein can bind to the DKK1 binding site of LRP5 and / or LRP6 proteins.

[0088] In a particular embodiment, the mutant Wnt7 protein disclosed herein is able to bind to the β-propeller-EGF-like domains 1 and 2 (P1E1P2E2) and / or the β-propeller-EGF-like domains 3 and 4 (P3E3P4E4) of the LRP protein.

[0089] In certain embodiments, the mutant Wnt7 protein disclosed herein can bind not only GPR124 and / or RECK proteins, but also Frizzled and / or LRP proteins.

[0090] According to the implementation method, if GPR124 and / or RECK are not present, the mutant Wnt7 protein disclosed herein cannot bind to FZD and / or LPR.

[0091] In this implementation, the absence of GPR124 and / or RECK proteins can refer to cells that express FZD and / or LRP proteins (FZD / LRP positive) but do not express GPR124 and / or RECK proteins, or cells with low levels of GPR124 and / or RECK protein expression (GRP124 / RECK negative).

[0092] In the implementation, GPR124 / RECK negative can refer to the absence or low expression of GPR124 and / or RECK in cells, or the absence or low expression of GPR124 and / or RECK proteins in cells, and / or the absence or low expression of GPR124 and / or RECK proteins on the cell surface.

[0093] In this implementation, GPR124 / RECK positive can refer to the expression of GPR124 and / or RECK in cells, or the presence of GPR124 and / or RECK proteins in cells, and / or the presence of GPR124 and / or RECK proteins on the cell surface.

[0094] In this implementation, FZD / LRP positivity can refer to the expression of FZD and / or LRP in cells, the presence of FZD and / or LRP proteins within cells, and / or the presence of FZD and / or LRP proteins on the cell surface.

[0095] In a particular implementation, cells expressing GPR124, RECK, FZD, and LRP on the plasma membrane are cells that naturally express all of GPR124, RECK, FZD, and LRP on their cell surface, such as brain endothelial cells.

[0096] In some embodiments, "presence of RECK and GPR124" can refer to the presence of RECK and GPR124 proteins at or near the cell membrane, preferably in close proximity to Frizzled and LRP proteins. Close proximity can promote the formation of the GPR124 / RECK / Frizzled / LRP receptor complex under suitable conditions, such as when the mutant Wnt7 protein taught herein is provided to cells externally. In embodiments, when used for gene or RNA therapy, the mutant Wnt7 protein taught herein can be expressed by the target cell population. On the other hand, the phrase "absence of RECK and / or GPR124" can refer to the absence or absence of RECK and / or GPR124 proteins at the cell membrane. RECK and / or GPR124 may be absent at or near the cell membrane when cells do not express, translate, or properly transport RECK and / or GPR124. The absence of RECK and / or GPR124 does not necessarily mean that RECK and / or GPR124 proteins are completely absent on the cell membrane. It could also mean, for example, that the amount of RECK and / or GPR124 protein is not detectable by conventional protein detection or quantification methods known to those skilled in the art, such as immunoblotting, immunocytochemistry, or immunofluorescence, or is below their sensitivity range.

[0097] In some embodiments, the mutant Wnt7 protein disclosed herein must first bind to RECK and / or GPR124 before it can further bind to FZD and / or LRP proteins.

[0098] In one embodiment, the mutant Wnt7 protein is capable of activating GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling, wherein in the absence of RECK and / or GPR124, the mutant Wnt7 protein does not activate Frizzled / LRP-mediated Wnt signaling.

[0099] In a particular embodiment, the mutant Wn7 protein activates GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling, but cannot activate Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124. This indicates that the mutant Wn7 protein can activate Wnt signaling in GPR124, RECK, FZD, and LRP-positive cells, but cannot activate Wnt signaling in FZ and LRP-positive and GPR124 and / or RECK-negative cells. The GPR124, RECK, FZ, and LRP-positive cells are otherwise substantially identical to FZ and LRP-positive but GPR124 and / or RECK-negative cells.

[0100] In a particular embodiment, the ability of the Wnt7 protein to activate GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling but not Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124 represents the ability of the mutant Wnt7 protein to activate classical Wnt signaling in the presence of RECK and GPR124 but not in the absence of RECK and / or GPR124.

[0101] The ability to activate Wnt signaling refers to the ability of the proteins disclosed herein to mimic, reproduce, or approximate the signal transduction function and / or activity of native Wnt ligands bound to FZD and LRP (e.g., bound to the FZD / LRP complex).

[0102] Activation of Wnt signaling can be appropriately determined and / or quantified by measuring the expression of one or more Wnt target genes, the expression of TCF reporter genes, β-catenin stabilization, LRP phosphorylation, and / or the transport of Axin from the cytoplasm to the cell membrane, as known in the art. For example, activation of Wnt signaling can be appropriately determined and / or quantified by measuring the expression of TCF genes (e.g., by RT-PCR or any other transcript detection method), the major output of Wnt signaling. For example, TCF reporter molecule assays (also known as TOP / FOP or TOPflash) can be used to assess transcriptional changes in TCF / LEF control genes. TCF reporter gene assays can be luciferase reporter gene assays. As another example, activation of Wnt signaling can be appropriately determined and / or quantified by measuring the expression of c-myc, n-myc, LEF1, or c-jun. Alternatively, activation of Wnt signaling can be determined by measuring the location, level, and / or phosphorylation status of β-catenin. A non-limiting example of this assay is the "β-catenin Redistribution Assay" (ThermoScientific), which provides recombinant U20S cells stably expressing human β-catenin fused to the C-terminus of enhanced green fluorescent protein (EGFP). This assay allows visualization and monitoring of the translocation of the GFP-β-catenin fusion protein from the membrane to the nucleus. Another method for determining Wnt signaling activation is to visualize Axin translocation, for example, using a GFP-Axin fusion protein.

[0103] In specific embodiments, such as those measured in assays described elsewhere herein, if the mutant Wnt7 protein disclosed herein enhances Wnt / β-catenin signaling by at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1000-fold, or at least 1 × 10⁻⁶ times compared to the baseline or background of Wnt / β-catenin signaling induced by neutral substances or negative controls, then... 4 Times, or at least 1×10 5 If the Wnt7 protein is counted multiple times, it can be considered that the Wnt7 protein can activate (classical) Wnt signaling.

[0104] In certain embodiments, such as those measured in assays described elsewhere herein, if the mutant Wnt7 protein disclosed herein enhances Wnt / β-catenin signaling by less than 10-fold, for example, particularly at least 5-fold or at most 2.5-fold, compared to a baseline or background of Wnt / β-catenin signaling induced by neutral substances or negative controls, or if the protein does not enhance or even reduces (e.g., by 2-fold, 5-fold, or 10-fold) Wnt / β-catenin signaling transduction, then the Wnt7 protein can be considered not to activate (classical) Wnt signaling.

[0105] In certain embodiments, the mutant Wnt7 protein disclosed herein is considered capable of activating the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling pathway (referred to as "targeting activity") if it induces at least 30% targeted signaling activity and less than 20% off-target activity. However, it does not activate the Frizzled / LRP-mediated Wnt signaling pathway in the absence of RECK and / or GPR124 (referred to as "off-target activity"). In embodiments, the degree to which the mutant Wnt7 protein activates the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling pathway can exceed 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, and all ranges and subranges therebetween, preferably exceeding 70%.

[0106] In embodiments, the mutant Wnt7 protein may activate the Frizzled / LRP-mediated Wnt signaling pathway to a degree 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, preferably less than 10%. Before comparing the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling activity (referred to as "Activity 1" in this paragraph) and the Frizzled / LRP-mediated Wnt signaling activity in the absence of RECK and / or GPR124 (referred to as "Activity 2" in this paragraph), the mutant Wnt7 protein-induced Activity 1 and Activity 2 may be normalized relative to the wild-type Wnt7a-induced Activity 1 and Activity 2, respectively, setting the latter, for example, to represent 100% activity.

[0107] In one embodiment, the mutant Wnt7 protein of the present invention functions through a Wnt7-specific RECK / GPR124 / Frizzled / LRP-mediated signaling pathway, wherein the mutant Wnt7 (Wnt7a or Wnt7b) specifically binds to RECK in an FZD-independent manner, and the RECK binding chaperone GPR124 bridges the RECK-bound Wnt7 to the FZD / LRP complex via an intracellular DVL scaffold, thereby assembling a Wnt7 ligand-specific RECK / GPR124 / FZD / LRP signaling pathway and activating classical Wnt signaling.

[0108] Those skilled in the art will further understand that, as envisioned herein, the GPR124 / RECK / FZD / LRP receptor complex may include other components that may or may not functionally modulate the complex. For example, the complex may include Disheveled (Dvl), which forms an intracellular scaffold capable of bridging GPR124 and Frizzled.

[0109] Activation of the Wnt signaling pathway can occur by promoting the tight association or proximity of Frizzled and LRP proteins on the cell membrane, thereby forming membrane-associated heterooligomers containing Frizzled and LRP proteins (ligand-driven Frizzled-LRP heterooligomer formation).

[0110] In some tissues expressing GPR124 and / or RECK, such as the brain's EC, a second Wnt7a / b specific receptor complex is formed to activate Wnt / β-catenin signaling. RECK stabilizes the ligand in a signaling-enabled hydrophobic conformation and delivers it to the Frizzled receptor via GPR124. Thus, RECK and GPR124 synergistically stimulate a Wnt7a / b specific response by assembling a higher-order GPR124 / RECK / FZD / LRP5 / 6 complex.

[0111] According to embodiments, in the presence of RECK and / or GPR124, the disclosed mutant Wnt7 protein induces heteropolymerization of Frizzled and LRP proteins. Preferably, the disclosed Wnt7 protein first binds to the RECK protein to induce heteropolymerization of Frizzled and LRP proteins, more preferably first binds to the RECK / GPR124 complex to induce heteropolymerization of Frizzled and LRP proteins. According to embodiments, in the absence of RECK and / or GPR124, the disclosed mutant Wnt7 protein cannot induce heteropolymerization of Frizzled and LRP proteins; more specifically, when the Wnt7 protein does not bind to RECK and / or does not bind to the GPR124 protein and / or does not bind to the GPR124 / RECK complex, it cannot induce heteropolymerization of Frizzled and LRP proteins.

[0112] Therefore, in certain embodiments, the mutant Wnt7 protein disclosed herein can bind Frizzled and LRP proteins, preferably at the cell membrane, in the presence of RECK and GPR124, but cannot bind to the proteins in the absence of RECK and / or GPR124.

[0113] In certain embodiments, the mutant Wnt7 protein 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 Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10. Preferably, the compounds disclosed herein are capable of specifically binding to at least Fzd4 and optionally specifically binding to one or more other Fzds; or they are capable of specifically binding to Fzd4, substantially excluding other Fzds. Fzd4 is considered a major member of the Fzd family in the endothelial cells of the central nervous system. More preferably, the compounds disclosed herein are capable of specifically binding to human Fzd4. The proteins disclosed herein can be selective for one or more preferred Frizzled proteins, for example, their specificity for one or more preferred Frizzled proteins is at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 1000 times, or at least 1 x 10^12 proteins greater than that for other non-preferred Frizzled proteins. 4 Times, or at least 1x10 5 times.

[0114] In certain embodiments, the mutant Wnt7 protein disclosed herein can bind to one or more different LRP proteins involved in Wnt signaling. Preferably, the protein disclosed herein can bind to LRP5 and / or LRP6, for example, any or each of LRP5 and LRP6. More preferably, the protein disclosed herein can bind to human LRP5 and / or human LRP6, for example, any or each of human LRP5 and human LRP6. The protein disclosed herein may be selective for one or more preferred LRP proteins, for example, its specificity for one or more preferred LRP proteins may be at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 1000 times, or at least 1 x 10^10 LRP proteins that is at least 1 x 10^10 times higher than that for other non-preferred LRP proteins. 4 Times, or at least 1x10 5 times.

[0115] In certain embodiments, the mutant Wnt7 protein disclosed herein can bind to both Frizzled and LRP proteins in addition to binding to GPR124 and / or RECK proteins.

[0116] RECK consists 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 the glycosylphosphatidylinositol (GPI) anchor site. The CK, CRD, and Kazal motifs are located extracellularly. Therefore, in embodiments, the proteins disclosed herein capable of binding RECK proteins may bind to the CK1, CK2, CK3, CK4, CK5, CRD, and / or one or more Kazal motifs of the RECK protein, preferably the CK4 and / or CK5 regions of the RECK protein.

[0117] In a particular embodiment, the mutant Wnt7 protein disclosed herein possesses 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 Wnt signaling activity mediated by GPR124 / RECK / Frizzled / LRP of the full-length wild-type Wnt7 protein. In a preferred embodiment, the protein disclosed herein possesses at least 70% of the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling activity of the full-length wild-type Wnt7 protein. In a particular embodiment, in the presence of RECK and / or GPR124, the mutant Wnt7 protein disclosed herein can activate GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling to a degree exceeding 30% of the activation level of the full-length wild-type Wnt7 protein. For example, over 30%, over 40%, over 50%, over 60%, over 70%, over 80%, over 90%, over 95%, over 99%, and all ranges and subranges in between.

[0118] In certain embodiments, in the absence of RECK and / or GPR124, the mutant Wnt7 protein of the present invention disclosed herein can activate Wnt signaling to a degree less than 20% of the activation level of the 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 therebetween.

[0119] In some embodiments, the mutant Wnt7 protein with an amino acid added to its C-terminus contains the following residues: glutamine (Q) residue at position 17 of SEQ ID NO: 1; isoleucine (I) residue at position 20 of SEQ ID NO: 1; proline (P) residue at position 25 of SEQ ID NO: 1; alanine (A) residue at position 27 of SEQ ID NO: 1; isoleucine (I) residue at position 28 of SEQ ID NO: 1; glutamate (E) residue at position 33 of SEQ ID NO: 1; methionine (M) residue at position 37 of SEQ ID NO: 1; leucine (L) residue at position 39 of SEQ ID NO: 1; glutamate (E) residue at position 41 of SEQ ID NO: 1; and so on. The following residues are present: phenylalanine (F) residue at position 44 of SEQ ID NO: 1; arginine (R) residue at position 50 of SEQ ID NO: 1; asparagine (N) residue at position 52 of SEQ ID NO: 1; valine (V) residue at position 68 of SEQ ID NO: 1; isoleucine (I) residue at position 129 of SEQ ID NO: 1; phenylalanine (F) residue at position 131 of SEQ ID NO: 1; lysine (K) residue at position 133 of SEQ ID NO: 1; phenylalanine (F) residue at position 135 of SEQ ID NO: 1; isoleucine (I) residue at position 141 of SEQ ID NO: 1; arginine (R) residue at position 146 of SEQ ID NO: 1; and arginine (N) residue at position 52 of SEQ ID NO: 1. Arginine (R) residue at position 158 of SEQ ID NO: 1; Lysine (K) residue at position 159 of SEQ ID NO: 1; Lysine (K) residue at position 181 of SEQ ID NO: 1; Arginine (R) residue at position 191 of SEQ ID NO: 1; Lysine (K) residue at position 198 of SEQ ID NO: 1; Lysine (K) residue at position 200 of SEQ ID NO: 1; Valine (V) residue at position 205 of SEQ ID NO: 1; Glutamic acid (E) residue at position 208 of SEQ ID NO: 1;The following residues are present: glutamic acid (E) residue at position 208 of SEQ ID NO: 1; arginine (R) residue at position 214 of SEQ ID NO: 1; lysine (K) residue at position 216 of SEQ ID NO: 1; proline (P) residue at position 218 of SEQ ID NO: 1; lysine (K) residue at position 222 of SEQ ID NO: 1; isoleucine (I) residue at position 223 of SEQ ID NO: 1; tyrosine (Y) residue at position 229 of SEQ ID NO: 1; proline (P) residue at position 232 of SEQ ID NO: 1; threonine (T) residue at position 235 of SEQ ID NO: 1; glutamic acid (E) residue at position 248 of SEQ ID NO: 1; and lysine (K) residue at position 216 of SEQ ID NO: 1. The arginine (R) residue at position 289 of SEQ ID NO: 1; the tryptophan (W) residue at position 291 of SEQ ID NO: 1; the threonine (T) residue at position 307 of SEQ ID NO: 1; and / or the lysine (K) residue at position 318 of SEQ ID NO: 1, are substituted by one or more (preferably no more than three, more preferably no more than two, more preferably one) other amino acid residues, such as, but not limited to, alanine (A) residues, arginine (R) residues or glutamine (Q) residues, more preferably by alanine (A) residues.

[0120] In some embodiments, the mutant Wnt7 protein with an amino acid added to its C-terminus contains the following residues: glutamine (Q) residue at position 17 of SEQ ID NO: 1; isoleucine (I) residue at position 20 of SEQ ID NO: 1; proline (P) residue at position 25 of SEQ ID NO: 1; isoleucine (I) residue at position 28 of SEQ ID NO: 1; glutamate (E) residue at position 33 of SEQ ID NO: 1; methionine (M) residue at position 37 of SEQ ID NO: 1; leucine (L) residue at position 39 of SEQ ID NO: 1; glutamate (E) residue at position 41 of SEQ ID NO: 1; phenylalanine (F) residue at position 44 of SEQ ID NO: 1; arginine (R) residue at position 50 of SEQ ID NO: 1; and so on. The following residues are present: valine (V) residue at position 68 of SEQ ID NO: 1; isoleucine (I) residue at position 129 of SEQ ID NO: 1; phenylalanine (F) residue at position 131 of SEQ ID NO: 1; lysine (K) residue at position 133 of SEQ ID NO: 1; phenylalanine (F) residue at position 135 of SEQ ID NO: 1; isoleucine (I) residue at position 141 of SEQ ID NO: 1; arginine (R) residue at position 146 of SEQ ID NO: 1; arginine (R) residue at position 158 of SEQ ID NO: 1; lysine (K) residue at position 181 of SEQ ID NO: 1; arginine (R) residue at position 191 of SEQ ID NO: 1; and lysine (K) residue at position 129 of SEQ ID NO: 1. Lysine (K) residue at position 198 of SEQ ID NO: 1; Lysine (K) residue at position 200 of SEQ ID NO: 1; Valine (V) residue at position 205 of SEQ ID NO: 1; Glutamic acid (E) residue at position 208 of SEQ ID NO: 1; Glutamic acid (E) residue at position 208 of SEQ ID NO: 1; Arginine (R) residue at position 214 of SEQ ID NO: 1; Lysine (K) residue at position 216 of SEQ ID NO: 1; Proline (P) residue at position 218 of SEQ ID NO: 1;Lysine (K) residue at position 222 of SEQ ID NO: 1; isoleucine (I) residue at position 223 of SEQ ID NO: 1; tyrosine (Y) residue at position 229 of SEQ ID NO: 1; proline (P) residue at position 232 of SEQ ID NO: 1; threonine (T) residue at position 235 of SEQ ID NO: 1; glutamic acid (E) residue at position 248 of SEQ ID NO: 1; arginine (R) residue at position 289 of SEQ ID NO: 1; tryptophan (W) residue at position 291 of SEQ ID NO: 1; threonine (T) residue at position 307 of SEQ ID NO: 1; and / or residues at positions corresponding to SEQ ID NO: 1. The lysine (K) residue at position 318 of SEQ ID NO: 1 is replaced by an alanine (A) residue; and / or the alanine (A) residue at position 27 of SEQ ID NO: 1 is replaced by an arginine (R) residue; and / or the asparagine (N) residue at position 52 of SEQ ID NO: 1 is replaced by a glutamine (Q) residue; and / or the lysine (K) residue at position 159 of SEQ ID NO: 1 is replaced by an alanine (A), serine (S), or leucine (L) residue.

[0121] In some embodiments, the mutant Wnt7 protein contains two or more (e.g., preferably two, preferably three, more preferably four) amino acid substitutions listed above.

[0122] Therefore, in a specific embodiment, the amino acid sequence of the mutant Wnt7 protein is identical to that of SEQ ID NO: The amino acid sequences listed in 1 have at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity, 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 glutamic acid, the residue at position 37 is not methionine, the residue at position 39 is not leucine, the residue at position 41 is not glutamic acid, 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, and the residue at position 146 is not glutamine. 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 glutamic acid, 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 glutamic acid, 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, 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, and the residue at position 191 is... The residues at positions 198, 200, 205, 208, 214, 216, 218, 222, 223, 229, 232, 235, 248, 289, 291, 307, and / or 318 are alanine.

[0123] In a particular embodiment, the mutant Wnt7a protein comprises the amino acid sequence listed in any one of SEQ ID NO: 5 to SEQ ID NO: 46, preferably SEQ ID NO: 25, or preferably SEQ ID NO: 13.

[0124] The following sequence is the amino acid sequence of the mature mutant Wnt7a protein, in which one amino acid residue has been replaced by another residue (shown in bold).

[0125] hWnt7a Q17A : LGASIICNKIPGLAPR ARAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 5)

[0126] hWnt7a I20A : LGASIICNKIPGLAPRQRA A CQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 6)

[0127] hWnt7a P25A : LGASIICNKIPGLAPRQRAICQSR ADAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 7)

[0128] hWnt7a A27R : LGASIICNKIPGLAPRQRAICQSRPD R IIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 8)

[0129] hWnt7a I28A :

[0130] LGASIICNKIPGLAPRQRAICQSRPDA AIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 9)

[0131] hWnt7a E33A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIG A GSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 10)

[0132] hWnt7a M37A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQ AGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 11)

[0133] hWnt7a L39A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMG A DECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 12)

[0134] hWnt7a E41A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLD ACQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 13)

[0135] hWnt7a F44A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQ A QFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 14)

[0136] hWnt7a R50A :

[0137] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNG AWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 15)

[0138] hWnt7a N52Q : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRW Q CSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 16)

[0139] hWnt7a V68A :

[0140] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELK AGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 17)

[0141] hWnt7a I129A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYG A GFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 18)

[0142] hWnt7a F131A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIG AAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 19)

[0143] hWnt7a K133A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFA A VFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 20)

[0144] hWnt7a F135A :

[0145] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKV AVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 21)

[0146] hWnt7a I141A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDARE A KQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 22)

[0147] hWnt7a R146A :

[0148] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNA ATLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 23)

[0149] hWnt7a R158A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAG A KILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 24)

[0150] hWnt7a K159A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGR A ILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 25)

[0151] hWnt7a K159L :

[0152] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGR L ILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 26)

[0153] hWnt7a K159S :

[0154] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGR S ILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 27)

[0155] hWnt7a K181A: LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTT A TCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 28)

[0156] hWnt7a R191A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQF A ELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 29)

[0157] hWnt7a K198A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVL ADKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 30)

[0158] hWnt7a K200A :

[0159] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKD A YNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 31)

[0160] hWnt7a V205A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEA A HVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 32)

[0161] hWnt7a E208A: LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHV A PVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 33)

[0162] hWnt7a R214A :

[0163] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRAS A NKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 34)

[0164] hWnt7a K216A: LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRN A RPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 35)

[0165] hWnt7a P218A :

[0166] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKR A TFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 36)

[0167] hWnt7a K222A: LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFL A IKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 37)

[0168] hWnt7a I223A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLK A KKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 38)

[0169] hWnt7a Y229A: LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLS A RKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 39)

[0170] hWnt7a P232A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRK A MDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 40)

[0171] hWnt7a T235A: LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMD A DLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 41)

[0172] hWnt7a E248A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC A EDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 42)

[0173] hWnt7a R289A: LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYA A VWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 43)

[0174] hWnt7a W291A : LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARV A QCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 44)

[0175] hWnt7a T307A: LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHN NEAGRKILEENMKLECKCHGVSSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCN A CSERTEMYTCK (SEQ ID NO: 45)

[0176] hWnt7a K318A :

[0177] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGR KILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTC A (SEQ ID NO: 46)

[0178] Table 1 shows the positions of amino acid substitutions in the amino acid sequence of the (mouse) Wnt7a precursor polypeptide and their corresponding positions in the amino acid sequence of the mature (mouse) Wnt7a polypeptide:

[0179] Table 1

[0180]

[0181] In a particularly preferred embodiment, in this type of mutant Wnt7a protein, the lysine (K) residue at position 159 of SEQ ID NO: 1 is replaced by one or more (preferably no more than three, more preferably no more than two, more preferably one) other amino acid residues, such as, but not limited to, alanine (A), serine (S), or leucine (L) residues. Those skilled in the art will understand that the K residue at position 159 of SEQ ID NO: 1 corresponds to the K residue at position 190 in the Wnt7a precursor polypeptide shown in Table 1.

[0182] Therefore, the present invention also relates to a mutant Wnt7 protein, wherein the amino acid sequence of the mutant Wnt7 protein has one amino acid substitution compared to the sequence of SEQ ID NO:1 or SEQ ID NO:2, and the mutant contains an amino acid addition of 1 to 8 amino acids in length at the C-terminus.

[0183] In one embodiment, the C-terminal amino acid additive is as described in any of the foregoing embodiments. In another or further embodiment, the amino acid substitution may be as described in any of the foregoing embodiments.

[0184] In a preferred embodiment, the C-terminal amino acid additive is one amino acid in length, preferably alanine.

[0185] In a preferred embodiment, as an amino acid substitution, the lysine (K) residue at position 159 of SEQ ID NO: 1 is substituted with an alanine (A), serine (S) or leucine (L) residue, more preferably with an alanine (A) residue.

[0186] In a preferred embodiment, the C-terminal amino acid additive is one amino acid in length, wherein the amino acid is selected from alanine and glycine, and the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO:1 is replaced by an alanine (A), serine (S) or leucine (L) residue, most preferably replaced by an alanine (A) residue.

[0187] In a second aspect, the present invention relates to nucleic acids encoding the mutant Wnt7 protein described in any of the above embodiments. As is well known in the art, the nucleic acid can be inserted into a nucleic acid expression cassette and / or vector.

[0188] Nucleic acid can refer to deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), wherein the RNA is preferably mRNA.

[0189] Therefore, another aspect relates to a nucleic acid expression cassette containing a nucleic acid encoding a mutant Wnt7 protein disclosed herein, which is operatively linked to a promoter and / or transcriptional and translational regulatory signals.

[0190] Preferably, the nucleic acid expression cassette may contain one or more open reading frames (ORFs) encoding the one or more proteins, polypeptides, or peptides.

[0191] The exact nature of the transcriptional and translational regulatory sequences or elements required for expression can vary between expression environments, but typically includes transcription terminators and optional enhancers.

[0192] In a particular embodiment, the nucleic acid expression cassette contains a nucleic acid encoding the mutant Wnt7 protein disclosed herein, which is operatively linked to one or more promoters, enhancers, ORFs, and / or transcription terminators.

[0193] Further aspects involve vectors, such as viral vectors, that contain nucleic acids encoding the mutant Wnt7 protein disclosed herein or the nucleic acid expression cassettes disclosed herein.

[0194] Important factors when selecting a specific vector include, but are not limited to: the selection of recipient cells; the ease of identification and screening between recipient cells containing and without the vector; the required number of vector copies in a specific recipient cell; whether it is desired for the vector to integrate into the chromosome or remain in an extrachromosomal state in the recipient cell; and whether it is desired to be able to "shuttle" the vector between recipient cells of different species.

[0195] Expression vectors can be autonomous or integrative. Nucleic acids can be introduced into cells using expression vectors (e.g., plasmids, bacteriophages, transposons, granules, or viral particles). Recombinant nucleic acids can be maintained extrachromosomally or integrated into the cell's chromosomal DNA. Expression vectors can contain selectable marker genes that encode proteins required for cell viability under selected conditions (e.g., URA3, which encodes an enzyme essential for uracil biosynthesis, or LEU2, which encodes an enzyme essential for leucine biosynthesis, or TRP1, which encodes an enzyme essential for tryptophan biosynthesis), to allow detection and / or selection of cells transformed with the target nucleic acid. Expression vectors can also contain autonomous replication sequences (ARS). ARS can contain a centromere (CEN) and an origin of replication (ORI). For example, an ARS can be ARS18 or ARS68.

[0196] An integration vector typically comprises a sequence of at least a first insertable DNA fragment, a selection marker gene, and a second insertable DNA fragment arranged sequentially. The first and second insertable DNA fragments are each about 200 nucleotides in length (e.g., about 250, 300, 350, 400, 450, 500, or 1000 or more) and have nucleotide sequences partially homologous to the genomic DNA of the cell species to be transformed. A nucleotide sequence containing the target nucleic acid is inserted into the vector between the first and second insertable DNA fragments, either before or after the marker gene. The integration vector may be linearized prior to transformation to facilitate the integration of the target nucleotide sequence into the cell genome. Before introducing the vector into the target cell, the vector may be grown (e.g., amplified) in bacterial cells (e.g., *Escherichia coli*). The vector DNA can be isolated from the bacterial cells by any method known in the art, resulting in the purification of the vector DNA from the bacterial environment. The purified vector DNA can be thoroughly extracted with phenol, chloroform, and ether to ensure that the plasmid DNA preparation is free of E. coli proteins, as these proteins may be toxic to mammalian cells.

[0197] In other specific embodiments, the vector containing the nucleic acid described herein is a viral vector, preferably a viral vector specifically directed to the central and / or peripheral nervous system (e.g., a brain-specific viral vector). In a further specific embodiment, the viral vector is a central nervous system (CNS) neuron-specific adeno-associated virus serotype 9 (AAV9) mutant.

[0198] In a preferred embodiment, the viral vector is a blood-brain barrier endothelial cell-specific viral vector. In a further preferred embodiment, the viral vector is a blood-brain barrier endothelial cell-specific capsid adeno-associated virus serotype 2 (AAV2) mutant.

[0199] In embodiments, the mutant Wnt7 protein, nucleic acid, nucleic acid expression cassette, and / or vector disclosed herein can be used in a method for activating classical Wnt signaling in the presence of RECK and GPR124 but in the absence of RECK and / or GPR124, the method comprising administering a therapeutically effective amount of at least one of the molecules to a mammal.

[0200] Pharmaceutical compositions and formulations

[0201] The pharmaceutical compositions disclosed herein comprise any of the mutant Wnt7 protein, nucleic acid, nucleic acid expression cassette or vector described in any of the embodiments herein, and optionally pharmaceutically acceptable vectors, diluents, excipients or adjuvants.

[0202] In some embodiments, the pharmaceutical composition may comprise a protein, such as the mutant Wnt7 protein disclosed herein. Such proteins may be suitably obtained by transforming a host cell or host organism with an expression construct encoding the protein and configured to express the protein in the host cell or host organism, followed by purification of the protein.

[0203] Therefore, a further aspect provides host cells containing nucleic acids, nucleic acid expression cassettes, or vectors taught herein.

[0204] In some embodiments, the host cell may be a bacterial cell, yeast cell, animal cell, or mammalian cell. The mutant Wnt7 protein can be isolated from these cells using any suitable method known to those skilled in the art.

[0205] In embodiments, the pharmaceutical compositions disclosed herein may also include a pharmaceutically acceptable carrier.

[0206] As used herein, the term "pharmaceutically acceptable" is consistent with the prior art and means that it is compatible with other components of a pharmaceutical composition and does not cause harm to the recipient.

[0207] As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (e.g., neutral buffered saline or phosphate buffered saline), solubilizers, colloids, dispersion media, carriers, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, aroma agents, thickeners, reagents for achieving reservoir effects, coating agents, antifungal agents, preservatives, antioxidants, isotonic regulators, absorption delay agents, etc. The use of such media and reagents in pharmaceutically active substances is well known in the art. Unless any conventional media or reagents are incompatible with the active substance, their use in therapeutic compositions may be considered.

[0208] Illustrative non-limiting carriers for formulating pharmaceutical compositions include, for example, oil-in-water or water-in-oil emulsions, aqueous compositions containing or not containing organic cosolvents suitable for intravenous (IV) use, liposomes or surfactant-containing vesicles, microspheres, microbeads and microparticles, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols and other carriers that are obvious to those skilled in the art.

[0209] In certain embodiments, the mutant Wnt7 protein, nucleic acid, nucleic acid expression cassette, or carrier of the pharmaceutical composition is provided in the form of liposomes or lipid nanoparticles.

[0210] The pharmaceutical compositions contemplated herein can be formulated for use essentially in any route of administration, such as, but not limited to, oral administration (e.g., oral or inhalation), intranasal administration (e.g., intranasal inhalation or intranasal mucosal administration), parenteral administration (e.g., subcutaneous, intravenous (IV), intramuscular, intraperitoneal, intrathecal, or intracisional injection or infusion), percutaneous or transmucosal administration (e.g., oral, sublingual, intranasal), local administration, rectal, vaginal, or tracheal injection, etc. In this way, the therapeutic effects obtainable by the methods and compositions, depending on the specific needs of a given application, can be, for example, systemic, local, tissue-specific, etc.

[0211] For example, for oral administration, the pharmaceutical composition may be formulated as pills, tablets, lacquered tablets, coated (e.g., sugar-coated) tablets, granules, hard gelatin capsules and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions.

[0212] For example, for oral or nasal aerosol or inhalation administration, the pharmaceutical composition may be formulated for administration as an aerosol or spray, for example, as a solution, suspension, or emulsion of the compound described herein or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable solvent (e.g., ethanol or water, or mixtures of such solvents). If desired, the formulation may also additionally contain other pharmaceutical excipients, such as surfactants, emulsifiers, stabilizers, and propellants.

[0213] Examples of carriers for transmural application depend on the specific route of administration, such as oral, sublingual, intranasal, etc., and are generally known in the art.

[0214] For example, for parenteral administration, the pharmaceutical composition can advantageously be formulated as a solution, suspension, or emulsion, using suitable solvents, diluents, solubilizers, or emulsifiers generally known in the art. Injectable solutions or suspensions can be formulated using suitable non-toxic, parenteral-acceptable diluents or solvents according to known techniques. The compounds of the present invention and their pharmaceutically acceptable salts can also be lyophilized, and the resulting lyophilized products can be used, for example, in the production of injectable or infusion formulations.

[0215] When aqueous formulations are preferred, they may contain one or more surfactants. For example, the composition may be in the form of a micellar dispersion containing at least one suitable surfactant, such as a phospholipid surfactant. Various types of phospholipids are known in the art. Typically, the molar ratio of surfactant to active substance in aqueous formulations is from about 10:1 to about 1:10, more typically from about 5:1 to about 1:5, but any effective amount of surfactant may be used in the aqueous formulation to best suit the specific target of interest.

[0216] In a preferred embodiment, a pharmaceutical composition comprising a mutant Wnt7 protein as taught herein or a nucleic acid encoding that protein is administered via a parenteral route. More preferably, the pharmaceutical composition taught herein is administered intravenously (e.g., by infusion) or intrathecally.

[0217] When administered rectally in suppository form, these formulations can be prepared by mixing the compounds of the present invention with a suitable non-irritating excipient (e.g., cocoa butter, synthetic glycerides, or polyethylene glycol), said formulation being solid at room temperature but liquefied and / or dissolved in the rectal lumen to release the drug.

[0218] In one embodiment, the pharmaceutical composition can be used in a method for activating classical Wnt signaling in the presence of RECK and GPR124, but not in the absence of RECK and / or GPR124; the method comprises administering a therapeutically effective amount of at least one of the mutant Wnt7 protein, nucleic acid, or vector to a mammal.

[0219] Those skilled in the art will recognize that the above description is exemplary and not exhaustive. In fact, many other formulation techniques and pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as are suitable dosages and treatment regimens for the use of the specific compositions described herein in a variety of treatment regimens.

[0220] It will be apparent to those skilled in the art that the aspects described in the “Molecular” section above for determining, analyzing and quantifying Wnt signaling activation are equally applicable here.

[0221] Treatment methods and the use of compounds or compositions

[0222] This document also discloses therapeutic uses and applications of the compounds and / or compositions disclosed herein. More particularly, this document discloses the use of the proteins, nucleic acids, expression cassettes, carriers, and / or pharmaceutical compositions described herein as medicines for human or veterinary use.

[0223] In the following paragraphs, the term "compound" is used to refer to any of the mutant Wnt7 protein, nucleic acid, expression cassette, or vector disclosed in any of the above embodiments.

[0224] The compounds and pharmaceutical compositions disclosed herein allow for the provision of agonists or modulators capable of stimulating Wnt / β-catenin signaling in brain endothelial cells. These agonists or modulators are substantially free from cross-reactivity with other Frizzled pathways and can be used as therapeutic agents, particularly for neurovascular diseases or central nervous system (CNS) diseases involving neurovascular dysfunction.

[0225] In certain embodiments, the compounds and pharmaceutical compositions disclosed herein are also suitable for treating or preventing diseases in which Wnt signaling abnormalities occur in cells, tissues and / or organs expressing RECK and / or GPR124.

[0226] In a particular embodiment, the neurovascular disease is selected from the group consisting of: ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, cerebral aneurysm, arteriovenous malformation (AVM), cavernous malformation, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformation, carotid artery stenosis, moyamoya disease, intracranial arteriosclerosis, and retinal vascular disease, including but not limited to Norrie's disease, familial exudative vitreoretinopathy, osteoporosis-pseudoglioma syndrome, diabetic retinopathy, and macular degeneration and combinations thereof.

[0227] In a particular embodiment, the CNS disease comprising neurovascular dysfunction is selected from the group consisting of: multiple sclerosis, ischemic stroke, brain cancer, glioblastoma, human monogenic neurological diseases (e.g., SLC2A1), epilepsy, neurodegenerative diseases, dementia, vascular dementia, HIV-1-related dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, infectious brain diseases, traumatic brain injury, migraine, chronic traumatic encephalopathy, neuroinflammation, neurocoronavirus infection, and combinations thereof.

[0228] "Neuroinflammatory" can refer to inflammation of the neurovascular system. Furthermore, neuroinflammatory can be triggered by various factors. For example, in a non-limiting embodiment, neuroinflammatory can be caused by injuries such as traumatic brain injury and / or spinal cord injury, infections such as viral, bacterial, or fungal infections, exposure to toxins or toxic metabolites, neurodegenerative diseases, autoimmune diseases, smoking or passive smoking, aging, or any combination thereof.

[0229] Neuroinflammation can be caused by infectious diseases. These infections may originate from bacteria, fungi, parasites, or viruses. In some cases, the infectious disease is a viral infection. Viral infections in certain situations include infections caused by one or more of the following: Herpesviridae (varicella-zoster virus (VZV) infection, herpes simplex virus (HSV-1 or HSV-2), cytomegalovirus (CMV)), Paramyxoviridae, coronaviruses (SARS-CoV-1, MERS-CoV or SARS-CoV-2, HCoVNL63, HCoV-229E, HCoV-OC43, HKU1 or their variants), and influenza viruses (influenza A virus group). Group 1 (H1N1), Group 2 (H2N2), Group 3 (H3N2), Group 5 (H5N1, H5N2, H5N8) or Group 7 (H7N7, H7N9), Influenza B virus, Influenza C virus, Zika virus, Japanese encephalitis virus, Epstein-Barr virus, Hepatitis B virus, Hepatitis C virus, Human Immunodeficiency Virus 1 (HIV-1), Human Papillomavirus (HPV), Human T-cell Leukemia Virus 1 (HTLV-1), and Kaposi's Sarcoma Herpesvirus (KSHV).

[0230] Neuroinflammation can be associated with pulmonary conditions such as acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS), which may be related to or caused by ventilator use, viral infections, sepsis, or systemic bacterial infections, as has been confirmed in subjects in need. The pulmonary conditions can be caused by infectious diseases resulting from bacterial, fungal, parasitic, or viral infections. Preferably, the infection is an infection causing respiratory problems or belongs to the respiratory tract. Preferably, the infection is a viral infection.

[0231] Neuroinflammation can be associated with bacterial infection. Such inflammation is mostly caused by blood-borne bacteria that cross the blood-brain barrier (BBB) ​​and eventually invade the brain parenchyma. Pathogens such as Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae are the main causes of bacterial meningitis.

[0232] In the context of this invention, prevention and / or treatment of neuroinflammation may include encephalitis. In some cases, said encephalitis is viral encephalitis, such as COVID-19-related encephalitis. Neuroinflammation may also be associated with optic nerve inflammation. Neuroinflammation may also be associated with acute disseminated encephalomyelitis, such as COVID-19-related acute disseminated encephalomyelitis. Neuroinflammation may also be associated with vitreoretinal ganglion cell inflammation.

[0233] In this embodiment, the compounds and compositions are particularly suitable for use on patients who are susceptible to or suffer from such diseases.

[0234] In certain embodiments, the mutant Wnt7a protein taught herein, the nucleic acid encoding the mutant Wnt7a protein, or the nucleic acid expression cassette containing the nucleic acid is used for gene therapy, particularly for gene therapy targeting blood-brain barrier endothelial cells.

[0235] Therefore, this article also provides methods for gene therapy, particularly methods for central and / or peripheral nervous system-targeted gene therapy for subjects in need of said gene therapy, said methods comprising: introducing, in particular, the nucleic acid expression cassette or vector described herein into said subject, particularly into the central and / or peripheral nervous system of said subject; and expressing, in the subject, particularly into the central and / or peripheral nervous system of said subject, a therapeutically effective amount of a mutant Wnt7a protein encoded by a nucleic acid taught herein.

[0236] In certain embodiments, the mutant Wnt7a protein or the nucleic acid encoding the mutant Wnt7a protein taught herein is used for mRNA therapy, particularly for blood-brain barrier endothelial cell-directed mRNA therapy.

[0237] Therefore, this document also provides methods for RNA therapy, preferably mRNA therapy, particularly methods for central and / or peripheral nervous system-targeted mRNA therapy, for a subject requiring said mRNA therapy, said methods comprising: introducing, into said subject, particularly into the central and / or peripheral nervous system of said subject, a nucleic acid encoding a mutant Wnt7a protein as taught herein; and expressing, in said subject, particularly in the central and / or peripheral nervous system of said subject, a therapeutically effective amount of the mutant Wnt7a protein encoded by the nucleic acid as taught herein.

[0238] One advantage of using RNA therapy is that RNA is generally believed not to integrate into the genome, and therefore does not pose a risk of insertional mutations.

[0239] This article can also utilize any other known methods for introducing nucleic acids into animal cells. The simplest method is to inject the nucleic acid directly into the target cells / tissue. Other methods include fusing recipient cells with bacterial protoplasts containing nucleic acids, using compositions such as calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids, or liposomes, or methods such as receptor-mediated endocytosis, bioparticle bombardment (“gene gun” approach), infection with viral vectors (i.e., derived from lentiviruses, adeno-associated viruses (AAVs), adenoviruses, retroviruses, or antivirals), electroporation, etc. Other techniques or methods suitable for delivering nucleic acid (NA) molecules to target cells include continuous delivery of NA molecules from poly(lactic-co-glycolic acid) polymer microspheres or direct injection of protected (stable) NA molecules into a micropump for product delivery. Another possibility is the use of implantable, biodegradable microspheres that release drugs. Encapsulation of NA, or delivery of NA in the form of various types of liposomes (immunoliposomes, PEGylated (immunoliposomes), cationic lipids and polymers, nanoparticles or dendritic polymers, poly(lactic acid-co-glycolic acid copolymer) polymer microspheres, implantable biodegradable drug-releasing microspheres, etc., is also envisioned; as well as co-injection of NA with a protective agent (e.g., the nuclease inhibitor ginsenoside tricarboxylic acid). It should be understood that combinations of different delivery modalities or methods described above can also be used.

[0240] In a specific embodiment, the compound is provided in the form of a carrier, such as liposomes, lipid nanoparticles, nanostructured lipid carriers, nanoemulsions, polymer nanoparticles, polymer micelles, or dendritic polymers. In a preferred embodiment, the carrier is a liposome or lipid nanoparticle (LNP).

[0241] In one embodiment, the carrier is a lipid-based carrier. The lipid-based carrier comprises one or more lipids. The one or more lipids may be in solid and / or liquid form. The lipid-based carrier may be a lipid nanoparticle (LNP), a lipid complex, a liposome, a phospholipid micelle, a solid lipid nanoparticle, a nanostructured lipid carrier, or a nanoemulsion. Lipid-based carriers to which this invention is applicable include, for example, cationic lipids, liposomes (especially cationic liposomes), micelles, and nanoparticles. Cationic lipids can form complexes with negatively charged nucleic acids. According to this invention, any cationic lipid can be used. Liposomes are bilayer membranes formed by the self-assembly of phospholipids and cholesterol, encapsulating an aqueous core that can contain hydrophilic molecules. Hydrophobic compounds can also be embedded in the lipid bilayer. Based on their size and lamellarity, liposomes can be classified as: (i) small monolayer vesicles (SUVs); (ii) large monolayer vesicles (LUVs); and (iii) multilayer vesicles (MLVs). Solid lipid nanoparticles (SLNs) are spherical with an average diameter of 10 nm to 1000 nm. They are used as colloidal nanoparticle drug delivery systems, in which the lipid drug carrier is solid at both room temperature and body temperature. Different solid lipids can be used to prepare SLNs, such as tripalmitoyl glycerol, cetyl alcohol, cetyl palmitate, glyceryl monostearate, trimyristate, tristearate, stearic acid, etc. SLNs comprise solid lipids (e.g., triglycerides, fatty acids, waxes, some glycerides, and PEGylated lipids), emulsifiers (e.g., polysorbates, poloxamer, and lecithin), and water. Nanostructured lipid carriers (NLCs) comprise a mixture of solid and liquid lipids, forming a partially crystalline lipid system with numerous advantages, such as increased drug loading, flexibility in drug release regulation, and improved stability.

[0242] In one embodiment, the lipid-based carrier is a lipid nanoparticle. Solid lipid nanoparticles (SLN, sLNP) or lipid nanoparticles (LNP) are nanoparticles composed of lipids and are suitable as delivery carriers for drug compounds, especially for the delivery of polynucleotides (e.g., RNA or DNA).

[0243] In a particular embodiment, the pharmaceutical composition described herein is administered to a subject by injection (e.g., intravenous injection) or transplantation of allogeneic cells transformed with a vector containing the said nucleic acid or nucleic acid expression cassette. Upon administration, the injected or transplanted allogeneic cells will transcribe and translate in vivo nucleic acids encoding the compounds taught herein.

[0244] The dosage or amount of the protein taught herein may optionally be combined with one or more other active compounds to be administered, depending on the individual circumstances, and is generally customarily adjusted to achieve optimal effect. Therefore, the unit dose and regimen depend on the nature and severity of the condition to be treated, as well as on a variety of factors such as the species of the subject, the sex, age, weight, general health, diet, administration method and time, immune status, and individual responsiveness of the compound used, its efficacy, metabolic stability, and duration of action, whether the treatment is acute, chronic, or prophylactic, or whether other active compounds are administered in addition to the compounds described in any of the above embodiments. To optimize therapeutic effects, the compounds taught herein may be administered initially with different administration regimens. Typically, appropriate screening tests can be used as part of a clinical testing procedure to monitor the level of the compound in tissues, for example, to determine the efficacy of a given treatment regimen. Dosing frequency is within the skill and clinical judgment of the healthcare professional (e.g., a physician, veterinarian, or nurse). Administration regimens are typically established through clinical trials that can establish optimal administration parameters. However, practitioners may modify such administration methods based on one or more of the factors described above (e.g., the subject's age, health, weight, sex, and medical condition). The frequency of administration can vary depending on whether the treatment is preventative or therapeutic.

[0245] The toxicity and therapeutic efficacy of the compounds described herein or the pharmaceutical compositions comprising such compounds as described in any of the above embodiments can be determined by known pharmaceutical procedures, such as in cell cultures or laboratory animals. These procedures can be used, for example, to determine the LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective to 50% of the population). The dose-to-therapeutic index, which is the ratio of LD50 to ED50, is the ratio of toxicity to therapeutic effect. Pharmaceutical compositions exhibiting a high therapeutic index are preferred. While pharmaceutical compositions exhibiting toxic side effects can be used, care should be taken to design delivery systems that target such compounds to the affected tissue sites to minimize potential damage to normal cells (e.g., non-target cells), thereby reducing side effects.

[0246] Data obtained from cell culture assays and animal studies can be used to determine a dose range suitable for appropriate subjects. The dose of such pharmaceutical compositions is typically within a range of circulating concentrations that include an ED50 with little or no toxicity. This dose can vary within this range depending on the dosage form and route of administration used. For pharmaceutical compositions used as described herein, the therapeutically effective dose can first be estimated from cell culture assays. Doses can be developed in animal models to achieve a range of circulating plasma concentrations that include the IC50 (i.e., the concentration of the pharmaceutical composition that achieves maximum symptom inhibition) determined in cell cultures. Such information can be used to more accurately determine the dose that is useful in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0247] In certain embodiments, a continuous delivery system, such as a (partially) implanted continuous delivery system, is used to administer the compounds taught herein. Those skilled in the art will understand that such a continuous delivery system may include reservoirs, pumps, and infusion devices (e.g., tubing systems) for containing reagents as taught herein. For example, a continuous delivery system may be a microosmotic pump system implanted in the brain.

[0248] In certain embodiments, the compounds disclosed herein are the main active ingredient or the only active ingredient of the pharmaceutical composition.

[0249] In another embodiment, the pharmaceutical composition described in any of the foregoing embodiments can be used in combination with a second therapy, preferably selected from surgery, chemotherapy, radiotherapy or immunotherapy.

[0250] On the other hand, it relates to pharmaceutical compositions comprising the compounds disclosed herein, the nucleic acids disclosed herein encoding the compounds, the nucleic acid expression cassettes disclosed herein, or the vectors disclosed herein, for use as a pharmaceutical remedy.

[0251] Other aspects involve pharmaceutical compositions comprising the mutant Wnt7 protein disclosed herein, the nucleic acid disclosed herein encoding the mutant Wnt7 protein, the nucleic acid expression cassette disclosed herein, or the vector disclosed herein, for the prevention or treatment of neurovascular diseases, or for the prevention or treatment of central nervous system (CNS) diseases including neurovascular dysfunction.

[0252] In a particular embodiment, the neurovascular disease is selected from the group consisting of: ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, cerebral aneurysm, arteriovenous malformation (AVM), cavernous malformation, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformation, carotid artery stenosis, moyamoya disease, intracranial atherosclerosis and combinations thereof, and retinal vascular diseases, such as Norrie's disease, familial exudative vitreoretinopathy, osteoporosis-pseudoglioma syndrome, diabetic retinopathy, macular degeneration and combinations thereof.

[0253] In one embodiment, the neurovascular disease is a retinopathy, wherein the retinopathy is a retinal vascular disease. In one embodiment, the retinal vascular disease is caused by inhibition of vascular development. In one embodiment, the retinopathy is caused by excessive angiogenesis. In one embodiment, the retinal vascular disease is selected from the group consisting of: familial exudative vitreoretinopathy, exudative vitreoretinopathy, Norrie's disease, Nome's disease, diabetic retinopathy (DR), age-related macular degeneration (AMD), retinopathy of preterm birth (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease.

[0254] In a particular embodiment, the CNS diseases including neurovascular dysfunction are selected from multiple sclerosis, ischemic stroke, brain cancer, glioblastoma, human monogenic neurological diseases (e.g., SLC2A1), epilepsy, neurodegenerative diseases, dementia, vascular dementia, HIV-1-related dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, infectious brain diseases, traumatic brain injury, migraine, chronic traumatic encephalopathy, neuroinflammation, neurocoronavirus infection, COVID-19, and combinations thereof.

[0255] "Neuritis" can refer to the aforementioned neurovascular inflammation.

[0256] On the other hand, a method is provided for preventing, slowing the progression of, or treating neurovascular diseases or central nervous system (CNS) diseases involving neurovascular dysfunction (such as, but not limited to, the diseases listed above) in a subject requiring treatment, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a mutant Wnt7 protein, wherein the mutant protein is a selective agonist of the Gpr124 and Reck-dependent Wnt / β-catenin pathway and has an amino acid addition of at least one to at most eight amino acids at its C-terminus, or a nucleic acid encoding such a mutant Wnt7 protein as described in any embodiment of this specification.

[0257] Obviously, the present invention also provides a method for treating neurovascular diseases or central nervous system (CNS) diseases involving neurovascular dysfunction, and relates to using a therapeutically effective amount of a pharmaceutical composition comprising a mutant Wnt7 protein, wherein the mutant protein is a selective agonist of the Gpr124 and Reck-dependent Wnt / β-catenin pathway and has an amino acid addition of at least one to at most eight amino acids at its C-terminus, or a nucleic acid encoding such a mutant Wnt7 protein, as described in any embodiment of this specification, for manufacturing a medicament for treating or slowing the progression of a neurovascular disease or central nervous system (CNS) disease involving neurovascular dysfunction in a subject. Further embodiments are as described above.

[0258] Those skilled in the art will recognize that the above description is exemplary and not exhaustive. In fact, many other formulation techniques and pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as are suitable dosages and treatment regimens for the use of the specific compositions described herein in a variety of treatment regimens.

[0259] synthesis

[0260] Adding an amino acid to the C-terminus of a protein can be performed using any method known in the art. For example, the amino acid can be introduced into the nucleic acid sequence encoding the protein by site-directed mutagenesis, or by PCR with a hanging primer and / or cloning into a vector. The protein can be prepared by translation of the vector or by de novo protein synthesis.

[0261] Example

[0262] The invention will now be further illustrated with reference to the following embodiments. The invention is not limited to the given embodiments or the implementations shown in the figures.

[0263] Materials and methods

[0264] Expression plasmid construct

[0265] Deletion, insertion, and substitution mutants of mouse Wnt7a (UniProtKB #P24383), mouse Wnt7b (UniProtKB #P28047), and human Wnt7a (UniProtKB #O00755), as well as C-terminal modifications (i.e., amino acid additions), were constructed using the Q5® site-directed mutagenesis kit (New England Biolabs®). All modified genes were cloned using the pCS2+ vector as a template. All constructs were confirmed by Sanger sequencing.

[0266] Super TOP-Flash Reporter Gene Testing

[0267] Dual luciferase assays were performed using the Super TOP-Flash HEK293 luciferase reporter cell line, which is known in the art.

[0268] In the targeted setting, cells were transiently transfected with Renal luciferase (0.5 ng), Wnt7 mutant (20 ng), Fz1 receptor (5 ng), Lrp5 (2.5 ng), Gpr124 (10 ng), and Reck (10 ng) using Lipofectamine™ 2000 transfection reagent (Invitrogen).

[0269] In the off-target setting, cells were transfected only with plasmids encoding Renin luciferase (0.5 ng), Wnt7 mutant (20 ng), Fz5 receptor (5 ng), and Lrp5 (2.5 ng).

[0270] The assay was performed 48 hours after transfection, and the activity was measured. The data shown (mean ± standard deviation (SD)) are summed from results of at least three independent experiments (each consisting of three replicates).

[0271] Example 1: Adding an amino acid to the C-terminus of the Wnt7a-K190A mutant in the absence of GPR124 / RECK to inhibit the growth of the mutant. The effects of off-target activation of the Wnt pathway

[0272] Add one to eight amino acids to the C-terminus of wild-type mouse Wnt7a or mouse Wnt7a mutant K190A. Figure 1 A). The obtained construct was co-transfected into HEK293 STF cells (off-target - GPR124-free / RECK) with Renal luciferase (0.5 ng), Lrp5 (2.5 ng), and Fz5 receptor (5 ng) constructs, and tested using a dual-luciferase assay. Briefly, after adding the plasmid, the total amount of DNA per well was adjusted to 100 ng using an empty pCS2 vector. Forty-eight hours after transfection, dual-luciferase detection was performed using a dual-luciferase reporter gene detection system (E1960, Promega) according to the manufacturer's instructions.

[0273] The data show relative luciferase activities summarized from at least five independent experiments (each experiment performed three times), and the data have been normalized relative to unmodified Wnt7a (without addition). Figure 1 B). Data shows that adding one to eight amino acids to the K190A mutant is sufficient to specifically activate Gpr124 / Reck, while some undesirable off-target activities still exist without the addition of amino acids.

[0274] After adding one to eight amino acids to the C-terminus of the mWnt7a-K190A mutant, the "targeting" activity remained at a high level.

[0275] Example 2: In the case of GPR124 / RECK (target) or GPR124 / RECK (off-target) conditions, The effect of adding an amino acid to the C-terminus of the Wnt7a-K190A mutant on the "targeted" and "off-target" activation of the classical Wnt pathway.

[0276] Subsequently, the effect of adding 20 arbitrary amino acids to the mWnt7a-K190A mutant on its agonistic properties was investigated. Twenty different amino acids were added to the C-terminus of the mouse Wnt7a-K190A mutant. The resulting constructs were co-transfected into HEK293 STF cells with either René luciferase, Lrp5 and Fz5 receptor constructs (off-target) or Fz1 receptor, Gpr124 and Reck constructs (target), and assayed using a dual-luciferase assay as described above. Results are shown in... Figure 2 middle.

[0277] It is noteworthy that adding just one amino acid to the C-terminus of the mWnt7a-K190A mutant protein can lead to undetectable off-target activity, which is exactly what is expected in clinical applications; however, without the addition, the mWnt7a-K190A mutant protein still exhibits some off-target activity.

[0278] Example 3: In the case of GPR124 / RECK (target) or GPR124 / RECK (off-target) conditions, The effect of adding one amino acid to the C-terminus of various Wnt7 mutant proteins on the "targeted" and "off-target" activation of the classical Wnt pathway.

[0279] exist Figure 3 In A, instances of the mWnt7a mutant with a single alanine residue added to the C-terminus did not exhibit off-target activity, or exhibited undetectable off-target activity. Furthermore, in Figure 3 In B, an example of an mWnt7a mutant with a single alanine added to the C-terminus is shown. The “targeting” activity of these mutants remains high and is practically unaffected.

[0280] Example 4: In vitro signal transduction activity of Wnt7a-K190A with various extensions

[0281] The effect of amino acid elongation on "target and off-target" signaling activity was determined by fusing the following three sequences into Wnt7a-K190A: GKPIPNPL, GGSGSGSGS, or AAAAAAAA and their variable-length (2-4-5-6-8 amino acid) variants. Figure 4 ).

[0282] In vitro signal transduction assays showed that adding these three sequences resulted in high “targeted” signal transduction activity, while the “off-target” activity was so low as to be undetectable.

[0283] These data indicate that the specific amino acid properties contained in the C-terminal additive are not important for achieving specific Gpr124 / Reck signaling.

[0284] This invention is not limited to the embodiments and / or the drawings shown. Rather, the method of this invention can be implemented in many different ways without departing from the scope of this invention.

Claims

1. A mutant Wnt7 protein, characterized in that, The mutant Wnt7 protein contains an amino acid addition of 1 to 8 amino acids in length at its C-terminus.

2. The mutant Wnt7 protein according to claim 1, wherein, The mutant Wnt7 protein is an agonist of the Gpr124 and Reck-dependent Wnt / β-catenin pathway, preferably a selective agonist.

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

2.

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

5. The mutant Wnt7 protein according to any one of the preceding claims, wherein, The amino acids in the C-terminal amino acid additive are selected from alanine, glycine, proline, serine, or combinations thereof.

6. The mutant Wnt7 protein according to any one of the preceding claims, wherein, The length of the amino acid additive at the C-terminus is one amino acid, wherein the amino acid is selected from alanine or glycine.

7. The mutant Wnt7 protein according to any one of the preceding claims, wherein, The amino acid sequence of the mutant Wnt7 protein, compared with the sequence shown in SEQ ID NO:1 or SEQ ID NO:2, includes one or more amino acid substitutions, preferably one amino acid substitution, and wherein the length of the amino acid addition at the C-terminus is one amino acid, wherein the amino acid is selected from alanine and glycine.

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

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

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

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

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

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

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

16. The nucleic acid of claim 11, the nucleic acid expression cassette of claim 12, or the vector of claim 13, for use in gene therapy, preferably for gene therapy targeting blood-brain barrier endothelial cells.

17. Use of the nucleic acid of claim 11 or the vector of claim 13 for RNA therapy, preferably for RNA therapy targeting blood-brain barrier endothelial cells.

18. The mutant Wnt7 protein of any one of claims 1 to 10, the nucleic acid of claim 11, the nucleic acid expression cassette of claim 12, or the vector of claim 13, for the prevention or treatment of neurovascular diseases, neuroinflammatory diseases, or central nervous system (CNS) diseases involving neurovascular dysfunction.

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

20. Use of 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, or the vector according to claim 13 for the treatment of glioblastoma.

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

22. The use of the mutant Wnt7 protein according to claim 21, wherein, The aforementioned retinal vascular diseases or conditions are caused by the inhibition of vascular development.

23. The use of the mutant Wnt7 protein according to claim 21, wherein, The aforementioned retinal vascular disease or condition is caused by excessive angiogenesis.

24. The use of the mutant Wnt7 protein according to claim 21, wherein, The retinal vascular diseases or conditions are selected from the group consisting of: familial exudative vitreoretinopathy (FEVR), exudative vitreoretinopathy, Norrie's disease, Nome's 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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