WO2023094581A1 - Compositions for the treatment of glioblastoma - Google Patents

Compositions for the treatment of glioblastoma Download PDF

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Publication number
WO2023094581A1
WO2023094581A1 PCT/EP2022/083243 EP2022083243W WO2023094581A1 WO 2023094581 A1 WO2023094581 A1 WO 2023094581A1 EP 2022083243 W EP2022083243 W EP 2022083243W WO 2023094581 A1 WO2023094581 A1 WO 2023094581A1
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residue
seq
alanine
amino acid
substituted
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PCT/EP2022/083243
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French (fr)
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Benoit Vanhollebeke
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Université Libre de Bruxelles
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention is directed to compositions and methods for treating subjects diagnosed with glioblastoma.
  • Glioblastoma multiforme is a common type of primary brain tumor in humans and is a very aggressive and devastating cancer, with a median survival of approximately one year. Glioblastoma has the worst prognosis of any central nervous system malignancy. Therapy for GBM is difficult due to its biological location in the brain. Current treatments can involve chemotherapy, radiation, radiosurgery, corticosteroids, antiangiogenic therapy, and surgery. Despite the development of new surgical and radiation techniques and the use of multiple antineoplastic drugs, a cure for malignant gliomas does not exist. Glioblastoma cells are often resistant to cytotoxic agents, and the high incidence of recurrence in a very short period of time in glioblastoma patients suggests that tumorigenic cells are capable of overtaking the treatments.
  • GBM is characterized by a dense vascular network exhibiting disrupted BBB properties (S. Watkins et al., Nat. Commun. 5, 1-15 (2014); S. W. Schneider et al., Acta Neuropathol. 2004 Mar;107(3):272-6).
  • endothelial Wnt signaling was reported to control vascular integrity in different brain tumor models.
  • EP 3 768 701 discloses therapeutic agents capable of activating G-protein coupled receptor (GPR)124/RECK/Frizzled/lipoprotein receptor- related protein (LRP)-mediated Wnt signaling, wherein said agents do not activate Frizzled/LRP -mediated Wnt signaling in the absence of RECK and/or GPR124. These agents are described to prevent or treat neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction.
  • GPR G-protein coupled receptor
  • LRP lipoprotein receptor-related protein
  • the present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages.
  • the present invention provides for a pharmaceutical composition for use according to claim 1 and dependent claims thereof.
  • Gprl24/Reck agonists blue dots
  • F Phenotypic scoring of stage 36 Xenopus laevis embryos injected into the ventral vegetal region of the 4-cell embryo with 15 pg of the indicated mRNA.
  • A-B GPR124-/-; RECK-/- HEK293 cells transiently expressing Wnt7a, Wnt7aNTD, or Wnt7aK190A together with Fz5, Reck, or the empty pCS2 vector were submitted to Wnt7a double immunostaining before (Wnt7a extracellular, green/black) and after (Wnt7a intracellular, pink) cell permeabilization.
  • Maximum intensity projections of confocal images are shown (A), and the ratio between the membrane-associated and the intracellular signal (B) was calculated as described in the method section.
  • C-D Western blot analysis of anti-HA coimmunoprecipitation assays after membrane- impermeable cross-linking in total lysates of RECK-/- HEK293T cells expressing HA- Reck (C) or HA-Fz5 (D) (bait) together with Wnt7a, Wnt7aNTD, or Wnt7aK190A (prey), with or without expression of untagged Reck (D).
  • E Gprl24/Reck/Fzl and Fz5 activity correlations across the Wnt7a single-residue collection.
  • A Characterization of the zebrafish 7-bp frameshift wnt7aaulb2 allele.
  • the protospacer adjacent motif (PAM) is underlined in blue.
  • B Dorsal views and quantification of 60 hpf Tg(kdrl:GFP) hindbrain CtAs (central arteries) and 72 hpf Tg(neurogl :GFP) trunk dorsal root ganglia (DRG, grey arrowheads) in WT and wnt7aa-/- zebrafish.
  • C Dorsal views of 30 hpf Tg(7xTCFXia.Siam:GFP);Tg(kdrl:ras- mCherry) hindbrains from WT and wnt7aa-/- embryos and quantification of the number of GFP+ (P-catenin signaling positive) endothelial cells in the perineural PHBCs (primordial hindbrain channels).
  • D Trunk DRG development in 72 hpf Tg(neurogl:GFP) wnt7aa morphant larvae injected at the one-cell stage with the indicated mRNA.
  • A-B Immunostaining of (A) sagittal brain sections for EGFP (green) and CD31- positive vessels (magenta, grey) and (B) coronal sections for CD31 (cyan, grey), Wnt7a (magenta, grey) together with a DAPI counterstain, two weeks after intravenous injections of the indicated AAVs.
  • C Staining of coronal brain slices of AAV-injected BAT-GAL mice for LEF1 (green), lacZ (magenta, fire), laminin (cyan, white) and DNA (DAPI, blue), in the hippocampal area dorsal to the dentate gyrus (DG, left) and in the parafascicular nucleus area (PFN, right).
  • mice GL261-implanted mice, injected intravenously at 2 dpi (days post implantation) with AAVs, were imaged by MRI to evaluate in vivo tumor volume between 21 and 24 dpi.
  • mice were euthanized for endpoint gross morphology assessment (left) and H8iE staining of serial sections (middle). Asterisks indicate hemorrhages (right).
  • C-G Coimmunostaining of coronal tumor or parenchymal (parench) sections for endothelial cells (CD31, cyan) (C), vascular basement membranes (laminin, grey) (D), LEF1 (magenta) together with the endothelial nuclear marker ERG (cyan) (E), GLUT1 (magenta) together with laminin (cyan) (F), or fibrinogen (magenta) together with laminin (cyan) (G).
  • H Correlation between endothelial Wnt activity (LEF1) and tumor volume, vessel density, BBB differentiation (GLUT1), and BBB leakage (fibrinogen extravasation) in tumors of AAV-EGFP mice (colored lines).
  • LEF1 Magnenta
  • LEF1 endothelial nuclear marker ERG
  • F GLUT1
  • BBB GLUT1
  • BBB leakage fibrinogen extravasation
  • FIG. 6 Gprl24/Reck agonists as BBB repair agents in glioblastoma and stroke models
  • A MRI monitoring of tumor volumes after implantation of 1.105 Tet-Off Dkkl GL261 cells, in the absence of doxycycline (+Dkkl, -dox), or the presence of doxycycline (-Dkkl, -i-dox). Doxycycline-exposed mice were injected intravenously with AAV-EGFP or AAV-K190A, as indicated.
  • B Quantification of fibrinogen leakage into the tumor. Data for WT GL261 are the same as in Fig.
  • FIG. 9 Frizzled specificity for Wnt7a/b signaling in the presence or absence of Gprl24/Reck (A) Gprl24/Reck-dependent and (B) -independent STF luciferase activity in GPR124- /-;RECK-/-; FZ1-10-/- HEK293T cells after co-transfection of Wnt7a or Wnt7b with all 10 Fz genes or the empty pCS2 vector .
  • Ligand secretion was evaluated qualitatively by means of anti-V5 dot blot analysis of HEK293T cellular supernatants collected 48 h after transfection with WT Wnt7a or the indicated signaling inactive Wnt7a variants. Only A86R and E89A were not detected in the supernatant.
  • Figure 14 Morphological defects induced by injection of low mRNA doses of Wnt7a or Wnt7aK190A in zebrafish embryos after ubiquitous expression of Gprl24/Reck
  • Data represent mean ⁇ SD.
  • FIG. 1 Cellular origins of Wnt7aK190A upon AAV-PHP.eB gene delivery
  • EGFP green was co-immunostained with (A) endothelial cells (isolectin, magenta), astrocytes (GFAP, white) and neurons (NeuN, cyan) or (B) endothelial cells (CD31, magenta) and pericytes (Desmin, white).
  • A endothelial cells
  • GFAP astrocytes
  • NeuroN cyan
  • B endothelial cells
  • CD31 magenta
  • pericytes Desmin, white
  • BAT-GAL mice were injected retro-orbitally with 4.1011 vg (viral genomes) of the indicated AAV viruses. After two weeks, coronal brain slices were stained for lacZ (magenta), laminin (cyan), and DNA (DAPI, blue). The percentage of LacZ-i- endothelial cells (ECs) and nonendothelial cells (non-ECs) in different brain regions is plotted on the right.
  • AON Anterior Olfactory Nucleus. Data represent mean ⁇ SD.
  • WT C57BL6 mice were injected retro-orbitally with 4.1011 vg (viral genomes) of the indicated AAV viruses.
  • RNAScope in situ hybridization of Axin2 was performed in coronal sections of the hippocampal dendate gyrus and the parafascicular nucleus of the thalamus.
  • the RNAScope images were analyzed using CellProfiler as described in the Methods section. ***P ⁇ 0.001; data represent mean ⁇ SD.
  • FIG. 21 LEF1 immunostaining after brain-wide Wnt7a or Wnt7aK190A gene delivery
  • WT C57BL6 mice were injected retro-orbitally with 4.1011 vg (viral genomes) of the indicated AAV viruses. 14 days after viral injection, LEF1 immunostaining (magenta, grey) was performed to assess Wnt activation in endothelial cells (ECs, laminin, cyan) and nonendothelial cells (non-ECs). The percentage of LEF1+ cells in different brain regions is plotted on the right. AON, Anterior Olfactory Nucleus. Data represent mean ⁇ SD.
  • Figure 22 Distribution of Wnt7aK190A-releasing EGFP+ cells in and around GL261 tumors after AAV-PHP.eB gene delivery
  • GL261 cells readily labeled by DAPI, are negative for the EGFP viral transduction marker, which is particularly high at the tumor margin.
  • A Growth curve of WT GL261 cells and Tet-Off Dkkl GL261 cells exposed or not to 1 pg.ml-1 doxycycline
  • B Western blot analysis of conditional Dkkl repression addition of doxycycline to the growth medium for 48 h. Dkkl was probed both in the cellular supernatant (left) and the cells (right). ATG9 is used as a loading control for the cellular extracts.
  • C Western blot analysis of Dkkl expression within tumors at 21 days post implantation in mice fed or not with a doxycycline-containing diet as described in the Methods section. Two independent tumors are shown for each condition. Tubulin is used as loading control.
  • FIG. 25 Hemorrhage score of Tet-Off Dkkl tumors after AAV-EGFP or AAV-K190A gene delivery
  • mice were harvested from mice injected or not with the indicated AAV virus. Intra- and peritumoral hemorrhages were categorized on vibratome sections as shown on the left, and scored in a double-blind manner.
  • the present invention concerns compositions and methods for treating glioblastoma in a subject. Definitions
  • a compartment refers to one or more than one compartment.
  • the terms "one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.
  • protein as used throughout this specification generally encompasses macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds.
  • the term may encompass naturally, recombinantly, semi-synthetically or synthetically produced proteins.
  • the term also encompasses proteins that carry one or more co- or post-expression-type modifications of the polypeptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, palmitoylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of proenzymes or pre-hormones into active forms, etc.
  • the term further also includes protein variants or mutants which carry one or more amino acid sequence variations vis-a-vis corresponding native proteins, such as, e.g., amino acid deletions, additions and/or substitutions.
  • the term contemplates both full-length proteins and protein parts or fragments, e.g., naturally- occurring protein parts that ensue from processing of such full-length proteins.
  • polypeptide as used throughout this specification generally encompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms “protein” and “polypeptide” may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced polypeptides.
  • polypeptides that carry one or more co- or post-expression-type modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, palmitoylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc.
  • the term further also includes polypeptide variants or mutants which carry one or more amino acid sequence variations vis-a-vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions, point mutations and/or substitutions.
  • the term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g., naturally- occurring polypeptide parts that ensue from processing of such full-length polypeptides.
  • peptide as used throughout this specification preferably refers to a polypeptide as used herein consisting essentially of 50 amino acids or less, e.g., 45 amino acids or less, preferably 40 amino acids or less, e.g., 35 amino acids or less, more preferably 30 amino acids or less, e.g., 25 or less, 20 or less, 15 or less, 10 or less or 5 or less amino acids.
  • a peptide, polypeptide or protein can be naturally occurring, e.g., present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom.
  • a peptide, polypeptide or protein can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesized.
  • a peptide, polypeptide or protein can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free translation or cell-free transcription and translation, or non-biological peptide, polypeptide or protein synthesis.
  • nucleic acid typically refers to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units.
  • a nucleoside unit commonly includes a heterocyclic base and a sugar group.
  • Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally- occurring nucleic acids, other naturally- occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatized bases.
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • U uracil
  • modified nucleobases include without limitation 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine.
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability and may be preferred base substitutions in for example antisense agents, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.
  • Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and/or 2- deoxyribose common in naturally- occurring nucleic acids, or arabinose, 2- deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as without limitation 2'-0-alkylated, e.g., 2'-0- methylated or 2'- O-ethylated sugars such as ribose; 2'-0-alkyloxyalkylated, e.g., 2'-0- methoxyethylated sugars such as ribose; or 2'-0,4'-C-alkylene-linked, e.g., 2'-0,4'- C-methylene- linked or 2'-0,4'-C-ethylene-linked sugars such as ribose; 2'-fluoro- arabinose, etc.).
  • Nucleic acid molecules comprising at least one ribonucleoside unit may be typically referred to as ribonucleic acids or RNA.
  • Such ribonucleoside unit(s) comprise a 2'-OH moiety, wherein -H may be substituted as known in the art for ribonucleosides (e.g., by a methyl, ethyl, alkyl, or alkyloxyalkyl).
  • ribonucleic acids or RNA may be composed primarily of ribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be ribonucleoside units.
  • Nucleic acid molecules comprising at least one deoxyribonucleoside unit may be typically referred to as deoxyribonucleic acids or DNA.
  • deoxyribonucleoside unit(s) comprise 2'-H.
  • deoxyribonucleic acids or DNA may be composed primarily of deoxyribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be deoxyribonucleoside units.
  • Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter alia phosphodiester linkages common in naturally- occurring nucleic acids, and further modified phosphate- or phosphonate-based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3'-N-carbamate, morpholino, borano, thioether, 3'-thioace
  • inter-nucleoside linkages may be phosphate-based linkages including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof.
  • the term "nucleic acid” also encompasses any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo- DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)).
  • Alkyl as used herein particularly encompasses lower hydrocarbon moieties, e.g., C1-C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1 -propenyl, 2-propenyl, and isopropyl.
  • Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof.
  • a modified nucleoside may include a modified heterocyclic base, a modified sugar moiety, a modified inter-nucleoside linkage or a combination thereof.
  • nucleic acid further preferably encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesised) DNA, RNA or DNA/RNA hybrids.
  • RNA is inclusive of RNAi (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), mi RNA (micro-RNA), tRNA (transfer RNA, whether charged or discharged with a corresponding acylated amino acid), and cRNA (complementary RNA).
  • a nucleic acid can be naturally occurring, e.g., present in or isolated from nature, e.g., produced natively or endogenously by a cell or a tissue and optionally isolated therefrom.
  • a nucleic acid can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesized.
  • a nucleic acid can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free transcription, or non-biological nucleic acid synthesis.
  • a nucleic acid can be double-stranded, partly double stranded, or single- stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.
  • any peptides, polypeptides, proteins or nucleic acids may particularly encompass such peptides, polypeptides, proteins or nucleic acids with a native sequence, i.e., ones of which the primary sequence is the same as that of the peptides, polypeptides, proteins or nucleic acids found in or derived from nature.
  • native sequences may differ between different species due to genetic divergence between such species.
  • native sequences may differ between or within different individuals of the same species due to normal genetic diversity (variation) within a given species.
  • native sequences may differ between or even within different individuals of the same species due to somatic mutations, or post-transcriptional or post-translational modifications.
  • the peptides, polypeptides, proteins or nucleic acids may be human, i.e., their primary sequence may be the same as a corresponding primary sequence of or present in naturally occurring human peptides, polypeptides, proteins or nucleic acids.
  • the qualifier "human” relates to the primary sequence of the respective peptides, polypeptides, proteins or nucleic acids, rather than to their origin or source.
  • such peptides, polypeptides, proteins or nucleic acids may be present in or isolated from samples of human subjects or may be obtained by other means (e.g., by recombinant expression, cell-free transcription or translation, or non-biological nucleic acid or peptide synthesis).
  • the peptides, polypeptides, proteins or nucleic acids may be wild-type. While most native peptides, polypeptides, proteins or nucleic acids may be considered wild-type, those carrying naturally-occurring mutations leading to partial or complete loss of function, which may contribute to or be causative of a disease phenotype, are generally excluded from the scope of the term "wild-type".
  • the reference to any peptides, polypeptides, proteins or nucleic acids may also encompass variants or fragments of such peptides, polypeptides, proteins or nucleic acids, particularly of naturally-occurring, native or wild-type forms thereof.
  • fragment as used throughout this specification with reference to a peptide, polypeptide, or protein generally denotes a portion of the peptide, polypeptide, or protein, such as typically an N- and/or C-terminally truncated form of the peptide, polypeptide, or protein.
  • a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the amino acid sequence length of said peptide, polypeptide, or protein.
  • a fragment may include a sequence of > 5 consecutive amino acids, or > 10 consecutive amino acids, or > 20 consecutive amino acids, or > 30 consecutive amino acids, e.g., > 40 consecutive amino acids, such as for example > 50 consecutive amino acids, e.g., > 60, > 70, > 80, > 90, > 100, > 200, >300, >400, > 500, > 600, > 700, > 800, > 900 or > 1000 consecutive amino acids of the corresponding full-length peptide, polypeptide, or protein.
  • fragment with reference to a nucleic acid (polynucleotide) generally denotes a 5'- and/or 3'-truncated form of a nucleic acid.
  • a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the nucleic acid sequence length of said nucleic acid.
  • a fragment may include a sequence of > 5 consecutive nucleotides, or > 10 consecutive nucleotides, or > 20 consecutive nucleotides, or > 30 consecutive nucleotides, e.g., > 40 consecutive nucleotides, such as for example > 50 consecutive nucleotides, e.g., > 60, > 70, > 80, > 90, > 100, > 200, > 300, > 400, > 500, > 600, > 700, > 800, > 900, > 1000, > 1500, > 2000, > 2500, > 3000, > 3500 or > 4000 consecutive nucleotides of the corresponding full-length nucleic acid.
  • the terms encompass fragments arising by any mechanism, in vivo and/or in vitro, such as, without limitation, by alternative transcription or translation, exo- and/or endo-proteolysis, exo- and/or endo-nucleolysis, or degradation of the peptide, polypeptide, protein or nucleic acid, such as, for example, by physical, chemical and/or enzymatic proteolysis or nucleolysis.
  • variant or mutant of a protein, polypeptide, peptide or nucleic acid generally refers to proteins, polypeptides or peptides the amino acid sequence of which, or nucleic acids the nucleotide sequence of which, is substantially identical (i.e., largely but not wholly identical) to the sequence of the protein, polypeptide, peptide, or nucleic acid, e.g., at least about 80% identical or at least about 85% identical, e.g., preferably at least about 90% identical, e.g., at least 91% identical, 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical to the sequence of the recited protein, polypeptide, peptide, or nucleic acid.
  • a variant may display such degrees of identity to a recited protein, polypeptide, peptide or nucleic acid when the whole sequence of the recited protein, polypeptide, peptide or nucleic acid is queried in the sequence alignment (i.e., overall sequence identity).
  • Sequence identity may be determined using suitable algorithms for performing sequence alignments and determination of sequence identity as know per se. Exemplary but non-limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al.
  • BLAST Basic Local Alignment Search Tool
  • An example procedure to determine the percent identity between a particular amino acid sequence and the amino acid sequence of a query polypeptide will entail aligning the two amino acid sequences using the Blast 2 sequences (BI2seq) algorithm, available as a web application or as a standalone executable programme (BLAST version 2.2.31+) at the NCBI web site (www.ncbi.nlm.nih.gov), using suitable algorithm parameters.
  • the output will not present aligned sequences.
  • the number of matches will be determined by counting the number of positions where an identical amino acid residue is presented in both sequences.
  • the percent identity is determined by dividing the number of matches by the length of the query polypeptide, followed by multiplying the resulting value by 100.
  • the percent identity value may, but need not, be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 may be rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 may be rounded up to 78.2. It is further noted that the detailed view for each segment of alignment as outputted by BI2seq already conveniently includes the percentage of identities.
  • a variant of a protein, polypeptide, peptide or nucleic acid may be a homologue (e.g., orthologue or paralogue) of said protein, polypeptide, peptide or nucleic acid.
  • homologue e.g., orthologue or paralogue
  • the term "homology” generally denotes structural similarity between two macromolecules from same or different taxons, wherein said similarity is due to shared ancestry.
  • a variant of a protein, polypeptide, or peptide may comprise one or more amino acid additions, deletions, point mutations or substitutions relative to (i.e., compared with) the corresponding protein or polypeptide.
  • a variant (substitution variant) of a protein, polypeptide, or peptide may comprise up to 70 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, 50, 60, or 70) conservative amino acid substitutions relative to (i.e., compared with) the corresponding protein or polypeptide; and/or a variant (substitution variant) of a protein, polypeptide, or peptide may comprise up to 20 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19) non-conservative amino acid substitutions relative to (i.e., compared with) the corresponding protein or polypeptide
  • a conservative amino acid substitution is a substitution of one amino acid for another with similar characteristics.
  • Conservative amino acid substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine.
  • the nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (i.e., basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.
  • a variant (deletion variant) of a protein, polypeptide, or peptide may lack up to 20 amino acid segments (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 segments) relative to (i.e., compared with) the corresponding protein or polypeptide.
  • the deletion segment(s) may each independently consist of one amino acid, two contiguous amino acids or three contiguous amino acids.
  • the deletion segments may be non-contiguous, or two or more or all of the deletion segments may be contiguous.
  • a variant of a nucleic acid may comprise one or more nucleotide additions, deletions, or substitutions relative to (i.e., compared with) the corresponding nucleic acid.
  • fragment or variant or “variant or fragment” of any peptide, polypeptide, protein or nucleic acid, also encompasses fragments of variants of such peptide, polypeptide, protein or nucleic acid, and variants of fragments of such peptide, polypeptide, protein or nucleic acid.
  • biologically active fragments and/or variants of the recited peptides, polypeptides or proteins are biologically active fragments and/or variants of the recited peptides, polypeptides or proteins.
  • biologically active is interchangeable with terms such as “functionally active” or “functional”, denoting that the fragment and/or variant at least partly retains the biological activity or intended functionality of the respective or corresponding peptide, polypeptide or protein.
  • Reference to the "activity" of a peptide, polypeptide or protein may generally encompass any one or more aspects of the biological activity of the peptide, polypeptide or protein, such as without limitation any one or more aspects of its biochemical activity, enzymatic activity, signaling activity, interaction activity, ligand activity, and/or structural activity, e.g., within a cell, tissue, organ or an organism.
  • a functionally active fragment or variant may retain at least about 20%, e.g., at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, e.g., at least 60%, more preferably at least about 70%, e.g., at least 80%, yet more preferably at least about 85%, still more preferably at least about 90%, and most preferably at least about 95% or even about 100% of the intended biological activity or functionality compared with the corresponding peptide, polypeptide or protein.
  • a functionally active fragment or variant may even display higher biological activity or functionality compared with the corresponding peptide, polypeptide or protein, for example may display at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% of the intended biological activity or functionality compared with the corresponding peptide, polypeptide or protein.
  • a functionally active fragment or variant of the peptide, polypeptide or protein may produce a signal which is at least about 20%, or at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, or at least 60%, more preferably at least about 70%, or at least 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% of the signal produced by the corresponding peptide, polypeptide or protein.
  • complex protein complex
  • polypeptide complex polypeptide complex
  • the terms broadly denote a cluster comprising two or more proteins or polypeptides.
  • the cluster may be stabilized by non-covalent bonds, more particularly non-covalent protein-protein interactions, wherein all or only part of the polypeptides present within the cluster physically interact.
  • a protein complex can be comprised entirely of peptides, polypeptides or proteins, or it may include other molecules or macromolecules such as carbohydrates, lipids, glycolipids, nucleic acids, oligonucleotides, nucleoproteins, nucleosides, nucleoside phosphates, enzyme cofactors, porphyrins, metal ions and the like.
  • the terms encompass without limitation protein complexes which may be obligate or non-obligate, transient or permanent, and/or homomultimeric or heteromultimeric.
  • the terms encompass without limitation protein complexes which may be located at the cell membrane (plasma membrane), extracellularly, within cytoplasm, within cellular organelles, or at membranes of cellular organelles.
  • Complexes located at the cell membrane typically contain at least one membrane-anchored or transmembrane protein.
  • the terms also encompass membrane microdomains and membrane-associated macromolecular organelle-like structures known as "signalosome" which compartmentalize a given signaling pathway.
  • signalosome membrane-associated macromolecular organelle-like structures
  • Such higher-order protein complexes can be at least partly stabilized by intracellular scaffolds.
  • the terms may thus also denote situations in which certain proteins or polypeptides are locally concentrated or accumulated at a given site of a cell membrane, such as to allow signal transduction through the cell membrane at said site mediated by said proteins or polypeptides.
  • Wnt signaling specifically refers to the mechanism by which a biologically active Wnt ligand exerts its effect upon a cell to modulate said cell's activity and/or actions.
  • Biologically active Wnt ligands modulate cell activity and/or action by binding to Wnt receptor(s), such as the FZD/LRP receptor complex. Once activated by binding of the Wnt ligand, the Wnt receptor(s) will activate one or more intracellular signaling pathways.
  • Wnt signaling pathways are classically recognized: the canonical (i.e., mediated by p-catenin activation as a transcriptional co-activator) Wnt pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt/calcium pathway. All three pathways are typically activated by binding of a Wnt ligand to a FZD receptor. However, the recruitment of the LRP receptor appears to be a prerequisite for inducing canonical (or p-catenin- dependent) Wnt signaling.
  • p-catenin is phosphorylated in the cytoplasm by Casein Kinase and glycogen synthase kinase-3 (GSK-3).
  • GSK-3 glycogen synthase kinase-3
  • APC adenomatous polyposis coli
  • Active canonical (or beta-catenin-dependent) Wnt signaling involves binding of Wnt ligands to a receptor complex of FZD and LRP on the cell surface.
  • FZD cooperates with LRP in such a way that binding of the Wnt protein leads to dimerization of the two receptors. It is theorized that this dimerization leads to a conformational change of the FZD and LRP receptors.
  • the cytoplasmic tail of LRP recruits and binds to the scaffold protein Axin in a phosphorylation-dependent manner and leads to formation of a complex involving DVL, Axin, and GSK3.
  • Multimers of receptor-bound DVL and Axin molecules might support the formation of the LRP-FZD dimer.
  • p-catenin phosphorylation is inhibited, releasing p- catenin from the degradation complex and allowing p-catenin to accumulate in the cytoplasm.
  • the accumulation of p-catenin in the cytoplasm allows to p-catenin to enter the nucleus and to interact with TCF/LEF transcription factors.
  • Wnt7 in the present context refers to both “Wnt7a” and/or “Wnt7b", which are part of the Wnt-family of proteins.
  • bind means that an agent binds to or influences one or more desired molecules or analytes substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related.
  • the terms do not necessarily require that an agent binds exclusively to its intended target(s).
  • an agent may be said to specifically bind to target(s) of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold or more greater, such as, e.g., at least about 1000-fold or more greater, at least about lxl0 4 -fold or more greater, or at least about lxl0 5 -fold or more greater, than its affinity for a nontarget molecule.
  • nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides, or to another nucleic acid sequence in a template-transcription product (e.g. RIMA or RNA analogue) relationship.
  • a template-transcription product e.g. RIMA or RNA analogue
  • nucleic acid expression cassettes refers to nucleic acid molecules, typically DNA, to which nucleic acid fragments, preferably the recombinant nucleic acid molecule as defined herein, may be inserted to be expressed, wherein said nucleic acid molecules comprise one or more nucleic acid sequences controlling the expression of the nucleic acid fragments.
  • nucleic acid sequences controlling the expression of the nucleic acid fragments include promoter sequences, open reading frames and transcription terminators.
  • An "open reading frame” or “ORF” refers to a succession of coding nucleotide triplets (codons) starting with a translation initiation codon and closing with a translation termination codon known per se, and not containing any internal in-frame translation termination codon, and potentially capable of encoding a protein, polypeptide or peptide. Hence, the term may be synonymous with "coding sequence” as used in the art.
  • operably linked is a linkage in which regulatory sequences and sequences sought to be expressed are connected in such a way as to permit said expression.
  • sequences such as, e.g., a promoter and an ORF
  • sequences may be said to be operably linked if the nature of the linkage between said sequences does not: (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter to direct the transcription of the ORF, (3) interfere with the ability of the ORF to be transcribed from the promoter sequence.
  • "operably linked” may mean incorporated into a genetic construct so that expression control sequences, such as a promoter, effectively control transcription I expression of a sequence of interest.
  • promoter is to be taken in its broadest context and includes transcriptional regulatory sequences required for accurate transcription initiation and where applicable accurate spatial and/or temporal control of gene expression or its response to, e.g., internal or external (e.g., exogenous) stimuli. More particularly, “promoter” may depict a region on a nucleic acid molecule, preferably DNA molecule, to which an RNA polymerase binds and initiates transcription. A promoter is preferably, but not necessarily, positioned upstream, i.e., 5', of the sequence the transcription of which it controls.
  • a promoter region may contain both the promoter per se and sequences which, when transcribed into RNA, will signal the initiation of protein synthesis (e.g., Shine-Dalgarno sequence).
  • a promoter sequence can also include "enhancer regions", which are one or more regions of DNA that can be bound with proteins (namely the trans-acting factors) to enhance transcription levels of genes in a gene-cluster.
  • the enhancer while typically at the 5' end of a coding region, can also be separate from a promoter sequence, e.g., can be within an intronic region of a gene or 3' to the coding region of the gene.
  • Promoters contemplated herein may be constitutive or inducible.
  • a constitutive promoter is understood to be a promoter whose expression is constant under the standard culturing conditions.
  • Inducible promoters are promoters that are responsive to one or more induction cues.
  • an inducible promoter can be chemically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a chemical inducing agent such as an alcohol, tetracycline, a steroid, a metal, or other small molecule) or physically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a physical inducer such as light or high or low temperatures).
  • An inducible promoter can also be indirectly regulated by one or more transcription factors that are themselves directly regulated by chemical or physical cues.
  • promoters include T7, U6, Hl, retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.
  • terminal or “transcription terminator” refer generally to a sequence element at the end of a transcriptional unit which signals termination of transcription.
  • a terminator is usually positioned downstream of, i.e., 3' of ORF(s) encoding a polypeptide of interest.
  • a transcription terminator may be advantageously positioned 3' to the most downstream ORF.
  • expression vector refers to nucleic acid molecules, typically DNA, to which nucleic acid fragments, preferably the recombinant nucleic acid molecule as defined herein, may be inserted and cloned, i.e., propagated.
  • a vector will typically contain one or more unique restriction sites, and may be capable of autonomous replication in a defined cell or vehicle organism such that the cloned sequence is reproducible.
  • a vector may also preferably contain a selection marker, such as, e.g., an antibiotic resistance gene, to allow selection of recipient cells that contain the vector.
  • Vectors may include, without limitation, plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PAC, BAC, linear nucleic acids, e.g., linear DNA, transposons, viral vectors, etc., as appropriate (see, e.g., Sambrook et al., 1989; Ausubel 1992).
  • Viral vectors may include inter alia retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno- associated viral vectors, for example, vectors based on HIV, SV40, EBV, HSV or BPV.
  • Expression vectors are generally configured to allow for and/or effect the expression of nucleic acids or open reading frames introduced thereto in a desired expression system, e.g., in vitro, in a cell, organ and/or organism.
  • expression vectors may advantageously comprise suitable regulatory sequences.
  • isolated with reference to a particular component (such as for instance a nucleic acid, protein, polypeptide or peptide) generally denotes that such component exists in separation from - for example, has been separated from or prepared and/or maintained in separation from - one or more other components of its natural environment. For instance, an isolated human or animal protein or complex may exist in separation from a human or animal body where it naturally occurs.
  • isolated as used herein may preferably also encompass the qualifier "purified”. As used herein, the term “purified” with reference to peptides, polypeptides, proteins, or nucleic acids does not require absolute purity.
  • nucleic acids are in a discrete environment in which their abundance (conveniently expressed in terms of mass or weight or concentration) relative to other analytes is greater than in the starting composition or sample.
  • a discrete environment denotes a single medium, such as for example a single solution, gel, precipitate, lyophilisate, etc.
  • Purified nucleic acids, proteins, polypeptides or peptides may be obtained by known methods including, for example, laboratory or recombinant synthesis, chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification, etc.
  • Purified peptides, polypeptides or proteins may preferably constitute by weight s 10%, more preferably > 50%, such as > 60%, yet more preferably > 70%, such as > 80%, and still more preferably > 90%, such as > 95%, > 96%, > 97%, > 98%, > 99% or even 100%, of the protein content of the discrete environment. Protein content may be determined, e.g., by the Lowry method (Lowry et al. 1951. J Biol Chem 193: 265), optionally as described by Hartree 1972 (Anal Biochem 48: 422-427).
  • Purity of peptides, polypeptides, or proteins may be determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Quantity of nucleic acids may be determined by measuring absorbance A260. Purity of nucleic acids may be determined by measuring absorbance A260/A280, or by agarose- or polyacrylamidegel electrophoresis and ethidium bromide or similar staining.
  • gene therapy refers to the introduction of an exogenous polynucleotide into a host cell for therapeutic or prophylactic purposes, irrespective of the method used for the introduction.
  • methods include a variety of well- known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as described elsewhere herein.
  • the introduced polynucleotide may be stably or transiently maintained in the host cell.
  • Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art.
  • the terms “treat” or “treatment” encompass the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction, as well as reducing the progression of a neurovascular disorder or a CNS disorder comprising neurovascular dysfunction.
  • CNS central nervous system
  • Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers in a patient, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like.
  • Treatment can also mean prolonging survival or improving well-being in a patient as compared to expected survival or well-being if not receiving treatment.
  • treatment also refers to prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • treat include alleviating, abating or ameliorating disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition.
  • central nervous system (CNS) disorder comprising neurovascular dysfunction or "CNS disease comprising neurovascular dysfunction” as used herein refer to a group of neurological disorders that affect the structure and/or function of the brain and/or spinal cord, which collectively form the CNS, that are caused by, contributed to, or typified by neurovascular dysfunction, or which lead to an abnormality of the structure and/or function of the blood vessels within or supplying the brain and/or spine.
  • CNS central nervous system
  • Non-limiting examples of central nervous system (CNS) disorders comprising neurovascular dysfunction are multiple sclerosis, ischemic stroke, brain cancer, glioblastoma, epilepsy, dementia, vascular dementia, HIV-1- associated dementia, Alzheimer's disease, Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, infectious brain diseases, traumatic brain injuries, migraine, chronic traumatic encephalopathy and retinal vascular disorders, including but not limited to Norrie disease, familial exudative vitreoretinopathy, osteoporosis- pseudoglioma syndrome, diabetic retinopathy and macular degeneration.
  • CNS central nervous system
  • neurovascular disorder or “neurovascular disease” as used herein refer to a disease or pathological alteration or condition which affects the cerebral vascular system and/or the vascular system supplying the spinal cord.
  • the terms encompass any abnormality of the blood vessels within or supplying the brain and/or spine. Abnormalities may be without limitation (i) narrowing of arteries which reduces blood flow to the brain that may lead to hypoxia, ischemia or stroke and/or (ii) weakening of arteries that may lead to brain aneurysms and increase the risk of intracranial bleeding.
  • Non-limiting examples of neurovascular diseases are ischemic stroke, hemorrhagic stroke, ischemia/reperfusion injury, brain aneurysms, arteriovenous malformations (AVMs), cavernous malformations, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformations, carotid artery stenosis, Moyamoya disease, intracranial atherosclerosis and retinal vascular disorders, including but not limited to Norrie disease, familial exudative vitreoretinopathy, osteoporosis-pseudoglioma syndrome, diabetic retinopathy and macular degeneration.
  • Blood-brain barrier refers to highly selective semipermeable properties of endothelial cells that prevents solutes in the circulating blood from non-selectively crossing into the extracellular fluid of the central nervous system where neurons reside.
  • the blood-brain barrier is formed by endothelial cells of the capillary wall, astrocyte end-feet ensheathing the capillary, and pericytes embedded in the capillary basement membrane. This system allows the passage of some small molecules by passive diffusion, as well as the selective and active transport of various nutrients, ions, organic anions, and macromolecules that are crucial to neural function.
  • the blood-brain barrier by the selectivity of tight junctions between the endothelial cells of brain capillaries, restricts the passage of pathogens, the diffusion of solutes in the blood, and large or hydrophilic molecules into the cerebrospinal fluid, while allowing the diffusion of hydrophobic molecules (O2, CO2, hormones) and small non-polar molecules.
  • Cells of the barrier actively transport metabolic products such as glucose across the barrier using specific transport proteins.
  • the barrier also restricts the passage of peripheral immune factors, like signalling molecules, antibodies, and immune cells, into the CNS, thus insulating the brain from damage due to peripheral immune events.
  • the BBB is composed of endothelial cells restricting passage of substances from the blood more selectively than endothelial cells of capillaries elsewhere in the body.
  • Glioblastoma glioblastoma multiforme
  • GBM grade IV astrocytoma
  • Glioblastomas represent 15% of all brain tumors. They can either start from normal brain cells or develop from an existing low-grade astrocytoma.
  • the diagnosis typically is made by a combination of a CT scan, MRI scan, and tissue biopsy. There is no known method of preventing the cancer. Treatment usually involves surgery, after which chemotherapy and radiation therapy are used.
  • temozolomide is frequently used as part of chemotherapy.
  • High-dose steroids may be used to help reduce swelling and decrease symptoms.
  • Surgical removal (decompression) of the tumor is linked to increased survival, but only by some months. Despite maximum treatment, the tumor always recurs. Such recurrent tumor is referred to as "recurrent glioblastoma”.
  • the typical duration of survival following diagnosis is 12-15 months, with fewer than 3-7% of people surviving longer than five years. Without treatment, survival is typically three months.
  • subject or “patient” can be used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals and primates.
  • Preferred subjects are human subjects.
  • the terms "subject” or “patient” include subjects in need of treatment, more particularly subjects that would benefit from treatment of a given condition, particularly a neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction.
  • Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and/or those in who said condition is to be prevented.
  • therapeutically active/effective amount refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a surgeon, researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated.
  • prolactically active/effective amount refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician.
  • therapeutically active amount is used to refer to both the “therapeutically active amount” and “prophylactically active amount” as described above, unless the distinction is clear from the context.
  • therapeutically and/or prophylactically effective doses of a compound, a nucleic acid encoding the compound, a nucleic acid expression cassette, or a pharmaceutical composition, as taught herein.
  • therapeutically effective dose refers to an amount of a compound, a nucleic acid encoding the compound, a nucleic acid expression cassette, or a pharmaceutical composition, as taught herein, that when administered brings about a positive therapeutic response with respect to treatment of a patient having a specific disease or disorder.
  • Wnt decoding module capable of discriminating Wnt ligands that are otherwise largely synonymous in their capacity to bind Frizzled, thereby helping cells interpret the complexity of Wnt signaling inputs in order to orchestrate tissue development and homeostasis.
  • selectivity is conferred by RECK, which mediates Wnt7-specific binding in a Frizzled-independent manner.
  • G-protein coupled receptor GPR124 a RECK binding partner, acts as a signaling-deficient transmembrane protein which is able to recruit intracellular Dishevelled (Dvl).
  • Dvl scaffolds bridge GPR124 and Frizzled, thereby assembling Wnt7 ligand-specific RECK/GPR124/Frizzled/lipoprotein receptor-related protein (LRP) signalosomes.
  • LRP lipoprotein receptor-related protein
  • novel agonists capable of activating Wnt signaling selectively in cells expressing RECK and GPR124, such agonists being useful as therapeutics, such as particularly for the treatment of neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction.
  • the invention allows to provide inter alia novel agonists capable of stimulating Wnt/0-catenin signaling in cerebral endothelial cells substantially without cross-reactivity with other Frizzled pathways, and useful as therapeutics, particularly for neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction.
  • glioblastoma multiforme also referred to as glioblastoma
  • GBM glioblastoma multiforme
  • said glioblastoma is recurrent glioblastoma.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically active amount of a compound chosen from Wnt7 polypeptide or a fragment thereof capable of activating G-protein coupled receptor (GPR)124/RECK/Frizzled/lipoprotein receptor-related protein (LRP)-mediated Wnt signaling, wherein said Wnt7 polypeptide or fragment thereof does not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof for use in the reduction in progression and/or treatment of glioblastoma in a subject.
  • said glioblastoma is recurrent glioblastoma.
  • Such pharmaceutical composition is particularly effective in preventing, reducing the progression of and in the treatment of glioblastoma, such as recurrent glioblastoma.
  • said compound is able to influence the permeability of the bloodbrain barrier (BBB) in subjects having glioblastoma or subjects prone to develop recurrent glioblastoma.
  • BBB bloodbrain barrier
  • the pharmaceutical composition for use in preventing or reducing the progression of glioblastoma of the present invention provides for a novel way for maintaining a functional BBB in subjects prone to develop glioblastoma or having glioblastoma, while the composition for use in the treatment of glioblastoma of the present invention provides for a novel way in -at least partly- restoring the BBB function in subjects having (recurrent) glioblastoma.
  • the compound is able to repair tight-junctional defects in glioblastoma vessels. By repairing said tight-junctional defects, the BBB impermeability is increased, thereby restoring the BBB functionality.
  • the compound is able to reduce vesicular transport in glioblastoma vessels. By reducing the transport, the BBB impermeability in increased, thereby restoring the BBB functionality.
  • the compound is able to restore the paracellular permeability and/or the transcellular permeability of the BBB in glioblastoma vessels, in particular to reduce the paracellular permeability and/or the transcellular permeability of the BBB, thereby increasing the BBB impermeability.
  • said compound is further able to control tumor angiogenesis in a subject suffering from glioblastoma.
  • the compound is able to repair tight-junctional defects in general, thereby restoring impaired BBB function in general.
  • references to any peptides, polypeptides, proteins or nucleic acids denote the respective peptides, polypeptides, proteins or nucleic acids as commonly known under the respective designations in the art. More particularly, the references to "WNT” and in particular to "WNT7", to "G-protein coupled receptor 124" (GPR124), "Reversion-inducing cysteine-rich protein with Kazal motifs” (RECK), “Frizzled” (FZD), or “lipoprotein receptor-related protein” (LRP) denote the respective peptides, polypeptides, proteins or nucleic acids, as apparent from the context, as commonly known under said designations in the art.
  • GPR124 G-protein coupled receptor 124"
  • RECK Reversion-inducing cysteine-rich protein with Kazal motifs
  • FZD Frrizzled
  • LRP lipoprotein receptor-related protein
  • the terms encompass the peptides, polypeptides, proteins or nucleic acids when forming a part of a living organism, organ, tissue or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources.
  • the terms also encompass the peptides, polypeptides, proteins or nucleic acids when produced by recombinant or synthetic means.
  • any peptide, polypeptide, protein or nucleic acid also encompasses modified forms of said peptide, polypeptide, protein or nucleic acid, such as forms bearing post-expression modifications including, for example, phosphorylation, glycosylation, palmitoylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, ubiquitination, oxidation of methionine to methionine sulphoxide or methionine sulphone, signal peptide removal, N-terminal Met removal, conversion of proenzymes or pre-hormones into active forms, and the like.
  • a broader definition is given above.
  • the pharmaceutical composition for use of the present invention comprises in particular a therapeutically active amount of a compound chosen from Wnt7 polypeptide or a fragment thereof, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof.
  • said Wnt7 is Wnt7a or Wnt7b.
  • the Wnt polypeptide or fragment thereof, or nucleic acid encoding for said Wnt7 polypeptide or fragment thereof as employed herein is of animal origin, preferably warm-blooded animal origin, more preferably vertebrate origin, yet more preferably mammalian origin, including human origin and nonhuman mammalian origin, still more preferably human origin.
  • human WNT7A gene is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) Gene ID 7476.
  • Human WNT7A mRNA (transcript variant 1) is annotated under NCBI Genbank accession number NM_004625.3. Nucleotides 306 (start codon) to 1355 (stop codon) of NM_004625.3 constitute the WNT7A coding sequence.
  • Human WNT7A protein sequence is annotated under NCBI Genbank accession number NP_004616.2, and Uniprot accession number 000755.2, and is further reproduced below (SEQ ID NO: 9):
  • the mature form of human Wnt7a protein (not comprising the signal peptide) comprises an amino sequence as further reproduced below (SEQ ID NO: 2):
  • human WNT7B gene is annotated under NCBI Genbank Gene ID 7477.
  • Human WNT7B mRNA (transcript variant 1) is annotated under NCBI Genbank accession number NM_058238.2.
  • Nucleotides 375 (start codon) to 1424 (stop codon) of NM_058238.2 constitute the WNT7B coding sequence.
  • Human WNT7B protein sequence is annotated under NCBI Genbank accession number NP_478679.1, and Uniprot accession number P56706.2, and is further reproduced below (SEQ ID NO: 10):
  • the compound as disclosed herein is or consists essentially of a fragment of a Wnt7 polypeptide, such as a fragment of a Wnt7a or Wnt7b polypeptide, preferably a fragment of a human Wnt7 polypeptide, such as a fragment of a human Wnt7a or human Wnt7b polypeptide.
  • the fragment of the Wnt7 polypeptide has at least 30%, and preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, preferably at least 100% or more, of the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length Wnt7 polypeptide.
  • a GPR124/RECK/FZD/LRP receptor complex broadly denotes a protein complex, particularly a membrane-associated protein complex, more particularly a plasma membrane-associated protein complex comprising at least one GPR124 polypeptide, at least one RECK polypeptide, at least one FZD polypeptide and at least one LRP polypeptide.
  • a GPR124/RECK/FZD/LRP receptor complex when located at the plasma membrane of a cell, is capable of activating Wnt/0-catenin signaling in said cell in response to extracellularly provided Wnt7 ligand.
  • a FZD/LRP receptor complex broadly denotes a protein complex, particularly a membrane-associated protein complex, more particularly a plasma membrane- associated protein complex comprising at least one FZD polypeptide and at least one LRP polypeptide.
  • a FZD/LRP receptor complex when located at the plasma membrane of a cell, is capable of activating Wnt/0-catenin signaling in said cell in response to extracellularly provided Wnt ligand, such as, but not limited to Wnt7 ligand.
  • LRP lipoprotein receptor-related protein
  • lipoprotein receptor-related protein encompasses any and all lipoprotein receptor-related proteins, also known in the art as low-density lipoprotein receptor- related proteins or prolow-density lipoprotein receptor-related proteins.
  • the terms denote LR.P5, LR.P6, or LR.P5 and LR.P6 (LRP5/6).
  • composition comprising a therapeutically active amount of a compound chosen from Wnt7 polypeptide or a fragment thereof capable of activating GPR124/RECK/Frizzled/LRP5/6-mediated Wnt signaling, wherein said Wnt7 polypeptide or fragment thereof does not activate Frizzled/LRP5/6-mediated Wnt signaling in the absence of RECK and/or GPR124, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof for use in the reduction in progression and/or treatment of glioblastoma in a subject.
  • sequences represented in sequence databases or in the present specification may be of precursors of the respective peptides, polypeptides, proteins or nucleic acids and may include parts which are processed away from mature molecules.
  • any peptides, polypeptides, proteins or nucleic acids encompasses such peptides, polypeptides, proteins or nucleic acids of any organism where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and non-human mammals, still more preferably of humans.
  • one or more and preferably all of WNT7, GPR124, RECK, FZD and LRP as employed herein is or are of animal origin, preferably warmblooded animal origin, more preferably vertebrate origin, yet more preferably mammalian origin, including human origin and non-human mammalian origin, still more preferably human origin.
  • the pharmaceutical composition for use of the present invention acts by Wnt7-specific RECK/GPR124/Frizzled/LRP-mediated signaling, in which Wnt7 (Wnt7a or Wnt7b) binds specifically to Reck in a FZD-independent manner and GPR124, a RECK binding partner, bridges RECK-bound Wnt7 to the FZD/LRP complex via intracellular DVL scaffolds, thereby assembling Wnt7-ligand specific RECK/GPR124/FZD/LRP signalosomes and activating canonical Wnt signaling.
  • Wnt7 Wnt7a or Wnt7b
  • a GPR124/RECK/FZD/LRP receptor complex as envisaged herein may include further component(s), which may or need not functionally modulate the complex.
  • the complex may include Dishevelled (Dvl), forming intracellular scaffolds capable of bridging GPR124 and Frizzled.
  • cells expressing GPR124, RECK, FZD and LRP polypeptides at its plasma membrane is a cell naturally expressing all GPR124, RECK, FZD and LRP polypeptides at the cell surface, such as a cerebral endothelial cell.
  • the capability of the compound chosen from Wnt7 polypeptide or a fragment thereof or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof to activate GPR124/RECK/Frizzled/LRP-mediated Wnt signaling, but not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124 denotes the capability of the compound to activate Wnt signaling in cells positive for GPR124, RECK, FZ and LRP, but not in cells positive for FZ and LRP and negative for GPR124 and/or RECK, wherein the cells positive GPR124, RECK, FZ and LRP and the cells positive for FZ and LRP and negative for GPR124 and/or RECK are otherwise substantially identical.
  • the capability of activating Wnt signaling refers to the ability of the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein to mimic, reproduce or approximate the signal transduction effect and/or activity of a natural Wnt ligand binding to FZD and LRP, such as to an FZD/LRP complex.
  • Activation of Wnt signaling may be suitably determined and/or quantitated by measuring the expression of one or more Wnt target genes, TCF reporter gene expression, beta-catenin stabilization, LRP phosphorylation, and/or translocation of Axin from cytoplasm to cell membrane as known in the art.
  • activation of Wnt signaling may be suitably determined and/or quantitated by measuring the expression of TCF gene (e.g., by RT-PCR or any other transcript detection method), a primary output of Wnt signaling (Nature, 1997, vol. 385(6619), 829-33).
  • a TCF reporter assay (also known as TOP/FOP or TOPflash) may be used to assess changes in the transcription of TCF/LEF controlled genes.
  • the TCF reporter assay may be a luciferase reporter assay.
  • activation of Wnt signaling may be suitably determined and/or quantitated by measuring the expression of c-myc, n-myc, LEF1, or c-jun.
  • activation of Wnt signaling may be determined by measuring the location, level and/or phosphorylation status of p-catenin.
  • a non-limiting example of such an assay is the "P-Catenin Redistribution Assay” (Thermo Scientific) which provides recombinant U20S cells stably expressing human p-catenin fused to the C- terminus of enhanced green fluorescent protein (EGFP).
  • the assay allows visualization and monitoring of the translocation of a GFP- p-catenin fusion protein from the membrane to the nucleus.
  • Another way of determining activation of Wnt signaling is the visualization of Axin translocation, for example with a GFP-Axin fusion protein.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein may be considered capable of activating (canonical) Wnt signaling if the compound enhances Wnt/p-catenin signaling at least 10-fold more, at least 20-fold more, at least 30-fold more, at least 40-fold more, at least 50-fold more, at least 100-fold more, at least 250-fold more, at least 500-fold more, at least 750-fold more, at least 1000-fold more, at least lxl0 4 -fold more, or at least lxl0 5 -fold more compared to Wnt/ p-catenin signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein may be considered to not activate (canonical) Wnt signaling if the compound enhances Wnt/p-catenin signaling less than 10-fold more, such as particularly at most 5-fold more or at most 2.5-fold more, or if the compound does not enhance or even reduces (e.g., 2-fold less or 5-fold less or 10- fold less) Wnt/p-catenin signaling compared to Wnt/ p-catenin signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein may be considered to activate the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling, but not activate Frizzled/LRP- mediated Wnt in the absence of RECK and/or GPR124, if the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity induced by said compound is at least 3.5-fold more, at least 5-fold more, at least 10-fold more, at least 15-fold more, at least 20-fold more, at least 25-fold more, at least 50-fold more, at least 100-fold more, at least 500-fold more, at least 1000-fold more, at least lxl0 4 -fold more, or at least lxl0 5 -fold more, preferably at least 50-fold more, than the Frizzled/LRP-mediated Wnt signaling activity induced by said compound, in absence of RECK and/or GPR124.
  • activity 1 and activity 2 induced by the compound may be normalized to activity 1 and activity 2 induced by wild-type Wnt7a, respectively, the latter for example set to represent 100% activity.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof may be considered to activate GPR124/RECK/Frizzled/LRP- mediated Wnt signaling, but not activate Frizzled/LRP -mediated Wnt signaling in the absence of RECK and/or GPR124, when the Wnt signaling activity induced by the same quantity of said compound under substantially identical conditions is at least 3.5-fold more, 5-fold more, at least 10-fold more, at least 15-fold more, at least 20- fold more, at least 25-fold more, at least 50-fold more, at least 100-fold more, at least 500-fold more, at least 1000-fold more, at least lxl0 4 -fold more, or at least
  • Activation of the Wnt signaling pathway may occur by promoting the close association or mutual proximity of the Frizzled and LRP polypeptides at the cell membrane, thereby forming membrane-associated hetero-oligomers comprising the Frizzled and LRP polypeptides.
  • the intracellular portion of the LRP polypeptide becomes accessible for phosphorylation, for example by CK1 and GSK-3, which greatly increases its affinity for Axin.
  • the intracellular portion of the Frizzled polypeptide is able to induce the phosphorylation and recruitment of DVL.
  • the resulting assembly of an activated LRP- FZD-DVL-Axin complex leads indirectly to the dissociation of the destruction complex of beta-catenin, thereby allowing beta-catenin to accumulate in the cytoplasm and translocate to the cell nucleus where beta-catenin may induce gene transcription.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein is capable of concurrently binding to Frizzled and LRP polypeptides at a cell membrane, in the presence of RECK and GPR124, but not in the absence of RECK and/or GPR124.
  • the compound as disclosed herein is capable of binding to one or more different Frizzled polypeptides, such as one or more Frizzled polypeptides selected from the group consisting of Fzd 1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and FzdlO.
  • the compound as disclosed herein may be capable of specifically binding to at least Fzd4, and optionally to one or more other Fzd; or may be capable of specifically binding to Fzd4 substantially to the exclusion of other Fzd.
  • Fzd4 is believed to be the dominant Fzd family member in endothelial cells of the central nervous system.
  • the compound as disclosed herein is capable of specifically binding to human Fzd4.
  • the compound as disclosed herein may be selective for the one or more preferred Frizzled polypeptides, for example having a specificity for the one or more preferred Frizzled polypeptides of at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 1000-fold, at least lxl0 4 -fold, or at least lxl0 5 -fold, compared to other non-preferred Frizzled polypeptides.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein is capable of binding to one or more different LRP polypeptides involved in Wnt signaling.
  • the compound as disclosed herein is capable of binding to LRP5 and/or LRP6, e.g., any one or each of LRP5 and LRP6.
  • the compound as disclosed herein is capable of binding to human LRP5 and/or LRP6, e.g., any one or each of human LRP5 and human LRP6.
  • the compound as disclosed herein may be selective for the one or more preferred LRP polypeptides, for example having a specificity for the one or more preferred LRP polypeptides of at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 1000-fold, at least lxl0 4 -fold, or at least lxl0 5 -fold, compared to other non-preferred LRP polypeptides.
  • the compound as disclosed herein is capable of binding to the GPR124 and/or the RECK polypeptide.
  • the compound as disclosed herein is capable of concurrently binding to Frizzled and LRP polypeptides, and in addition to the GPR124 and/or the RECK polypeptide.
  • the compound as disclosed herein is capable of binding to the RECK polypeptide.
  • the compound as disclosed herein is capable of concurrently binding to Frizzled, LRP and RECK polypeptides.
  • RECK is composed of five N-terminal cysteine-knot (CK) motifs or regions (i.e. CK1, CK2, CK3, CK4 and CK5), a cysteine-rich domain (CRD) and three Kazal motifs preceding a Glycosylphosphatidylinositol (GPI)-anchor site.
  • the CK motifs, the CRD and the Kazal motifs are located extracellularly. Accordingly, the compound capable of binding to the RECK polypeptide as disclosed herein may bind the CK1 motif, CK2 motif, CK3 motif, CK4 motif, CK5 motif, CRD, and/or one or more of the Kazal motifs of the RECK polypeptide.
  • the compound as disclosed herein is capable of binding to the CK4 and/or CK5 regions of RECK polypeptide.
  • the CK4 motif spans from amino acid C216 to C263, and the CK5 motif spans from amino acid C292 to C338 of the amino acid sequence of the human RECK protein annotated under NCBI Genbank accession number NP_066934.1 as disclosed elsewhere herein.
  • the CK4 motif of human RECK comprises, consists essentially of or consists of the amino acid sequence CCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQC (SEQ ID NO: 3) and the CK5 motif of human RECK comprises, consists essentially of or consists of the amino acid sequence
  • the compound as disclosed herein is capable of binding to the amino acid sequence of SEQ ID NO: 3 and/or the amino acid sequence of SEQ ID NO: 4.
  • the Frizzled receptor is a G protein-coupled receptor protein and ranges in length from about 500 to about 700 amino acids.
  • the N-terminus is predicted to be extracellular and comprises a cysteine rich domain (CRD) of approximately 120 amino acids followed by a hydrophilic linker region of approximately 40-100 amino acids.
  • CCD cysteine rich domain
  • the Frizzled receptor also comprises seven hydrophobic domains that are predicted to form transmembrane alpha-helices.
  • the intracellular C-terminal domain has a variable length and, though the intracellular domain is overall not well conserved among different family members, it comprises a proximal KTXXXW amino acid motif (SEQ ID NO: 5), wherein X can be any amino acid, which is highly conserved in Frizzled polypeptides and which is required for canonical Wnt signaling.
  • the Frizzled CRD domain comprises a motif of 10 invariantly spaced cysteines and is largely conserved between the known Frizzled family members, but also in several other proteins, such as RECK, secreted frizzled related proteins (SFRPs), receptor tyrosine kinases (RTKs), and collagen ol XVIII.
  • the CRD domain is important for ligand (e.g. Wnt) binding to the Frizzled polypeptide, for example by recognition and/or binding of cis-unsaturated fatty acyl groups present in the ligand contact site 1 or the residues located at the Wnt ligand "index" contact site 2.
  • ligand e.g. Wnt
  • Frizzled polypeptide for example by recognition and/or binding of cis-unsaturated fatty acyl groups present in the ligand contact site 1 or the residues located at the Wnt ligand "index" contact site 2.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein is capable of binding to the cysteine-rich domain (CRD) of the Frizzled polypeptide.
  • CRD cysteine-rich domain
  • the compound as disclosed herein is capable of binding to the cis-unsaturated fatty acyl group- binding domain located within the CRD domain of the Frizzled polypeptide or the "index" contact site within the CRD domain.
  • the N-terminal extracellular domain of LRP is composed of four YWTD (SEQ ID NO: 6) repeat domains (or beta-propeller domains), four epidermal growth factor (EGF)- like domains and an LDLR-like domain (LDLRLD).
  • a single YWTD repeat domain comprises six tandem YWTD sequences. The first two, most N-terminal YWPD repeat domains are predicted to bind to Wnt ligands, and the second two sets of YWPD repeat domains are predicted to bind to Dickkopf (DKK).
  • DKK Dickkopf
  • N-terminal extracellular domain is followed by a single membrane-spanning segment and a cytoplasmic tail harboring between one and three NPXY motifs (SEQ ID NO: 7), wherein X can be any amino acid (e.g. in LRP1, LRP2, LRP4, APOER2, LDLR, LRP9), or between one and five PPPSP motifs (SEQ ID NO: 8) (e.g. in LRP5 and LRP6).
  • X can be any amino acid (e.g. in LRP1, LRP2, LRP4, APOER2, LDLR, LRP9), or between one and five PPPSP motifs (SEQ ID NO: 8) (e.g. in LRP5 and LRP6).
  • DKK Dickkopf
  • DKK1 RefSeq Protein: NP_036374.1; GenelD: 22943
  • DKK2 RefSeq Protein: NP_055236.1; GenelD: 27123
  • DKK3 RefSeq Protein: NP_001317149.1; GenelD: 27122
  • DKK4 RefSeq Protein: NP_055235.1; GenelD: 27121.
  • the terms may particularly denote DKK1.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein is capable of binding to the extracellular domain of the LRP polypeptide.
  • the compound as disclosed herein is capable of binding to the DKK-binding site of the LRP polypeptide. In more particular embodiments, the compound as disclosed herein is capable of binding to the DKKl-binding site of the LRP5 and/or LRP6 polypeptide.
  • the compound as disclosed herein is capable of binding to the Wnt-binding site of the LRP polypeptide. In more particular embodiments, the compound as disclosed herein is capable of binding to the Wnt-binding site of the LRP5 and/or LRP6 polypeptide.
  • the compound as disclosed herein is capable of binding to the DKK-binding site and the Wnt-binding site of the LRP polypeptide.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein is capable of binding to 0-propeller-EGF-like domains 1 and 2 (P1E1P2E2) and/or p-propeller-EGF-like domains 3 and 4 (P3E3P4E4) of the LRP polypeptide.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof or nucleic acid encoding such polypeptide or fragment as disclosed herein is or consists essentially of a fragment of a Wnt7 polypeptide, such as a fragment of a Wnt7a or Wnt7b polypeptide, preferably a fragment of a human Wnt7 polypeptide, such as a fragment of a human Wnt7a or human Wnt7b polypeptide.
  • the fragment of the Wnt7 polypeptide has at least 30%, and preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, preferably at least 100% or more, of the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length Wnt7 polypeptide.
  • the fragment of the Wnt7 polypeptide has a GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity which is higher (e.g., 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold or 2-fold higher or even higher) than the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length Wnt7 polypeptide.
  • N-terminal domain of Wnt7a is capable of binding to RECK and activating Wnt signaling mediated by the GPR124/RECK/Frizzled/LRP receptor complex, while not activating Wnt signaling mediated by the Frizzled/LRP receptor complex in the absence of RECK and/or GPR124.
  • the fragment of the Wnt7 polypeptide has Frizzled/LRP-mediated Wnt signaling activity in the absence of RECK and/or GPR124 which is at least 10-fold less, or at least 100-fold less, or at least 1000-fold less, or at least 1 x 10 4 -fold less, or at least 1 x 10 5 -fold less, or at least 1 x 10 6 -fold less than the Frizzled/LRP-mediated Wnt signaling activity of the full-length Wnt7 polypeptide in the absence of RECK and/or GPR124.
  • said fragment of the Wnt7 polypeptide is or consists essentially of the N-terminal domain (NTD) of the Wnt7 polypeptide, such as the NTD of a Wnt7a or Wnt7b polypeptide, preferably the NTD of human Wnt7 polypeptide, such as human Wnt7a or Wnt7b.
  • NTD N-terminal domain
  • the N-terminal domain of the human Wnt7a or Wnt7b polypeptide typically ranges from the Leucine (L) residue at the position corresponding to position 1 in SEQ ID NO: 1 (Wnt7a) or SEQ ID NO: 11 (Wnt7b) to the cysteine (C) residue at the position corresponding to position 247 in SEQ ID NO: 1 (Wnt7a) or SEQ ID NO: 11 (Wnt7b).
  • said fragment of the Wnt7 polypeptide comprises at least the NTD of the Wnt7 polypeptide and does not comprise the C-terminal domain (CTD) of the Wnt7 polypeptide.
  • CTD C-terminal domain
  • the fragment of Wnt7 polypeptide or nucleic acid encoding such fragment as disclosed herein comprises, consists essentially of, or consists of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, contiguous amino acids of the NTD of human Wnt7a polypeptide
  • the compound as disclosed herein comprises, consists essentially of or consists of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, contiguous amino acids of the amino acid sequence: LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFG KELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSA DIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMK
  • the compound chosen from Wnt7 polypeptide or a fragment thereof or nucleic acid encoding such polypeptide or fragment as disclosed herein comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 11, preferably SEQ ID NO: 1, or an amino acid sequence having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, sequence identity to the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 11, preferably SEQ ID NO: 1.
  • the nucleic acid encoding the compound as taught herein preferably encodes a precursor form of the compound including an N-terminal signal peptide sequence. Accordingly, the nucleic acid may encode a fragment of the precursor polypeptide of Wnt7 (i.e. including the signal peptide), such as the precursor polypeptide of human Wnt7a or Wnt7b.
  • the nucleic acid encodes an compound comprising, consisting essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 11 or an amino acid sequence having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 11, wherein said amino acid sequence is preceded N-terminally by a signal peptide having an amino acid sequence MNRKARRCLGHLFLSLGMVYLRIGGFSSVVA (SEQ ID NO: 12) or MHRNFRKWIFYVFLCFGVLYVKLGALSSVVA (SEQ ID NO: 13), respectively.
  • the nucleic acid encoding the compound as taught herein may be comprised within a vector providing for a signal peptide.
  • the signal peptide may be a homologous or heterologous signal peptide, depending on the host cell used for production of the compound as taught herein.
  • a protease cleavage site motif may be present C-terminally of said signal peptide and N-terminally of the compound as taught herein.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof or nucleic acid encoding such polypeptide or fragment as disclosed herein is a variant of a Wnt7 polypeptide, such as a variant of a Wnt7a or Wnt7b polypeptide.
  • the compound as disclosed herein is a variant of the NTD of a Wnt7a or Wnt7b polypeptide.
  • the N-terminal domain of human Wnt7a comprises an amino acid sequence referred to as SEQ ID NO: 1, while the mature form of human Wnt7a protein (not comprising the signal peptide) comprises an amino sequence referred to as SEQ ID NO: 2.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof or nucleic acid encoding such polypeptide or fragment as disclosed herein is a mutated Wnt7 polypeptide, such as a mutated of a Wnt7a or Wnt7b polypeptide.
  • the compound as disclosed herein is a mutated NTD of a Wnt7a or Wnt7b polypeptide, the NTD of Wnt7a or Wnt7b polypeptide which is a fragment of the Wnt7a or Wnt7b polypeptide.
  • the NTD of the Wnt7a or Wnt7b polypeptide comprises two or more (e.g., preferably two, preferably three, more preferably four) of the amino acid substitutions listed above.
  • the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein comprises, consists essentially of or consists of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, wherein the residue at position 17 is not glutamine, the residue at position 20 is not isoleucine, the residue at position 25 is not proline, the residue at position 27 is not alanine, the residue at position 28 is not isoleucine, the residue at position 33 is not glutamate, the residue at position 37 is not methionine, the residue at position 39 is not leucine, the residue at position 41 is not glutamate, the residue at position 44 is not phenylalanine, the residue at position 50 is not arginine, the residue at position 52 is
  • the compound is a fragment of Wnt7 polypeptide as disclosed herein and is a variant of the NTD of human Wnt7a comprising, consisting essentially of or consisting of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the residue at position 17 is not glutamine, the residue at position 20 is not isoleucine, the residue at position 25 is not proline, the residue at position 27 is not alanine, the residue at position 28 is not isoleucine, the residue at position 33 is not glutamate, the residue at position 37 is not methionine, the residue at position 39 is not leucine, the residue at position 41 is not glutamate, the residue at position 44 is not phenylalanine, the residue at position 50 is not arginine, the residue at position 17
  • Table 1 gives an overview of the position of amino acid substitutions in the amino acid sequence of the (mouse) Wnt7a precursor polypeptide and their corresponding position in the amino acid sequence of the mature (mouse) Wnt7a polypeptide:
  • the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by one or more (preferably not more than three, preferably not more than two, more preferably one) other amino acid residue, such as preferably but without limitation by an alanine (A) residue, a serine (S) or a leucine (L) residue.
  • K residue at the position corresponding to position 159 in SEQ ID NO: 1 or SEQ ID NO: 2 relates to the K residue at position 190 in the Wnt7a precursor polypeptide, as shown in Table 1.
  • the compound as disclosed herein comprises, consists essentially of or consists of an amino acid sequence as set forth in SEQ ID NO: 14, 54, 55, 59, 92 or 93, preferably SEQ ID NO: 14 or 59, more preferably SEQ ID NO: 14.
  • the variant of the Wnt7 polypeptide has at least 35%, preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, preferably 100%, of the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length wildtype Wnt7 polypeptide.
  • the variant of the Wnt7 polypeptide has at least 70% of the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length wild-type Wnt7 polypeptide.
  • the variant of the Wnt7 polypeptide has a GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity which is higher (e.g. 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold or 2-fold higher or even higher) than the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length wild-type Wnt7 polypeptide.
  • the compound as disclosed herein is soluble in water.
  • the solubility of the compound can be increased by using a Frizzled- specific antibody or fragment thereof, instead of a palmitoylated Frizzled-binding domain of a Wnt polypeptide.
  • the compound as disclosed herein can be a protein, polypeptide or a peptide.
  • said compound is a nucleic acid encoding the Wnt7 protein or fragment as disclosed herein. Said nucleic acid may be inserted into a nucleic acid expression cassette and/or vector, as is well-known in the art.
  • a further aspect relates to a nucleic acid expression cassette comprising the nucleic acid encoding the compound as disclosed herein, operably linked to a promoter and/or transcriptional and translational regulatory signals.
  • the nucleic acid expression cassette may comprise one or more open reading frames (ORF) encoding said one or more proteins, polypeptides or peptides.
  • ORF open reading frames
  • the precise nature of transcriptional and translational regulatory sequences or elements required for expression may vary between expression environments, but typically include a transcription terminator, and optionally an enhancer.
  • the nucleic acid expression cassette comprises the nucleic acid encoding the compound as disclosed herein, operably linked to one or more promoters, enhancers, ORFs and/or transcription terminators.
  • a further aspect relates to vector comprising the nucleic acid encoding the compound as disclosed herein, or the nucleic acid expression cassette as disclosed herein, such as a viral vector.
  • Factors of importance in selecting a particular vector include inter alia: choice of recipient cell, ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in particular recipient cells; whether it is desired for the vector to integrate into the chromosome or to remain extra-chromosomal in the recipient cells; and whether it is desirable to be able to "shuttle" the vector between recipient cells of different species.
  • Expression vectors can be autonomous or integrative.
  • a nucleic acid can be in introduced into a cell in the form of an expression vector such as a plasmid, phage, transposon, cosmid or virus particle.
  • the recombinant nucleic acid can be maintained extrachromosomally or it can be integrated into the cell chromosomal DNA.
  • Expression vectors can contain selection marker genes encoding proteins required for cell viability under selected conditions (e.g., URA3, which encodes an enzyme necessary for uracil biosynthesis, or LEU2, which encodes an enzyme required for leucine biosynthesis, or TRP1, which encodes an enzyme required for tryptophan biosynthesis) to permit detection and/or selection of those cells transformed with the desired nucleic acids.
  • Expression vectors can also include an autonomous replication sequence (ARS).
  • the ARS may comprise a centromere (CEN) and an origin of replication (ORI).
  • the ARS may be ARS18 or ARS68.
  • Integrative vectors generally include a serially arranged sequence of at least a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment.
  • the first and second insertable DNA fragments are each about 200 (e.g., about 250, about 300, about 350, about 400, about 450, about 500, or about 1000 or more) nucleotides in length and have nucleotide sequences which are homologous to portions of the genomic DNA of the cell species to be transformed.
  • a nucleotide sequence containing a nucleic acid of interest for expression is inserted in this vector between the first and second insertable DNA fragments, whether before or after the marker gene.
  • Integrative vectors can be linearized prior to transformation to facilitate the integration of the nucleotide sequence of interest into the cell genome.
  • the vectors Prior to introducing the vectors into a cell of interest, the vectors can be grown (e.g., amplified) in bacterial cells such as Escherichia coli (E. coli).
  • E. coli Escherichia coli
  • the vector DNA can be isolated from bacterial cells by any of the methods known in the art, which result in the purification of vector DNA from the bacterial milieu.
  • the purified vector DNA can be extracted extensively with phenol, chloroform, and ether, to ensure that no E. coli proteins are present in the plasmid DNA preparation, since these proteins can be toxic to mammalian cells.
  • a compound may comprise a protein, polypeptide or peptide. Such may be suitably obtained through expression by host cells or host organisms, transformed with an expression construct encoding and configured for expression of said protein, polypeptide or peptide in said host cells or host organisms, followed by purification of the protein, polypeptide or peptide.
  • a further aspect provides a host cell comprising the nucleic acid, nucleic acid expression cassette or vector as taught herein.
  • the host cell may be a bacterial cell, a yeast cell, an animal cell, or a mammalian cell.
  • host cell and "host organism” may suitably refer to cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals. Contemplated as host cells are inter alia unicellular organisms, such as bacteria (e.g., E.
  • Contemplated as host organisms are inter alia multi-cellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrate animals, still more preferably mammals, yet more preferably primates; particularly contemplated are such animals and animal categories which are non-human.
  • Such protein, polypeptide or peptide may be suitably isolated.
  • nucleic acid can be directly injected into the target cell I target tissue.
  • Other methods include fusion of the recipient cell with bacterial protoplasts containing the nucleic acid, the use of compositions like calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids or liposomes or methods like receptor- mediated endocytosis, biolistic particle bombardment ("gene gun” method), infection with viral vectors (i.e.
  • nucleic acid molecules derived from lentivirus, adeno-associated virus (AAV), adenovirus, retrovirus or antiviruses), electroporation, and the like.
  • AAV adeno-associated virus
  • Other techniques or methods which are suitable for delivering nucleic acid molecules to target cells include the continuous delivery of an NA molecule from poly (lactic-Co- Glycolic Acid) polymeric microspheres or the direct injection of protected (stabilized) NA molecule(s) into micropumps delivering the product.
  • Another possibility is the use of implantable drug-releasing biodegradable microspheres.
  • NA encapsulation of NA or providing NA in various types of liposomes (immunoliposomes, PEGylated (immuno) liposomes), cationic lipids and polymers, nanoparticles or dendrimers, poly (lactic-Co-Glycolic Acid) polymeric microspheres, implantable drug-releasing biodegradable microspheres, etc.; and co-injection of NA with protective agent like the nuclease inhibitor aurintricarboxylic acid.
  • liposomes immunoliposomes
  • cationic lipids and polymers nanoparticles or dendrimers
  • poly (lactic-Co-Glycolic Acid) polymeric microspheres implantable drug-releasing biodegradable microspheres, etc.
  • protective agent like the nuclease inhibitor aurintricarboxylic acid
  • the compound is provided in a liposome or lipid nanoparticle.
  • lipid nanoparticle refers to a particle having at least one dimension in the order of nanometers (e.g., 1-1,000 nm) and comprises a plurality of lipid molecules physically associated with each other by intermolecular forces.
  • the lipid nanoparticles may be, e.g., microspheres (including unilamellar and multilamellar vesicles, e.g. "liposomes”), a dispersed phase in an emulsion, micelles or an internal phase in a suspension.
  • An active agent or therapeutic agent such as a nucleic acid or polypeptide, is encapsulated or provided in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.
  • LNPs are commonly formulated with two or more excipients: (i) a sterol, which enhances the stability of the LNP bilayer and promotes membrane fusion; (ii) optionally a phospholipid, which fortifies the LNP bilayer structure and also aids in endosomal escape; and (iii) a lipid-polyethylene glycol (PEG) conjugate, which inserts into the LNP bilayer and provides a PEG coating that reduces LNP aggregation, reduces nonspecific binding of proteins due to sterically hindrance, and reduces nonspecific endocytosis by immune cells.
  • a LNP may further comprise one of more buffering agents.
  • the vector comprising the nucleic acid as described herein is a viral vector, preferably a viral vector specifically directed towards the central and/or peripheral nervous system (e.g., a brain-specific viral vector).
  • the viral vector is a central nervous system (CNS) neuron-specific adeno-associated virus serotype 9 (AAV9) mutant.
  • the viral vector is a blood brain barrier endothelial cellspecific viral vector.
  • the viral vector is a blood brain barrier endothelial cell-specific capsid adeno-associated virus serotype 2 (AAV2) mutant.
  • the pharmaceutical composition for use, comprising a therapeutically active amount of a compound chosen from Wnt7 polypeptide or a fragment thereof or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof as described in any of the previous embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.
  • carrier or “excipient” includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like.
  • buffers such as, e.g., neutral buffered saline or phosphate buffered saline
  • solubilisers such as, e.g., EDTA or glutathi
  • Illustrative, non-limiting carriers for use in formulating the pharmaceutical compositions include, for example, oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents suitable for intravenous (IV) use, liposomes or surfactant-containing vesicles, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to one of ordinary skill in the art.
  • compositions as intended herein may be formulated for essentially any route of administration, such as without limitation, oral administration (such as, e.g., oral ingestion or inhalation), intranasal administration (such as, e.g., intranasal inhalation or intranasal mucosal application), parenteral administration (such as, e.g., subcutaneous, intravenous (I.V.), intramuscular, intraperitoneal, intrathecal or intracisternal injection or infusion), transdermal or transmucosal (such as, e.g., oral, sublingual, intranasal) administration, topical administration, rectal, vaginal or intratracheal instillation, and the like.
  • oral administration such as, e.g., oral ingestion or inhalation
  • intranasal administration such as, e.g., intranasal inhalation or intranasal mucosal application
  • parenteral administration such as, e.g.
  • compositions may be formulated in the form of pills, tablets, lacquered tablets, coated (e.g., sugar-coated) tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions.
  • preparation of oral dosage forms may be is suitably accomplished by uniformly and intimately blending together a suitable amount of the compound as disclosed herein in the form of a powder, optionally also including finely divided one or more solid carrier, and formulating the blend in a pill, tablet or a capsule.
  • Exemplary but non-limiting solid carriers include calcium phosphate, magnesium stearate, talc, sugars (such as, e.g., glucose, mannose, lactose or sucrose), sugar alcohols (such as, e.g., mannitol), dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • Compressed tablets containing the pharmaceutical composition can be prepared by uniformly and intimately mixing the compound as disclosed herein with a solid carrier such as described above to provide a mixture having the necessary compression properties, and then compacting the mixture in a suitable machine to the shape and size desired.
  • Moulded tablets maybe made by moulding in a suitable machine, a mixture of powdered compound moistened with an inert liquid diluent.
  • Suitable carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc.
  • compositions may be formulated with illustrative carriers, such as, e.g., as in solution with saline, polyethylene glycol or glycols, DPPC, methylcellulose, or in mixture with powdered dispersing agents, further employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilising or dispersing agents known in the art.
  • illustrative carriers such as, e.g., as in solution with saline, polyethylene glycol or glycols, DPPC, methylcellulose, or in mixture with powdered dispersing agents, further employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilising or dispersing agents known in the art.
  • Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the compound as taught herein or their physiologically tolerable salts in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents.
  • a pharmaceutically acceptable solvent such as ethanol or water, or a mixture of such solvents.
  • the formulation can also additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant.
  • delivery may be by use of a single-use delivery device, a mist nebuliser, a breath-activated powder inhaler, an aerosol metered-dose inhaler (MDI) or any other of the numerous nebuliser delivery devices available in the art.
  • MDI aerosol metered-dose inhaler
  • mist tents or direct administration through endotracheal tubes may also be used.
  • Examples of carriers for administration via mucosal surfaces depend upon the particular route, e.g., oral, sublingual, intranasal, etc.
  • illustrative examples include pharmaceutical grades of mannitol, starch, lactose, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate and the like, with mannitol being preferred.
  • illustrative examples include polyethylene glycol, phospholipids, glycols and glycolipids, sucrose, and/or methylcellulose, powder suspensions with or without bulking agents such as lactose and preservatives such as benzalkonium chloride, EDTA.
  • the phospholipid 1,2 dipalmitoyl-sn-glycero-3- phosphocholine is used as an isotonic aqueous carrier at about 0.01-0.2% for intranasal administration of the compound of the subject invention at a concentration of about 0.1 to 3.0 mg/ml.
  • compositions may be advantageously formulated as solutions, suspensions or emulsions with suitable solvents, diluents, solubilisers or emulsifiers, etc.
  • suitable solvents are, without limitation, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose, invert sugar, sucrose or mannitol solutions, or alternatively mixtures of the various solvents mentioned.
  • the injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • suitable non-toxic, parenterally-acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • suitable dispersing or wetting and suspending agents such as sterile, bland, fixed oils, including synthetic mono- or dig
  • a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP Water for Injection (WFI).
  • Other illustrative carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral vegetable oil-in-water emulsion.
  • Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solution, 5% dextrose in WFI and 0.01-0.1% triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a 1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol and the balance an acceptable isotonic solution such as 5% dextrose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI and 1 to 10% squalene or parenteral vegetable oil-in-water emulsions.
  • PBS phosphate buffered saline
  • aqueous formulations may comprise one or more surfactants.
  • the composition can be in the form of a micellar dispersion comprising at least one suitable surfactant, e.g., a phospholipid surfactant.
  • phospholipids include diacyl phosphatidyl glycerols, such as dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoyl phosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines, such as dimyristoyl phosphatidylcholine (DPMC), dipalmitoyl phosphatidylcholine (DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acids, such as dimyristoyl phosphatidic acid (DPMA), dipahnitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); and diacyl phosphatidyl ethanolamines such as dimyristoyl phosphatidyl ethanolamine (DPME), dipalmitoyl phosphatid
  • a surfactant:active substance molar ratio in an aqueous formulation will be from about 10: 1 to about 1 : 10, more typically from about 5: 1 to about 1 :5, however any effective amount of surfactant may be used in an aqueous formulation to best suit the specific objectives of interest.
  • these formulations When rectally administered in the form of suppositories, these formulations may be prepared by mixing the compounds according to the invention with a suitable nonirritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidity and/or dissolve in the rectal cavity to release the drug.
  • a suitable nonirritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidity and/or dissolve in the rectal cavity to release the drug.
  • Suitable carriers for microcapsules, implants or rods are, for example, copolymers of glycolic acid and lactic acid.
  • the pharmaceutical composition comprising the compound or nucleic acid encoding the compound, as taught herein is administered parenterally. More preferably, the pharmaceutical composition as taught herein is administered intravenously, for example by infusion, or intrathecally.
  • the pharmaceutical composition for use comprising the compound, the nucleic acid encoding the compound, or the nucleic acid expression cassette comprising the nucleic acid as taught herein is used in gene therapy, in particular in blood brain barrier endothelial cell-directed gene therapy, most in particular in glioblastoma-directed gene therapy.
  • a method for gene therapy in particular central and/or peripheral nervous system-directed gene therapy, in a subject in need of said gene therapy comprising: introducing in the subject, in particular in the central and/or peripheral nervous system of the subject, a nucleic acid expression cassette or a vector as described herein; and expressing a therapeutically effective amount of the compound encoded by the nucleic acid as taught herein in the subject, in particular the central and/or peripheral nervous system of the subject.
  • the pharmaceutical composition for use comprising the compound or the nucleic acid encoding the compound as taught herein is used in mRNA therapy, in particular in blood brain barrier endothelial cell-directed mRNA therapy, most in particular in glioblastoma-directed mRNA therapy.
  • a method for mRNA therapy in a subject suffering from glioblastoma, in particular central and/or peripheral nervous system-directed mRNA therapy, in a subject in need of said mRNA therapy comprising : introducing in the subject, in particular in the central and/or peripheral nervous system of the subject, a nucleic acid encoding the compound as taught therein; and expressing a therapeutically effective amount of the compound encoded by the nucleic acid as taught herein in the subject, in particular the central and/or peripheral nervous system of the subject.
  • mRNAs do not integrate into the genome and therefore do not have the risk of insertional mutagenesis.
  • the pharmaceutical composition as taught herein is administered to the subject by the injection (e.g., intravenously) or transplantation of allogeneic cells transformed with the vector comprising the nucleic acid or the nucleic acid expression cassette as taught herein.
  • the injected or transplanted allogenic cells will transcribe and translate the nucleic acid encoding the compound as taught herein in vivo.
  • the dosage or amount of the compound as taught herein, optionally in combination with one or more other active compounds to be administered depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect.
  • the unit dose and regimen depend on the nature and the severity of the disorder to be treated, and also on factors such as the species of the subject, the sex, age, body weight, general health, diet, mode and time of administration, immune status, and individual responsiveness of the human or animal to be treated, efficacy, metabolic stability and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the compound described in any of the embodiments above.
  • the compound as taught herein can be first administered at different dosing regimens.
  • levels of the compound in a tissue can be monitored using appropriate screening assays as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen.
  • the frequency of dosing is within the skills and clinical judgement of medical practitioners (e.g., doctors, veterinarians or nurses).
  • the administration regime is established by clinical trials which may establish optimal administration parameters. However, the practitioner may vary such administration regimes according to the one or more of the aforementioned factors, e.g., subject's age, health, weight, sex and medical status.
  • the frequency of dosing can be varied depending on whether the treatment is prophylactic or therapeutic.
  • Toxicity and therapeutic efficacy of the compound as described herein or pharmaceutical composition for use of the invention as described in any of the embodiments, comprising the same can be determined by known pharmaceutical procedures in, for example, cell cultures or experimental animals. These procedures can be used, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Pharmaceutical compositions that exhibit high therapeutic indices are preferred. While pharmaceutical compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to normal cells (e.g., non-target cells) and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in appropriate subjects.
  • the dosage of such pharmaceutical compositions lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the pharmaceutical composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the pharmaceutical composition which achieves a half-maximal inhibition of symptoms
  • levels in plasma can be measured, for example, by high performance liquid chromatography.
  • a typical dosage e.g., a typical daily dosage or a typical intermittent dosage, e.g., a typical dosage for every two days, every three days, every four days, every five days, every six days, every week, every 1.5 weeks, every two weeks, every three weeks, every month, or other
  • a typical dosage may range from about 10 pg/kg to about 500 pg/kg body weight of the subject, per dose, depending on the factors mentioned above, e.g., such as 20-400 pg/kg or 20-200 pg/kg or 20-100 pg/kg or 40-80 pg/kg body weight of the subject.
  • the compound as taught herein may be administered at about 5 pg/kg, or at about 10 pg/kg, or at about 15 pg/kg, or at about 20 pg/kg, or at about 25 pg/kg, or at about 30 pg/kg, or at about 35 pg/kg, or at about 40 pg/kg, or at about 45 pg/kg, or at about 50 mg/kg, or at about 60 pg/kg, or at about 70 pg/kg, or at about 80 pg/kg, or at about 90 pg/kg, or at about 100 pg/kg, or at about 200 pg/kg, or at about 300 pg/kg, or at about 400 pg/kg, or at about 500 pg/kg per dose.
  • the compound as taught herein is administered using a sustained delivery system, such as a (partly) implanted sustained delivery system.
  • a sustained delivery system may comprise a reservoir for holding the compound as taught herein, a pump and infusion means (e.g., a tubing system).
  • the sustained delivery system may be a mini- osmotic pump system implanted in the brain.
  • the compound as disclosed herein is the main or only active ingredient of the pharmaceutical composition.
  • the pharmaceutical composition for use as described in any of the embodiments is combined with a second therapy, preferably chosen from surgery, chemotherapy, radiotherapy or immunotherapy.
  • a further aspect relates to the pharmaceutical composition
  • the pharmaceutical composition comprising the compound as disclosed herein, the nucleic acid encoding the compound as disclosed herein the nucleic acid expression cassette as disclosed herein or the vector as disclosed herein, for use as a medicament.
  • a further aspect provides a method of preventing, reducing in progression or treating glioblastoma in a subject in need thereof, comprising administering to a subject a therapeutically active amount of pharmaceutical composition comprising a compound chosen from Wnt7 polypeptide or a fragment thereof capable of activating G-protein coupled receptor (GPR)124/RECK/Frizzled/lipoprotein receptor-related protein (LRP)-mediated Wnt signaling, wherein said Wnt7 polypeptide or fragment thereof does not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof, as described in any of the embodiments throughout this specification.
  • GPR G-protein coupled receptor
  • LRP lipoprotein receptor-related protein
  • the current invention equally provides methods for the treatment of glioblastoma, as well as is directed to the use of a therapeutically active amount of pharmaceutical composition
  • a therapeutically active amount of pharmaceutical composition comprising a compound chosen from Wnt7 polypeptide or a fragment thereof capable of activating G-protein coupled receptor (GPR)124/RECK/Frizzled/lipoprotein receptor-related protein (LRP)-mediated Wnt signaling, wherein said Wnt7 polypeptide or fragment thereof does not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof, as described in any of the embodiments throughout this specification for the manufacture of a medicament for the treatment or reduction of progression of glioblastoma in a subject. Further embodiments are as described above.
  • the NTD coding sequences correspond to amino-acids 1-278 of Wnt7a and Wnt7b, and the CTD part of the chimeric ligands to 279-360 (Wnt2), 281-351 (Wnt4), 310-380 (Wnt5a), 294-364 (Wnt6), 347-417 (WntlOa), 284-354 (Wntll), 294-364 (Wntl6) and 261-358 (XWnt8a).
  • the collection of active human WNT-V5 ligands (Addgene #43807 to #43825 from the Xi He laboratory) was used in the Wnt/Fz pairwise STF experiments of Fig. 8.
  • Firefly luciferase activities (derived from a genomic transgene in STF cells or ectopically expressed from the transfected M50 Super 8x TOPFIash plasmid, Addgene plasmid #12456 in other cells) were normalized to Renilla luciferase activities (pTK-Renilla vector transfection).
  • PLA proximity ligation assay
  • IF immunofluorescence
  • IP immunoprecipitation
  • mice monoclonal antibody against V5 R960-25, Thermo Fisher Scientific; RRID: AB_2556564
  • rabbit polyclonal antibodies against Wnt7a Wnt7a
  • purified rabbit polyclonal anti-HA antibodies H6908, Sigma-Aldrich, RRID:AB_260070
  • rabbit polyclonal anti-Glut-1 07-1401, Millipore, RRID:AB_1587074
  • rabbit polyclonal anti-Fibrinogen (ab34269, Abeam, RRID:AB_732367) at 1 :300
  • rabbit polyclonal anti-Mfsd2A (a gift from D.
  • Isolectin GS-IB4 Alexa 594-Conjugate (121413, Invitrogen) at 1 :200 and Streptavidin Alexa 647conjugate (S21374, Invitrogen) at 1 : 1000.
  • Isolectin GS-IB4 Alexa 594-Conjugate (121413, Invitrogen) at 1 :200
  • Streptavidin Alexa 647conjugate S21374, Invitrogen
  • HRP conjugated secondary antibodies Promega
  • HEK293T cells were obtained from ATCC (CRL-3216), and HEK293 STF cells were kindly provided by Jeremy Nathans (John Hopkins). They were grown in DMEM/F12 1 : 1 medium (Lonza) supplemented with 10% fetal bovine serum (Biowest, S1810) and maintained at 37°C in a humidified incubator equilibrated with 5% CO2.
  • HEK293(T) cells were transfected with lipofectamine 2000 (Invitrogen) for STF assays or with Polyethylenimine (PEI max, Polyscience) for IP and IF at 70% confluency 24 h after plating.
  • HEK293(T) cells were seeded on poly-lysine (0.1 mg.ml-1) coated 12 mm coverslips.
  • the CRISPR/Cas9- engineered GPR124-/-;RECK-/- mutant HEK293 STF line has been described before, together with the FZ1-10-/- HEK293T clone used to build the FZl-10-/-;GPR124-/- ;RECK-/- HEK293T line.
  • GPR124 5'-GCATCCGCTGGTACCACAAC-3'; SEQ ID NO: 96
  • RECK 5'-ATTGTTGATGGTCTCATCGA-3'; SEQ ID NO: 97
  • CRISPR/Cas9 guide sequences were designed using the http://crispr.mit.edu website and cloned into pSpCas9(BB)-2A-GFP.
  • the top 1% of GFP+ cells were isolated by FACS (Arialll, BD Biosciences) 48 h after transfection and distributed in 96-well plates for clonal expansion and lesion determination by Sanger sequencing.
  • Dual luciferase assays were performed 2 days after transfection in 96-well plates using DualLuciferase Reporter Assay system (E1960, Promega) according to the manufacturers' protocol: after extraction in passive lysis buffer, the ratio of Firefly and Renilla luciferases activities of the cell lysates were measured.
  • the co-culture assay was performed by seeding transfected reporter cells with Wnt7 ligands expressing HEK293T cells at a 1 : 1 ratio 1 day after transfection and 1 day prior to luciferase measurements.
  • the amount of plasmid DNA transfected per well was optimized for each expression vector as follows: Renilla luciferase (0.5 ng), Wnt ligands (20 ng), Fz receptors (5 ng), Lrp5 (2.5 ng), Gprl24 (10 ng), Reck (10 ng). The total amount of DNA was adjusted to 100 ng per well with the empty pCS2 vector. Proximity ligation assay
  • the Duolink PLA technology (Sigma-Aldrich) was used to infer protein-protein interaction between HA-Reck and Wnt-V5 variants in situ. Two days after transfection, cells were fixed with 4% PFA for 10 min, blocked 30 min at 37°C with the Duolink blocking solution, incubated with primary antibodies anti-HA and anti- RECK for 1 h at RT, with anti-rabbit PLUS and anti-mouse MINUS PLA probes for 1 h at 37°C, with the Duolink Ligation solution for 30 min at 37°C and finally with the Duolink Amplification solution (Red) for 100 min at 37°C. Two PBS washes were included between each step.
  • PLA signal was quantified using Image! PLA-associated channel was thresholded to create a binary mask in which PLA dots were detected using the Particle Analysis plugin of ImageJ set to quantify the area and the mean signal intensity of each particle. Their Nearest Neighbor Distance (NND) was additionally computed using the ImageJ BAR plugin. To sort for membrane-localized signal, we filtered out the particles whose area was ⁇ 5pm2 or NND > 4pm.
  • PLA signal was calculated per field of view as ( Ax/,)/ N where A is the area of the particle, I its mean intensity corrected for background and N the area fraction occupied by the nuclei signal in the field (measured on DAPI thresholded pictures), indicative of the number of cells.
  • GPR124-/-;RECK-/- HEK293 cells were blocked in PBS/BSA 2% for 20 min at 4°C, incubated with primary antibody against Wnt7a for 1 h at 4°C, fixed in 4% paraformaldehyde for 10 min at RT, permeabilized with 0.1% saponin in PBS for 5 min, blocked again in PBS/BSA 2% for 1 h and sequentially incubated with primary anti-V5 and Alexaconjugated secondary antibodies for 1 h at RT. Antibodies were diluted in PBS/BSA 2% and cells were washed three times with PBS after each step.
  • Membrane accumulation of the ligand was quantified using ImageJ as follows: maximum intensity projection of the signal obtained after permeabilization (V5 intracellular signal) was used to detect expressing cells and to measure their mean signal intensity. For each cell, the Z plane associated with the strongest membrane-associated signal (Wnt7 extracellular signal) was used to measure its mean signal intensity along a line of circa 2 pm width (30 pixels) along the cell surface. We also included in this analysis cells that were only positive for membrane signal. The ratio between extracellular and intracellular signals after background correction was calculated per cell, normalized to the ratio corresponding to a double negative cell in the same field of view and averaged per field. Co-Immunoprecipitation, dot blot, and western blot
  • RECK-/- HEK293T were incubated with the membrane impermeable crosslinker DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate), Sigma-Aldrich) at 0.2 mM in HBS (20 mM HEPES, pH 7.4, 150 mM NaCI, 1 mM CaCI2, 0.5 mM MgCI2) for 2 h at 4°C, two days after transfection in 6- well plates.
  • DTSSP membrane impermeable crosslinker
  • HBS 20 mM HEPES, pH 7.4, 150 mM NaCI, 1 mM CaCI2, 0.5 mM MgCI2
  • mice Wnt7a structure was modeled based on the crystal structure of Xenopus Wnt8a in complex with mouse Fz8 CRD (PDBID:4F0A) as described previously.
  • transgenic and mutant lines used in this study are Tg(kdrl:EGFP)s843, Tg(kdrl:HRASmCherry)s896, Tg(7xTCF-Xla.Siam:GFP)ia4, Tg(-
  • the targeting sgRNA construct was cloned into pT7-gRNA (Addgene plasmid #46759) using these primers: wnt7aa-fwd 5'-TAGGTGGAACTGCTCGGCCCTC-3' (SEQ ID NO: 98), wnt7aa-rev 5'-AAACGAGGGCCGAGCAGTTCCA-3' (SEQ ID NO: 99).
  • sgRNA was transcribed from pT7-gRNA using the MEGAshortscript T7 transcription kit (Ambion) and injected at 50 pg into one-cell stage embryos, together with 150 pg of nls-zCas9-nls mRNA transcribed from pT3TS-nCas9n (Addgene plasmid # 46757) using the mMessage mMachine T3 Kit (Ambion).
  • wnt7aa TTCCATTTGACCCTACTTACCCAAT (SEQ ID NO: 100)
  • wnt7ab AACCCCTACAAATGACCACAGAACT
  • wnt7ba AAGCATTCGCTCAAACCTACAGGTA
  • wnt7bb ACAGATTGAAACACTTACAGGTAGC
  • Synthetic mRNAs were transcribed from pCS2 plasmids after Notl digestion using the mMessage mMachine SP6 Kit (Ambion) and injected into one-cell stage zebrafish embryos at the concentration indicated in the Figures.
  • Transient mosaic endothelial overexpression was obtained by co-injection of 10 pg of Tol2 transposase mRNA and 10 pg of the pT2-kdrl-mWnt7a(WT, K190A or NTD)-P2A-NLS-eBFP DNA constructs.
  • Probes were transcribed with T7 RIMA polymerase (Roche) and detected using anti- DIG AP (1 : 5000) and NBT/BCIP substrate (Roche). Embryos were sectioned (Vibratome, 50 pm) after embedding in 4% ultrapure low melting point agarose (Thermo Fisher Scientific).
  • CtAs, DRG neurons and TCF-positive endothelial cells were quantified on confocal Z-stack recordings of live, anesthetized (low doses of tricaine), agarose-embedded (1% low melting point agarose) transgenic zebrafish.
  • Tg(kdrl: EGFP);Tg(gatala :dsRed) embryos were treated from 34 hpf onwards with 1 pM atorvastatin (Sigma-Aldrich). Embryos were graded based on the extent of intracerebral bleeding under a bright-field stereoscope.
  • brains were arbitrary divided into 4 areas (left and right, forebrain/midbrain and hindbrain) in which the level of hemorrhages was scored in a double-blind manner as 0 (no hemorrhage), 1 (small hemorrhage) or 2 (large hemorrhage).
  • the phenotype was considered as mild if the cumulated score of the 4 zones was ⁇ 1, moderate if it was ⁇ 4 and severe if it was >4. Confocal pictures were taken for illustration. Tracer leakage assays were performed by microinjection of 1 nl of 10 mg.ml-1 Alexa Fluor 488 Dextran 10.000 MW (Invitrogen) in PBS in the common cardinal vein at 54 hpf.
  • Embryos were mounted dorsoventrally and individually imaged at 30 min post-injection using a Zeiss LSM900 inverted laser scanning microscope. Quantification was performed using ImageJ by generating maximum intensity projection of stack volumes around 20 pm thick, including the dorsal arch of the CtAs about 40 pm above the PHBC. Projections were positioned minimally 15 pm below the dorsally located ventricle to avoid fluorescence signal contamination from tracer accumulated in this structure. Mean fluorescence intensity was calculated in the area covering the hindbrain parenchyma. The relative fluorescence intensity was determined by dividing the mean parenchymal tracer intensity by the maximal intensity of the tracer signal in the CtA lumen.
  • a custom polyclonal rabbit antibody was generated against zebrafish Glutl/Slc2ala (Eurogentec, Belgium).
  • Two KLH-MBS-conjugated peptides (Slc2ala 45-59: NETWHNRYSEYIPPT (SEQ ID NO: 112) and 471485: ADKYNRDDLNTLGAD (SEQ ID NO: 113)), designed to minimize cross-reactivity with the paralogue Slc2alb, were used for immunization.
  • Glut-1 immunostaining manually dechorionated zebrafish embryos were fixed with 4% paraformaldehyde overnight at 4°C, permeabilized with 20 pg.ml-1 Proteinase K in PBS 0.5% TritonX-100 for 30 min at 37°C, fixed again with 4% paraformaldehyde 20 min at RT, blocked 1 h at RT with 10% normal goat serum, 0.5% TritonXIOO, 1% DMSO in PBS and sequentially stained overnight at 4°C with primary and secondary antibodies diluted in blocking solution. Embryos were washed several times with PBS 0.5% TritonX-100 for 5 min after each step and for 30 min after the staining steps.
  • Xenopus laevis embryos obtained from adult frogs by hormone-induced egg-laying and in vitro fertilized using standard methods, were microinjected with 15 pg mRNA of Wnt7a variants into the ventral vegetal region of four-cell stage embryos. Experiments were conducted in accordance with European and national ethical and animal welfare guidelines (protocol approval number: CEBEA-IBMM-2017-22: 19).
  • Locomotor activity was recorded by videotracking at P20, 18 days after AAV injection. Pools of four mice were familiarized for 10 min in open field locomotor activity boxes (14 cm x 36 cm) before being recorded for 3 min. Horizontal ambulation was analyzed at 3 Hz using the Manual Tracking plugin from ImageJ. The cumulated travelled distance was calculated and normalized to the non-injected condition.
  • Mouse GL261 glioblastoma cells (DSMZ no. ACC 802) were cultured in DMEM- GlutaMAX-I medium (Invitrogen) containing 10% fetal bovine serum (Biowest, S1810). The previously generated Dkkl Tet-Off GL261 cells were cultured in the same media supplemented or not with 1 pg.ml-1 doxycycline for 5 days before implantation.
  • mice were placed into a stereotactic device and 2 pl of PBS containing 105 living GL261 cells were injected at 0.25 pl.min-1 into the striatum using the coordinates relative to bregma: 0.5 (anterior-posterior), 2 (mediolateral), and 3.5 (dorsoventral) using a Hamilton Microliter #75N injection syringe with a 26Ga needle. The needle is left in place for at least 2 min before being slowly removed. At the end of the procedure, the incision was sutured and mice were allowed to recover two days before being injected with AAVs. Alternatively, mice were injected with AAVs 2 weeks before GL261 cell implantation.
  • mice were fed with control or 1 g.kg-1 doxycycline hyclate containing diet (Safe Diets, France) starting 2 weeks before implantation until the end of the experiment. Mice were individually monitored at 7, 14 and/or 21 days post implantation (dpi) using Magnetic Resonance Imaging (MRI) performed on a 9.4T Biospec or on a IT ICON operating under Paravision 5.1 software (Bruker). MR signal was obtained using a mouse head transmit/receive volume coil (23 mm inner diameter).
  • MRI Magnetic Resonance Imaging
  • Animals were anesthetized in a chamber with isoflurane (2.53%) vaporized in oxygen (2 L.min-1) and were transferred to a dedicated imaging cradle, in which isoflurane delivery was adapted (1.5-2% in 0.4 L.min-1 oxygen flow). Their body temperature was maintained by warm water circulating in a blanket, and their respiratory rate was monitored during the whole imaging session, in accordance with CMMI protocol number 2011- 07 (LA1500589).
  • a T2-weighted 2D TurboRARE (Rapid Acquisition with Relaxation Enhancement) sequence was used to visualize the brain on 15 slices (0.5 mm thickness (no gap), 83 microns plane resolution, repetition time (TR): 3000 ms, effective echo time (TE): 60 ms, number of averages: 16, acquisition time: 9 min 36 sec, fat saturation). Tumors were manually contoured on each slice by drawing region of interest (ROI), from which volumes were obtained with the VivoQuant software (InviCRO, version 2.0). For histology, mice were euthanized at 25 dpi and brains were embedded into OCT before freezing at -80°C.
  • mice For sulfo-NHS-biotin permeability assay, anesthetized mice were perfused intracardially at 22-24 days post implantation with 10 ml of 0.5 mg.ml-1 EZ-LinkSulfo-NHS-Biotin (Thermo Fisher Scientific, 21217) followed by 10 ml of 1%PFA/PBS at a flow rate of 2-2.5 ml.min-1.
  • the dissected brains were fixed overnight in 4% PFA/PBS at 4 °C before being rinsed in PBS, embedded in 4% agarose and cut in 50 pm thick sections using a vibratome.
  • Floating sections were blocked/permeabilized in PBS 0.5% TritonX-100, 10% normal goat serum for 2 h at RT, incubated with primary antibodies against CD31 overnight at 4°C and 4 h at RT, with secondary antibodies additionally containing Alexa 647-conjugated streptavidin 2 h at RT, with five PBS 0.1% TritonX-100 washes after each staining step. After PBS and distilled water washes, sections were mounted in Dako medium (Agilent).
  • Antibodies were diluted in the blocking solution and DAPI (1 pg.ml-1) was added to the secondary antibodies. Slides were mounted in DAKO medium (Agilent) and imaged using a confocal microscope. LacZ and LEF-1 immunostainings were used to assess 0-catenin activity in brain from BAT-Gal mice after AAV injection. Staining was performed on similarly located sections and the number of lacZ or LEF- 1 positive nuclei (co-localizing with DAPI signal) were counted.
  • Tumor vessel phenotypes were analyzed on slides from the center of the tumor. Quantifications were performed using ImageJ on maximum intensity projections from 5 different fields of view and averaged per mouse. For analysis of vessel coverage, thresholded pictures of CD31 staining were used to measure the portion of the area occupied by vessels (area fraction measurement option). Laminin dispersion was calculated as SDi/ i in which SDi is the standard deviation of intensity and i the mean intensity of the signal. For LEF-1 expression analysis, thresholded DAPI, ERG and laminin signals were used to isolate endothelial and nonendothelial nuclei using the Particle Analysis plugin.
  • Endothelial specific mean signal intensity of LEF-1 in nuclei was measured and normalized to the nuclear LEF-1 signal outside of the vessels after background subtraction. Quantification of Claudin-5, Mfsd2A and Caveolin-1 endothelial expressions were performed by detecting vessel structures using the Particle Analysis plugin on thresholded CD31/laminin pictures and measuring their mean respective signal intensity after background subtraction. The same procedure was used to measure GLUT1 mean endothelial intensity per surface unit and this value was multiplied by the cell area (vessel area divided by the number of nucleus) to get the total GLUT1 signal per cell. For assessing fibrinogen leakage, the detected vessel areas were scaled up by 125% before measuring fibrinogen intensity in this area.
  • the ratio between the area of the thresholded desmin and CD31 signals was calculated.
  • Sulfo-NHS-Biotin leakage was quantified as the intensity of the streptavidin signal in the area around the vessel after background subtraction. This area was defined as the region corresponding to a 150% scale up of the thresholded CD31 signal from which the initial vessel area was subtracted. Only vessels whose inside streptavidin signal was bigger than the background were taken into account.
  • Epitope retrieval was achieved with the RNAScope Target Retrieval Reagent for 15 min at 100°C and sections were let to dry at 60°C. Tissues were then submitted to a series of incubation at 40°C in the HybEZ Oven followed by two washes in RNAScope Wash Buffer IX: 15 min with the Protease Plus solution, 2 h with the probe, 30 min with the APM1 solution, 15 min with the APM2 solution, 30 min with the APM3 solution, 15 min with the APM4 solution. The two last amplification steps were performed at RT : 30 min with the APM5 solution and 15 min with the APM6 solution. Sections were stained with a 1 : 1 ratio of RNAScope DAB-A and DAB-B solutions for 10 min at RT and counterstained with 50% Hematoxylin for 2 min at RT before dehydration and mounting.
  • mice were retro-orbitally injected with 10 mg HRP (Sigma-Aldrich) in 200 pl of PBS that was let to circulate 30 min. After cervical dislocation, brains were harvested and fixed in 2% glutaraldehyde/4%PFA in 0.1 M cacodylate buffer for 1 h at RT followed by 5 h at 4°C. Brains were embedded in 4% agarose in 0.1 M cacodylate buffer and cut in 50 pm thick sections using a vibratome agarose. DAB revelation was performed by incubating floating sections with the DAB substrate (SIGMAFAST tablets, D4293-5SET, Sigma-Aldrich) for 20 min at RT.
  • DAB substrate SIGMAFAST tablets, D4293-5SET, Sigma-Aldrich
  • BBB blood-brain barrier
  • NVU neurovascular unit
  • the endothelial monolayer limits paracellular permeability by linking adjacent cell membranes through tight junctions, represses transcellular traffic by downregulating vesicular transport, constrains immune cell trafficking, and uses ATP-fueled transporters to efflux a wide range of undesirable small molecules back into the bloodstream.
  • BBB dysfunction along with varying degrees of cerebrovascular hyperpermeability, neurovascular uncoupling, or blood flow dysregulation, has been linked to stroke, gliomas, epilepsy, traumatic brain injury, and neurodegenerative disorders.
  • BBB breakdown leakage of neurotoxic plasma components, infiltration of immune cells, and CNS milieu alterations contribute to neuronal demise and worsen disease outcome.
  • clinically-approved BBB-protective strategies are currently lacking.
  • endothelial Wnt/0- catenin signaling acts as a master regulator of BBB physiology in response to neural- derived Wnt7a/b ligands.
  • Wnt/0-catenin signaling initiates the BBB differentiation cascade at the earliest steps of CNS vascular invasion, and then maintains BBB function in adults.
  • Recent evidence suggests that inhibition of Wnt signaling by conditional deletion of 0-catenin signaling in ECs causes BBB breakdown and accelerates disease progression in stroke, glioblastoma, and multiple sclerosis in mice.
  • recombining a constitutively active form of 0-catenin in the CNS endothelium is protective in models of brain cancer and stroke.
  • identifying safe modalities to enhance Wnt/0-catenin signaling selectively at the BBB constitutes a promising therapeutic avenue for a range of neurological disorders.
  • Wnt7a/b are the endogenous ligands controlling p-catenin-dependent BBB maturation, they constitute, in principle, legitimate therapeutic agents to repair the dysfunctional BBB.
  • Wnt signaling exerts pleiotropic functions across a range of tissues and organs, in both health and disease. More so, the structural modalities of Wnt/Frizzled (Fz) interactions a priori disqualify natural Wnt ligands as safe therapeutics.
  • Wnt7a/b indeed belongs to a multigenic family of 19 closely related secreted glycoproteins that fold into an unusual two-domain structure reminiscent of a human hand.
  • the N-terminal domain NTD, Fig.
  • Wnt7a/b ligands in vivo is thus predicted to have multiple adverse outcomes, including altered organogenesis, stem cell expansion, and tumorigenesis. Accordingly, Wnt7a overexpression is incompatible with proper vertebrate development: when expressed in Xenopus embryos, Wnt7a causes axis duplication, a classical dysmorphic outcome of exacerbated Wnt/0-catenin signaling (fig. 7A).
  • Wnt7a and other "canonical" Wnt ligands induce posteriorization of the anterior nervous system in zebrafish embryos, resulting in the loss of forebrain and eye structures (fig. 7B).
  • This promiscuous signaling mode together with the widespread Frizzled expression patterns, and the difficulty in producing active Wnt proteins at bulk levels, has contributed to hampering the clinical development of Wnt-signaling based therapies.
  • Wnt7a/b-specific receptor complex to activate Wnt/p-catenin signaling for brain angiogenesis and BBB regulation.
  • This receptor complex is composed of the GPI-anchored glycoprotein Reck and the adhesion G-protein coupled receptor Gprl24.
  • Reck after physically binding to the linker peptide of Wnt7a/b NTD (Fig. 1A, pink residues), stabilizes the ligand in a signaling-competent lipophilic conformation and delivers it to Fz receptors via the transmembrane tethering function of Gprl24.
  • Reck and Gprl24 synergistically stimulate Wnt7a/b-specific responses by assembling higher-order Gprl24/Reck/Fz/Lrp5/6 complexes.
  • Wnt7a/b thus activates two distinct types of membrane receptor complexes.
  • One has broad tissue distribution and binds non-discriminately to Wnt7a/b and other Wnt ligands via two contacts sites within the Fz CRD, and a less well-known interaction with Lrp5/6 (Fig. 1A, hereafter termed systemic "off-target” complex).
  • the other complex is enriched at the level of the BBB ECs and is highly specific for Wnt7a/b.
  • Reck provides an additional contact point by binding at least in part to the divergent linker peptide of Wnt7a/b (Fig 1A, BBB "on-target").
  • Wnt signaling induced by 17 Wnt ligands including Wnt7a and Wnt7b, in pairwise combination with each of the ten Fz receptors in WT, GPR124-/-;RECK- /- and Gprl24/Reck-overexpressing Wnt/p-catenin reporting super top flash (STF) HEK293 cells (fig. 8).
  • WT and GPR124-/-;RECK-/- cells revealed a near-identical Wnt/Fz signaling landscape with multiple Wnt ligands signaling through several Fz receptors and most Fz responding to multiple Wnt ligands, as anticipated from the poorly discriminative Wnt/Fz binding mechanism.
  • Wnt7a and Wnt7b signaled preferentially through Fz5 and Fz8 in WT and GPR124-/;RECK-/- cells (fig. 8A, B).
  • Wnt7a/b signals were selectively and potently stimulated upon Gprl24 and Reck over-expression even in the absence of a coexpressed Fz (fig. 8C). This illustrates that the endogenous pool of Fz receptors is sufficient to support Wnt7a/b signaling in HEK cells.
  • Reck binds Wnt7a/b exclusively within the N-terminal domain, and chimeric Wnt ligands composed of Wnt7a NTD fused to a CTD derived from another Wnt were competent for Reck binding, which correlated with Gprl24/Reck signaling (fig. 10).
  • Wnt7aNTD The signaling properties of Wnt7aNTD further demonstrated that altering the structure of Wnt ligands can modulate their signaling specificity, and more specifically, that Wnt7a can be engineered into a Gprl24/Reckspecific ligand.
  • Wnt7aNTD is only ⁇ 30% active compared to the parental Wnt7a ligand.
  • Wnt7aNTD is only ⁇ 30% active compared to the parental Wnt7a ligand.
  • the enhanced Wnt7a signaling robustness associated with the presence of the BBB specific co-receptors resulted in the identification of 25 single-residue variants displaying highly specific Gprl24/Reck activity (so-called "agonists" >70% on-target, ⁇ 10% off-target), scattered over the entire Wnt7a structure (Fig. ID residues highlighted in blue, fig. 12B). Reciprocal Fz5-selective variants were not found.
  • Wnt7a signaling in this setting is Gprl24-independent, as revealed by the analysis of zygotic and maternal-zygotic gprl24s984 mutants (fig. 7C).
  • the morphological alterations induced, selectively, by Wnt7a mRNA injections may result either from a mere quantitative increase in Wnt signaling, as detected in cell culture using universal TOP-flash reporters, or from a qualitative difference between Gprl24/Reck dependent and -independent signaling.
  • the selectivity of the uncovered agonists results from their incapacity to bind, and therefore activate, Fz receptors in the absence of Gprl24/Reck.
  • Wnt7a immunolocalized to the surface of RECK-/- ;GPR124-/- cells transiently transfected with Reck or Fz5 Wnt7aNTD and Wnt7aK190A labeled the membrane in the presence of Reck, but not Fz5 (Fig. 2A, B).
  • the agonists bound HA-tagged Reck as efficiently as Wnt7a in antiHA co-immunoprecipitation experiments (Fig.
  • Reck has been shown to extend Wnt7a activity half-life by maintaining it in a monomeric, hydrophobic state.
  • Wnt7a-deficiency model in zebrafish. Although the Gprl24/Reck complex was first discovered in zebrafish by its essential role in cerebrovascular and dorsal root ganglia development, the exact nature of the Wnt7a/b ligand isoform implicated in these processes has not been explored. During mouse embryogenesis, Wnt7a and Wnt7b cooperate to control brain angiogenesis and BBB formation.
  • Wnt7a and Wnt7b are duplicated in the zebrafish genome (wnt7aa, wnt7ab, wnt7ba, and wnt7bb), and we used morpholino-mediated gene knock-down to identify the BBB-relevant ligand(s). Only wnt7aa morphants showed the anticipated phenotypes (fig. 16A) and, therefore, we generated a wnt7aa frame-shift allele (wnt7aaulb2) through CRISPR/Cas9 mutagenesis (Fig. 3A).
  • wnt7aaulb2 mutants displayed fully penetrant cerebrovascular defects in the 60 hours post fertilization (hpf) hindbrain (Fig. 3B), associated with down-regulated Tg(7xTCF-Xla.Siam:GFP) Wnt/0-catenin reporter expression in CNS-invading vessels (Fig. 3C).
  • hpf post fertilization
  • Fig. 3B mice fully penetrant cerebrovascular defects in the 60 hours post fertilization (hpf) hindbrain
  • Fig. 3C The development of ngnl :GFP-positive DRG neurons was also compromised (Fig. 3B), consistent with wnt7aa expression in the zebrafish brain and trunk region (fig. 16B).
  • the Gprl24/Reck-dependent DRG neurogenesis defects resulting from wnt7aa deficiency could be partially corrected by injecting 100 pg of Wnt7aNTD or Wnt7aK190A mRNA at the one-cell stage.
  • the parental Wnt7a could not restore DRG development as it triggered severe morphological defects (Fig. 1G and 3D).
  • Wnt7aa-/- cerebrovascular phenotypes could not be rescued by transient mRNA expression, presumably due to the late onset of angiogenic sprouting in zebrafish hindbrain (36 hpf). Therefore, to test if Wnt7aNTD and Wnt7aK190A can also function as bona fide BBB instructive ligands, we assessed their activity by mosaic transgenic endothelial expression, using a kdrl (vegfr2) promoter.
  • Wnt7aK190A restored the formation of central arteries (CtAs) in wnt7aa-/- embryos to a level comparable to Wnt7a, and these vessels expressed the glucose transporter-1 (Glutl), a marker of BBB maturation (Fig. 3E). Consistent with its partial activity in vitro, Wnt7aNTD was also competent for Gprl24/Reck-dependent brain angiogenesis and barriergenesis, although less potently than Wnt7aK190A. This difference is compatible with a more limited intrinsic signaling potential (Fig. IE), but we cannot exclude reduced protein expression or processing in vivo.
  • Fig. IE intrinsic signaling potential
  • ATV treatment also resulted in the accumulation of intracardially injected 10 kDa Dextran into the hindbrain (Fig. 3G).
  • Fig. 3G To test whether Wnt7aK190A expression could counteract these more subtle ATV- induced BBB defects, we examined Dextran-injected embryos with hemispheric transgenic endothelial expression of Wnt7aK190A, allowing to contrast CNS leakage among the two hemispheres of a single animal. Wnt7aK190A expression was sufficient to reduce the leakage to control levels (Fig. 3H).
  • AAV-PHP.eB viruses expressing either EGFP alone (AAV-EGFP), Wnt7a-P2AEGFP (AAV-Wnt7a), or Wnt7aK190A-P2A-EGFP (AAV-K190A) under the control of the constitutive CAG promoter.
  • Wnt7a fusions to EGFP via the cleavable P2A are active in vitro, and that upon intravenous injection of 4.1011 vg (viral genomes) in 8week old mice, CD31-positive brain vessels are surrounded by EGFP+ cells (Fig. 4A), exposing endothelial cells to local sources of Wnt7a or Wnt7aK190A.
  • AAV-PHP.eB capsid drives expression of the transgenes in ⁇ 25% of cortical and striatal ECs, in ⁇ 30% of astrocytes, and in ⁇ 45% of NeuN-i- neurons (fig. 18A).
  • Over 95% of the examined Desmin-i- pericytes were negative for EGFP.
  • EGFP+/Desmin+ double positive cells were seemingly detected at rare occasions, although we could not exclude endothelial contributions. Pericytes are thus, at best, marginal sources of Wnt7a in this approach (fig. 18B).
  • Wnt7a and Wnt7aK190A showed a discrete distribution, with preferential accumulation at the level of CD31-positive brain vessels, as well as some scattered parenchymal cells (Fig. 4B, asterisks).
  • the distribution, and hence potential activity of the ligands likely reflects the expression patterns of their receptors, with unbound ligands presumably getting cleared through the glymphatic system. Accordingly, Wnt7a, with its broader receptor repertoire, appeared to exhibit a more significant non- vascular distribution (Fig. 4B, asterisks).
  • mice with the largest tumors started to exhibit disease symptoms, including faulty postural syndromes and abnormal gait. This timepoint was therefore chosen for terminal analysis and tissue harvesting.
  • Control tumors showed more prominent intratumoral microvascular hemorrhages (asterisks) and edema than AAV-K190A tumors (Fig. 5B), suggesting cerebrovascular differences between the groups.
  • the tumor-associated vasculature showed features of vessel normalization, i.e. reduced CD31+ vascular density (Fig. 5C), reduced vessel diameter (Fig. 5C), and more compact and seemingly smoother distribution of laminin (Fig. 5D).
  • Cell densities were comparable in AAV-EGFP and AAV-K190A tumors (6,425 versus 7,141 cells.
  • Dkkl+ (-dox) GL261 tumors were compared with Dkkl- (+dox) tumors of mice injected with AAV-EGFP (control) or AAV-K190A (Wnt activation).
  • Dkkl- tumors grew and responded to AAV-K190A as WT GL261 cells, with 95% of the AAV-K190A- treated tumors smaller than 40 mm3 at 20-24 dpi and the control cohort exhibiting more variable volumes (40-80 mm3) (Fig. 6A).
  • Dkkl-i- tumors grew even bigger, up to 160 mm3 (Fig 6A).
  • Endothelial Wnt activity markers (LEF1 and GLUT1) were the highest in the Wnt stimulated Dkkl-/K190A tumors and the lowest in Wnt inhibitory Dkkl-i- tumors, with Dkkl-/EGFP tumors showing intermediate values (fig. 24A, 24B).
  • hemorrhage (fig. 25), vascular density (fig. 24C), and fibrinogen leakage (Fig. 6B) followed the opposite trend, being gradually reduced by the step-wise increase in endothelial Wnt signaling.
  • Wnt7aK190A restores endothelial Wnt signaling, reduces vascular density and normalizes the BBB pleiotropically, affecting both the transcellular and paracellular permeability routes, thereby slowing tumor progression.
  • Wnt7a ligands can be engineered into highly-specific Gprl24/Reck agonists, thereby disclosing a novel class of BBB therapeutics. This level of specificity was deemed unreachable for Wnt-derived proteins by virtue of their promiscuous interaction mode with widely expressed Fz receptors.
  • Gprl24/Reck agonists In addition to the strict signaling specificity of the Gprl24/Reck agonists, we suspect that homeostatic feedback loops maintain Wnt signaling activities within carefully controlled physiological activity windows. Accordingly, we detected significant reinforcement of endothelial Wnt signaling only in dysfunctional glioma vessels, while the signaling levels of healthy parenchyma vessels remained unaffected.
  • the Gprl24/Reck agonists described in this study constitute BBB therapeutic molecules, in particular for use in the treatment of glioblastoma. In glioblastoma, intra-tumoral BBB leakage leads to morbid vasogenic brain edema. GBM is therefore well-positioned for targeted vascular therapies, and systemic administration of bevacizumab, an anti-VEGF antibody, is used as second-line treatment for recurrent or non-responsive glioblastomas.

Abstract

The current invention relates to a pharmaceutical composition comprising a therapeutically active amount of a compound chosen from Wnt7 polypeptide or a fragment thereof capable of activating G-protein coupled receptor (GPR)I24/RECK/Frizzled/Iipoprotein receptor-related protein (LRP)-mediated Wnt signaling, wherein said Wnt7 polypeptide or fragment thereof does not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof for use in the reduction in progression and/or treatment of glioblastoma in a subject.

Description

COMPOSITIONS FOR THE TREATMENT OF GLIOBLASTOMA
FIELD OF THE INVENTION
The present invention is directed to compositions and methods for treating subjects diagnosed with glioblastoma.
BACKGROUND
Glioblastoma multiforme (GBM) is a common type of primary brain tumor in humans and is a very aggressive and devastating cancer, with a median survival of approximately one year. Glioblastoma has the worst prognosis of any central nervous system malignancy. Therapy for GBM is difficult due to its biological location in the brain. Current treatments can involve chemotherapy, radiation, radiosurgery, corticosteroids, antiangiogenic therapy, and surgery. Despite the development of new surgical and radiation techniques and the use of multiple antineoplastic drugs, a cure for malignant gliomas does not exist. Glioblastoma cells are often resistant to cytotoxic agents, and the high incidence of recurrence in a very short period of time in glioblastoma patients suggests that tumorigenic cells are capable of overtaking the treatments.
GBM is characterized by a dense vascular network exhibiting disrupted BBB properties (S. Watkins et al., Nat. Commun. 5, 1-15 (2014); S. W. Schneider et al., Acta Neuropathol. 2004 Mar;107(3):272-6). Interestingly, endothelial Wnt signaling was reported to control vascular integrity in different brain tumor models. EP 3 768 701 (also published as US 2021/309704) discloses therapeutic agents capable of activating G-protein coupled receptor (GPR)124/RECK/Frizzled/lipoprotein receptor- related protein (LRP)-mediated Wnt signaling, wherein said agents do not activate Frizzled/LRP -mediated Wnt signaling in the absence of RECK and/or GPR124. These agents are described to prevent or treat neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction.
It is the aim of the current invention to provide compositions and methods for treating subjects diagnosed with glioblastoma. SUMMARY OF THE INVENTION
The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention provides for a pharmaceutical composition for use according to claim 1 and dependent claims thereof.
DESCRIPTION OF FIGURES
Figure 1. Engineering Wnt7a ligands into highly specific Gprl24/Reck activators
(A) Backbone model of murine Wnt7a based on the crystal structure of CRD-bound XWnt8a, and schematics of the receptor complexes mediating Wnt7a/b/0-catenin signaling. (B) Models of Wnt7a, the Wnt7aS206A palmitoylation mutant, and the single-domain Wnt7a variants (N-terminal domain, NTD blue; C-terminal domain, CTD, grey) and their relative STF (super top flash) luciferase activity in the presence of Fz5 (grey) or Gprl24/Reck/Fzl (green). (C) Position and color-coded Gprl24/Reck/Fzl or Fz5 STF activities of 147 single-residue Wnt7a variants. Gprl24/Reck agonists (blue dots) were defined as variants with >70% Gprl24/Reck/Fzl and <10% Fz5 activity. (D) Front, lateral, and back views of the Wnt7a surface model with integrated color-coded activities of all 147 single-residue variants. Agonistic mutations are highlighted in blue. (E) Beta-catenin signaling assays using STF cells transfected with any of the 10 Fz receptors or Gprl24/Reck, together with Wnt7a, Wnt7aNTD, Wnt7aK190A, or the empty pCS2 vector. (F) Phenotypic scoring of stage 36 Xenopus laevis embryos injected into the ventral vegetal region of the 4-cell embryo with 15 pg of the indicated mRNA. (G) Phenotypic scoring of 3 dpf zebrafish larvae injected at the one-cell stage with Wnt7a, Wnt7aNTD, or Wnt7aK190A mRNA at the indicated doses. ***P<0.001; data represent mean ± SD.
Figure 2. Gprl24/Reck agonists are unable to bind Fz autonomously
(A-B) GPR124-/-; RECK-/- HEK293 cells transiently expressing Wnt7a, Wnt7aNTD, or Wnt7aK190A together with Fz5, Reck, or the empty pCS2 vector were submitted to Wnt7a double immunostaining before (Wnt7a extracellular, green/black) and after (Wnt7a intracellular, pink) cell permeabilization. Maximum intensity projections of confocal images are shown (A), and the ratio between the membrane-associated and the intracellular signal (B) was calculated as described in the method section. (C-D) Western blot analysis of anti-HA coimmunoprecipitation assays after membrane- impermeable cross-linking in total lysates of RECK-/- HEK293T cells expressing HA- Reck (C) or HA-Fz5 (D) (bait) together with Wnt7a, Wnt7aNTD, or Wnt7aK190A (prey), with or without expression of untagged Reck (D). (E) Gprl24/Reck/Fzl and Fz5 activity correlations across the Wnt7a single-residue collection. (F) Relative STF luciferase activity in the presence of Gprl24/Reck/Fzl (green) or Fz5 (grey) in response to Wnt7a or Wnt7aK190A secreted either by the STF reporter cells (autocrine monoculture setting) or by co-cultured HEK293T cells (paracrine coculture setting). (G, H, I) Gprl24/Reck/Fzl (green) or Fz5 (grey) STF activities with combined agonistic mutations (G), activity-increasing mutations (H), or alternative substitutions of Wnt7aK190 (I). ***P<0.001; ns: non-significant; data represent mean ± SD.
Figure 3. Gprl24/Reck agonists stimulate brain angiogenesis, blood-brain barrier induction and prevent hemorrhagic stroke in zebrafish
(A) Characterization of the zebrafish 7-bp frameshift wnt7aaulb2 allele. The protospacer adjacent motif (PAM) is underlined in blue. (B) Dorsal views and quantification of 60 hpf Tg(kdrl:GFP) hindbrain CtAs (central arteries) and 72 hpf Tg(neurogl :GFP) trunk dorsal root ganglia (DRG, grey arrowheads) in WT and wnt7aa-/- zebrafish. (C) Dorsal views of 30 hpf Tg(7xTCFXia.Siam:GFP);Tg(kdrl:ras- mCherry) hindbrains from WT and wnt7aa-/- embryos and quantification of the number of GFP+ (P-catenin signaling positive) endothelial cells in the perineural PHBCs (primordial hindbrain channels). (D) Trunk DRG development in 72 hpf Tg(neurogl:GFP) wnt7aa morphant larvae injected at the one-cell stage with the indicated mRNA. Injection of 100 pg of Wnt7a is associated with profound developmental defects (t) precluding DRG development. (E) Dorsal views of 48 hpf WT and wnt7aa-/- Tg(kdrl:GFP) hindbrain cerebrovasculatures, with transgenic endothelial expression of Wnt7a, Wnt7aNTD, or Wnt7aK190A. BFP is used as a transgenesis marker, and Glutl immunostaining to assess BBB differentiation. (F) Intracerebral (IC) hemorrhage score of 54 hpf Tg(kdrl:GFP);Tg(gatala:dsRed) embryos treated from 34 hpf onwards with 1 pM atorvastatin (ATV), with or without transgenic endothelial expression of Wnt7aK190A. Red arrowheads point to extravasated erythrocytes. (G) BBB leakage of Alexa Fluor 488 10 kDa Dextran upon ATV exposure in 54 hpf WT embryos. (H) Quantification of normalized brain parenchymal 10 kDa Dextran tracer intensity upon ATV exposure, with or without hemispheric transgenic endothelial expression of Wnt7aK190A in 54 hpf embryos. *P<0.05; **P<0.01; ***P<0.001; data represent mean ± SD. Figure 4. Absence of ectopic Wnt activities or behavioral defects after brainwide AAVmediated gene delivery of Gprl24/Reck agonists in the mouse.
(A-B) Immunostaining of (A) sagittal brain sections for EGFP (green) and CD31- positive vessels (magenta, grey) and (B) coronal sections for CD31 (cyan, grey), Wnt7a (magenta, grey) together with a DAPI counterstain, two weeks after intravenous injections of the indicated AAVs. (C) Staining of coronal brain slices of AAV-injected BAT-GAL mice for LEF1 (green), lacZ (magenta, fire), laminin (cyan, white) and DNA (DAPI, blue), in the hippocampal area dorsal to the dentate gyrus (DG, left) and in the parafascicular nucleus area (PFN, right). Nuclear LacZ-positive cells or signal intensities were quantified at 7, 14, and 28 days post injection (dpi). HPF, hippocampal formation, TH, Thalamus; HY, hypothalamus, (D) RNAScope in situ hybridization of Axin2 in coronal sections of AAV-injected WT mice at 14 dpi. (E) Spontaneous open field locomotor activity traces of P20 mice injected retro-orbitally at P2 with 4.1010 vg of the indicated AAV-PHP.eB viruses along with a group of uninjected control mice. Spontaneous locomotor activity is quantified as the total distance traveled for 3 min in the open field test. *<0.05; **P<0.01; ***P<0.001; ns: non-significant; data represent mean ± SD.
Figure 5. A single "hit-and-run" gene delivery of Gprl24/Reck agonists achieves neurovascular-specific Wnt/p-catenin activation and vessel normalization in glioblastoma multiforme
(A) GL261-implanted mice, injected intravenously at 2 dpi (days post implantation) with AAVs, were imaged by MRI to evaluate in vivo tumor volume between 21 and 24 dpi. (B) At 25 dpi, mice were euthanized for endpoint gross morphology assessment (left) and H8iE staining of serial sections (middle). Asterisks indicate hemorrhages (right). (C-G) Coimmunostaining of coronal tumor or parenchymal (parench) sections for endothelial cells (CD31, cyan) (C), vascular basement membranes (laminin, grey) (D), LEF1 (magenta) together with the endothelial nuclear marker ERG (cyan) (E), GLUT1 (magenta) together with laminin (cyan) (F), or fibrinogen (magenta) together with laminin (cyan) (G). (H) Correlation between endothelial Wnt activity (LEF1) and tumor volume, vessel density, BBB differentiation (GLUT1), and BBB leakage (fibrinogen extravasation) in tumors of AAV-EGFP mice (colored lines). (J) Same as (I) in AAV-K190A injected mice. *<0.05; **P<0.01; ***P<0.001; ns: nonsignificant; data represent mean ± SD.
Figure 6. Gprl24/Reck agonists as BBB repair agents in glioblastoma and stroke models (A) MRI monitoring of tumor volumes after implantation of 1.105 Tet-Off Dkkl GL261 cells, in the absence of doxycycline (+Dkkl, -dox), or the presence of doxycycline (-Dkkl, -i-dox). Doxycycline-exposed mice were injected intravenously with AAV-EGFP or AAV-K190A, as indicated. (B) Quantification of fibrinogen leakage into the tumor. Data for WT GL261 are the same as in Fig. 5C (C) Representative images and quantification of leakage of transcardiallyperfused sulfo-NHS-biotin within the tumor (Turn.) and the healthy parenchyma (Par.). (D) Electron micrographs of tumor and cortical sections of HRP-injected mice. White and black asterisks indicate tracer within the vessel lumen and vascular basement membrane, respectively. Arrowheads label the junctions where the progression of the tracer within the intercellular cleft is blocked (E-G) Coimmunostaining of CD31 (cyan) together with Claudin-5 (magenta, grey) (E), Mfsd2a (magenta, grey) (F), or Desmin (magenta, grey) (G). *<0.05; **P<0.01; ***P<0.001; ns: non-significant; data represent mean ± SD.
Figure 7. Developmental defects associated with Wnt7a expression in Xenopus and zebrafish
(A) Phenotypic scoring of stage 36 Xenopus laevis embryos injected or not in the ventral vegetal region of the 4-cell embryo with 15 pg of Wnt7a mRNA. The orange arrowhead highlights axis duplication. Data for Wnt7a injection are the same as in Fig. IF. (B) Gross morphology of 3 dpf WT zebrafish larvae injected at the one-cell stage with 20 pg mRNA of the indicated Wnt ligand. Wnt5 and Wntll are used as negative control "non-canonical" Wnt ligands. The percentage of embryos showing posteriorization of the anterior nervous system resulting in eye and forebrain loss (orange arrowhead) or not (unaffected, green arrowhead) are scored. (C) Phenotypic scoring of WT, gprl24-/- and maternal zygotic gprl24-/- (Mgprl24-/-) zebrafish larvae injected with 20pg of Wnt7a mRNA as in B.
Figure 8. Gene family-wide assessment of Gprl24/Reck impact on Wnt/Fz relationships
Heat map representation of average luciferase activities (n=3) 48 h after transfection of 17 of the 19 Wnt ligands in pairwise combination with each of the ten Fz receptors in WT (A), GPR124-/;RECK-/- (B) and Gprl24/Reck-overexpressing HEK293 STF cells (C).
Figure 9. Frizzled specificity for Wnt7a/b signaling in the presence or absence of Gprl24/Reck (A) Gprl24/Reck-dependent and (B) -independent STF luciferase activity in GPR124- /-;RECK-/-; FZ1-10-/- HEK293T cells after co-transfection of Wnt7a or Wnt7b with all 10 Fz genes or the empty pCS2 vector . *P<0.05; **P<0.01; ***P<0.001; data represent mean ± SD.
Figure 10. Chimeric Wnt7NTD/WntXCTD ligands mediate Gprl24/Reck- dependent signaling
Chimeric V5-flagged constructs consisting of Wnt7a NTD and a CTD derived from other Wnts (A) were expressed together with HA-Reck in GPR124-/-;RECK-/- HEK293 cells and their secretion (dotplot, B, top right), Reck-associated PLA signal (B, C, orange bars) and STF relative luciferase activities in the presence of Gprl24/Reck/Fzl (C, green bars) or Fz5 (C, grey bars) were assessed. Data for Wnt7a NTD are the same as in Fig. IB. *<0.05; **P<0.01; ***P<0.001; data represent mean ± SD.
Figure 11. Beta-catenin signaling assay in STF cells of Wnt7a variants mutated for residues involved in the site 2 Wnt7a/Fz interaction
Sequence alignment of Xenopus Wnt8 and mouse Wnt7a index (A, top) and structural model of Wnt7a based on the crystal coordinates of XWnt8a (20) (A, bottom). The orange Wnt7a residues predicted to be implicated in contacts with Fz residues (the predicted interacting amino acids of Fz5 or Fz8 are in black) within the site 2 interface were mutated to alanines. The relative STF luciferase activity of the corresponding variants was measured on Gprl24/Reck/Fzl (green) or Fz5 (grey)
(B). Data represent mean ± SD.
Figure 12. Beta-catenin signaling assay of single-residue Wnt7a variants in STF cells
(A) Frequency distribution of the relative STF luciferase activity of the 147 Wnt7a variants on Gprl24/Reck/Fzl (green) or Fz5 (grey) signaling. (B) Relative STF luciferase activity of Gprl24/Reck agonists on Gprl24/Reck/Fzl (green) or Fz5 (grey). Data represent mean ± SD.
Figure 13. Anti-V5 dot blot analysis of Wnt7a variants secretion
Ligand secretion was evaluated qualitatively by means of anti-V5 dot blot analysis of HEK293T cellular supernatants collected 48 h after transfection with WT Wnt7a or the indicated signaling inactive Wnt7a variants. Only A86R and E89A were not detected in the supernatant. Figure 14. Morphological defects induced by injection of low mRNA doses of Wnt7a or Wnt7aK190A in zebrafish embryos after ubiquitous expression of Gprl24/Reck
Phenotypic scoring of 3 dpf zebrafish larvae injected at the one-cell stage with Wnt7a or Wnt7aK190A mRNA at the indicated doses, in combination or not with 100 pg of Reck and Gprl24 mRNA.
Figure 15. Autocrine versus paracrine activity of the Gprl24/Reck agonists Relative Gprl24/Reck/Fzl-dependent STF luciferase activity of the Gprl24/Reck agonists in mono-culture (autocrine, green) and co-culture settings (paracrine, blue) as described in Fig. 2F. Wnt7a variants are either secreted by the reporter cells (mono-culture) or by a population of cocultured cells (1: 1). Data represent mean ± SD.
Figure 16. Functional and gene expression analysis of the zebrafish wnt7a/b paralogues
(A) Quantification of 60 hpf Tg(kdrl:GFP) hindbrain CtAs and 72 hpf Tg(neurogl:GFP) trunk DRG in the indicated zebrafish wnt7 morphants (4 ng each).
(B) Lateral and dorsal bright-field images of whole-mount in situ hybridization for wnt7aa, wnt7ab, wnt7ba and wnt7bb in 30 hpf WT embryos. (Right) transverse sections of the hindbrain and the trunk taken at levels indicated on the left. The otic vesicles are marked by an asterisk.
Figure 17. Evolutionary cross-reactivity of Wnt7a/b, Reck and Gprl24
Relative luciferase activity of HEK293 STF cells after co-transfection with different mouse, human, or zebrafish Wnt7 isoforms in combination with orthologues of murine or zebrafish Gprl24 and Reck. Data represent mean ± SD.
Figure 18. Cellular origins of Wnt7aK190A upon AAV-PHP.eB gene delivery
Two weeks after intravenous injection of 4.1011 vg of AAV-PHP.eB-CAG- Wnt7aK190A-P2AEGFP, EGFP (green) was co-immunostained with (A) endothelial cells (isolectin, magenta), astrocytes (GFAP, white) and neurons (NeuN, cyan) or (B) endothelial cells (CD31, magenta) and pericytes (Desmin, white). For pericyte colocalization assessment, three-color-intensity profiles along a line were made using the 'plot profile' option in ImageJ for each channel (EGFP, Desmin, CD31). The asterisks in the profiles below indicate presumptive colocalization between the EGFP and Desmin. Data represent mean ± SD. Figure 19. Brain-wide analysis of Wnt activation after AAV-PHP.eB gene delivery of Wnt7a or Wnt7aK190A in BAT-GAL mice
At 8 weeks of age, BAT-GAL mice were injected retro-orbitally with 4.1011 vg (viral genomes) of the indicated AAV viruses. After two weeks, coronal brain slices were stained for lacZ (magenta), laminin (cyan), and DNA (DAPI, blue). The percentage of LacZ-i- endothelial cells (ECs) and nonendothelial cells (non-ECs) in different brain regions is plotted on the right. AON, Anterior Olfactory Nucleus. Data represent mean ± SD.
Figure 20. Absence of ectopic Wnt activation after brain-wide Wnt7aK190A gene delivery
At 8 weeks of age, WT C57BL6 mice were injected retro-orbitally with 4.1011 vg (viral genomes) of the indicated AAV viruses. 32 days after viral injection, RNAScope in situ hybridization of Axin2 was performed in coronal sections of the hippocampal dendate gyrus and the parafascicular nucleus of the thalamus. The RNAScope images were analyzed using CellProfiler as described in the Methods section. ***P<0.001; data represent mean ± SD.
Figure 21. LEF1 immunostaining after brain-wide Wnt7a or Wnt7aK190A gene delivery
At 8 weeks of age, WT C57BL6 mice were injected retro-orbitally with 4.1011 vg (viral genomes) of the indicated AAV viruses. 14 days after viral injection, LEF1 immunostaining (magenta, grey) was performed to assess Wnt activation in endothelial cells (ECs, laminin, cyan) and nonendothelial cells (non-ECs). The percentage of LEF1+ cells in different brain regions is plotted on the right. AON, Anterior Olfactory Nucleus. Data represent mean ± SD.
Figure 22. Distribution of Wnt7aK190A-releasing EGFP+ cells in and around GL261 tumors after AAV-PHP.eB gene delivery
(A) Confocal tile scan of a coronal section through a GL261 glioblastoma tumor developing in a AAV-PHP.eB-CAG-Wnt7aK190A-P2A-EGFP injected mice 24 days post implantation. GL261 cells, readily labeled by DAPI, are negative for the EGFP viral transduction marker, which is particularly high at the tumor margin. (B) anti- EGFP (green, white) and anti-CD31 (endothelial cells, magenta, white) immunostaining within the core of the tumor. (C) Immunostaining of EGFP (viral transduction marker, green, white), S1OO0 (astrocytes, magenta, white), GFAP (astrocytes, magenta, white), NeuN (neurons, blue, white), Ibal (microglial cells, magenta, white) within the tumor glial scar. Figure 23. In vitro and in vivo characterization of the Tet-Off Dkkl GL261 cell line
(A) Growth curve of WT GL261 cells and Tet-Off Dkkl GL261 cells exposed or not to 1 pg.ml-1 doxycycline (B) Western blot analysis of conditional Dkkl repression addition of doxycycline to the growth medium for 48 h. Dkkl was probed both in the cellular supernatant (left) and the cells (right). ATG9 is used as a loading control for the cellular extracts. (C) Western blot analysis of Dkkl expression within tumors at 21 days post implantation in mice fed or not with a doxycycline-containing diet as described in the Methods section. Two independent tumors are shown for each condition. Tubulin is used as loading control.
Figure 24. In vivo characterization of the Tet-Off Dkkl GL261 tumors
(A) Co-immunostaining of coronal tumor sections for LEF1 (magenta) together with the endothelial nuclear marker ERG (cyan) (B) GLUT1 (magenta, grey) together with laminin (cyan) and (C) endothelial cells (CD31, cyan). Quantifications were done as described in Fig. 5 and data for WT GL261 (in transparency) are the same as in Fig. 5C, E, F.
Figure 25. Hemorrhage score of Tet-Off Dkkl tumors after AAV-EGFP or AAV-K190A gene delivery
At 25 days post implantation of 1.105 Tet-Off Dkkl GL261 cells, brains were harvested from mice injected or not with the indicated AAV virus. Intra- and peritumoral hemorrhages were categorized on vibratome sections as shown on the left, and scored in a double-blind manner.
Figure 26. Caveolin-1 immunostaining in Tet-Off Dkkl GL261 tumors
Co-immunostaining of endothelial cells (CD31, cyan) and Caveolin-1 (magenta, grey) in Tet-Off Dkkl GL261 tumors harvested from mice injected or not with the indicated AAV virus. *P<0.05; data represent mean ± SD.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns compositions and methods for treating glioblastoma in a subject. Definitions
Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.
As used herein, the following terms have the following meanings:
"A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.
"About" or "approximately" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-20% or less, preferably +/-10% or less, more preferably +/-5% or less, even more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.
"Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.
Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints. The expression "% by weight", "weight percent", "%wt" or "wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.
Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.
Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.
Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.
Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination. The term "protein" as used throughout this specification generally encompasses macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced proteins. The term also encompasses proteins that carry one or more co- or post-expression-type modifications of the polypeptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, palmitoylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of proenzymes or pre-hormones into active forms, etc. The term further also includes protein variants or mutants which carry one or more amino acid sequence variations vis-a-vis corresponding native proteins, such as, e.g., amino acid deletions, additions and/or substitutions. The term contemplates both full-length proteins and protein parts or fragments, e.g., naturally- occurring protein parts that ensue from processing of such full-length proteins.
The term "polypeptide" as used throughout this specification generally encompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms "protein" and "polypeptide" may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced polypeptides. The term also encompasses polypeptides that carry one or more co- or post-expression-type modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, palmitoylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes polypeptide variants or mutants which carry one or more amino acid sequence variations vis-a-vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions, point mutations and/or substitutions. The term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g., naturally- occurring polypeptide parts that ensue from processing of such full-length polypeptides.
The term "peptide" as used throughout this specification preferably refers to a polypeptide as used herein consisting essentially of 50 amino acids or less, e.g., 45 amino acids or less, preferably 40 amino acids or less, e.g., 35 amino acids or less, more preferably 30 amino acids or less, e.g., 25 or less, 20 or less, 15 or less, 10 or less or 5 or less amino acids. A peptide, polypeptide or protein can be naturally occurring, e.g., present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom. A peptide, polypeptide or protein can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesized. Without limitation, a peptide, polypeptide or protein can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free translation or cell-free transcription and translation, or non-biological peptide, polypeptide or protein synthesis.
The term "nucleic acid" as used throughout this specification typically refers to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units. A nucleoside unit commonly includes a heterocyclic base and a sugar group. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally- occurring nucleic acids, other naturally- occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatized bases. Exemplary modified nucleobases include without limitation 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine. In particular, 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability and may be preferred base substitutions in for example antisense agents, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and/or 2- deoxyribose common in naturally- occurring nucleic acids, or arabinose, 2- deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as without limitation 2'-0-alkylated, e.g., 2'-0- methylated or 2'- O-ethylated sugars such as ribose; 2'-0-alkyloxyalkylated, e.g., 2'-0- methoxyethylated sugars such as ribose; or 2'-0,4'-C-alkylene-linked, e.g., 2'-0,4'- C-methylene- linked or 2'-0,4'-C-ethylene-linked sugars such as ribose; 2'-fluoro- arabinose, etc.). Nucleic acid molecules comprising at least one ribonucleoside unit may be typically referred to as ribonucleic acids or RNA. Such ribonucleoside unit(s) comprise a 2'-OH moiety, wherein -H may be substituted as known in the art for ribonucleosides (e.g., by a methyl, ethyl, alkyl, or alkyloxyalkyl). Preferably, ribonucleic acids or RNA may be composed primarily of ribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be ribonucleoside units. Nucleic acid molecules comprising at least one deoxyribonucleoside unit may be typically referred to as deoxyribonucleic acids or DNA. Such deoxyribonucleoside unit(s) comprise 2'-H. Preferably, deoxyribonucleic acids or DNA may be composed primarily of deoxyribonucleoside units, for example, > 80%, > 85%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99% or even 100% (by number) of nucleoside units constituting the nucleic acid molecule may be deoxyribonucleoside units. Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter alia phosphodiester linkages common in naturally- occurring nucleic acids, and further modified phosphate- or phosphonate-based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3'-N-carbamate, morpholino, borano, thioether, 3'-thioacetal, and sulfone intemucleoside linkages. Preferably, inter-nucleoside linkages may be phosphate-based linkages including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof. The term"nucleic acid" also encompasses any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo- DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)). "Alkyl" as used herein particularly encompasses lower hydrocarbon moieties, e.g., C1-C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1 -propenyl, 2-propenyl, and isopropyl. Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof. A modified nucleoside may include a modified heterocyclic base, a modified sugar moiety, a modified inter-nucleoside linkage or a combination thereof.
The term "nucleic acid" further preferably encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesised) DNA, RNA or DNA/RNA hybrids. RNA is inclusive of RNAi (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), mi RNA (micro-RNA), tRNA (transfer RNA, whether charged or discharged with a corresponding acylated amino acid), and cRNA (complementary RNA). A nucleic acid can be naturally occurring, e.g., present in or isolated from nature, e.g., produced natively or endogenously by a cell or a tissue and optionally isolated therefrom. A nucleic acid can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesized. Without limitation, a nucleic acid can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free transcription, or non-biological nucleic acid synthesis. A nucleic acid can be double-stranded, partly double stranded, or single- stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.
The reference to any peptides, polypeptides, proteins or nucleic acids may particularly encompass such peptides, polypeptides, proteins or nucleic acids with a native sequence, i.e., ones of which the primary sequence is the same as that of the peptides, polypeptides, proteins or nucleic acids found in or derived from nature. A skilled person understands that native sequences may differ between different species due to genetic divergence between such species. Moreover, native sequences may differ between or within different individuals of the same species due to normal genetic diversity (variation) within a given species. Also, native sequences may differ between or even within different individuals of the same species due to somatic mutations, or post-transcriptional or post-translational modifications. Any such variants or isoforms of peptides, polypeptides, proteins or nucleic acids are intended herein. Accordingly, all sequences of peptides, polypeptides, proteins or nucleic acids found in or derived from nature are considered "native".
The peptides, polypeptides, proteins or nucleic acids may be human, i.e., their primary sequence may be the same as a corresponding primary sequence of or present in naturally occurring human peptides, polypeptides, proteins or nucleic acids. In certain embodiments the qualifier "human" relates to the primary sequence of the respective peptides, polypeptides, proteins or nucleic acids, rather than to their origin or source. For example, such peptides, polypeptides, proteins or nucleic acids may be present in or isolated from samples of human subjects or may be obtained by other means (e.g., by recombinant expression, cell-free transcription or translation, or non-biological nucleic acid or peptide synthesis).
The peptides, polypeptides, proteins or nucleic acids may be wild-type. While most native peptides, polypeptides, proteins or nucleic acids may be considered wild-type, those carrying naturally-occurring mutations leading to partial or complete loss of function, which may contribute to or be causative of a disease phenotype, are generally excluded from the scope of the term "wild-type". The reference to any peptides, polypeptides, proteins or nucleic acids may also encompass variants or fragments of such peptides, polypeptides, proteins or nucleic acids, particularly of naturally-occurring, native or wild-type forms thereof.
The term "fragment" as used throughout this specification with reference to a peptide, polypeptide, or protein generally denotes a portion of the peptide, polypeptide, or protein, such as typically an N- and/or C-terminally truncated form of the peptide, polypeptide, or protein. Preferably, a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the amino acid sequence length of said peptide, polypeptide, or protein. For example, insofar not exceeding the length of the full-length peptide, polypeptide, or protein, a fragment may include a sequence of > 5 consecutive amino acids, or > 10 consecutive amino acids, or > 20 consecutive amino acids, or > 30 consecutive amino acids, e.g., > 40 consecutive amino acids, such as for example > 50 consecutive amino acids, e.g., > 60, > 70, > 80, > 90, > 100, > 200, >300, >400, > 500, > 600, > 700, > 800, > 900 or > 1000 consecutive amino acids of the corresponding full-length peptide, polypeptide, or protein.
The term "fragment" with reference to a nucleic acid (polynucleotide) generally denotes a 5'- and/or 3'-truncated form of a nucleic acid. Preferably, a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the nucleic acid sequence length of said nucleic acid. For example, insofar not exceeding the length of the full-length nucleic acid, a fragment may include a sequence of > 5 consecutive nucleotides, or > 10 consecutive nucleotides, or > 20 consecutive nucleotides, or > 30 consecutive nucleotides, e.g., > 40 consecutive nucleotides, such as for example > 50 consecutive nucleotides, e.g., > 60, > 70, > 80, > 90, > 100, > 200, > 300, > 400, > 500, > 600, > 700, > 800, > 900, > 1000, > 1500, > 2000, > 2500, > 3000, > 3500 or > 4000 consecutive nucleotides of the corresponding full-length nucleic acid.
The terms encompass fragments arising by any mechanism, in vivo and/or in vitro, such as, without limitation, by alternative transcription or translation, exo- and/or endo-proteolysis, exo- and/or endo-nucleolysis, or degradation of the peptide, polypeptide, protein or nucleic acid, such as, for example, by physical, chemical and/or enzymatic proteolysis or nucleolysis.
The term "variant" or "mutant" of a protein, polypeptide, peptide or nucleic acid generally refers to proteins, polypeptides or peptides the amino acid sequence of which, or nucleic acids the nucleotide sequence of which, is substantially identical (i.e., largely but not wholly identical) to the sequence of the protein, polypeptide, peptide, or nucleic acid, e.g., at least about 80% identical or at least about 85% identical, e.g., preferably at least about 90% identical, e.g., at least 91% identical, 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical to the sequence of the recited protein, polypeptide, peptide, or nucleic acid. Preferably, a variant may display such degrees of identity to a recited protein, polypeptide, peptide or nucleic acid when the whole sequence of the recited protein, polypeptide, peptide or nucleic acid is queried in the sequence alignment (i.e., overall sequence identity). Sequence identity may be determined using suitable algorithms for performing sequence alignments and determination of sequence identity as know per se. Exemplary but non-limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), for example using the published default settings or other suitable settings (such as, e.g., for the BLASTN algorithm: cost to open a gap = 5, cost to extend a gap = 2, penalty for a mismatch = -2, reward for a match = 1, gap x_dropoff = 50, expectation value = 10.0, word size = 28; or for the BLASTP algorithm: matrix = Blosum62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci., 89: 10915-10919), cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3).
An example procedure to determine the percent identity between a particular amino acid sequence and the amino acid sequence of a query polypeptide will entail aligning the two amino acid sequences using the Blast 2 sequences (BI2seq) algorithm, available as a web application or as a standalone executable programme (BLAST version 2.2.31+) at the NCBI web site (www.ncbi.nlm.nih.gov), using suitable algorithm parameters. An example of suitable algorithm parameters include: matrix = Blosum62, cost to open a gap = 11, cost to extend a gap = 1, expectation value = 10.0, word size = 3). If the two compared sequences share homology, then the output will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the output will not present aligned sequences. Once aligned, the number of matches will be determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity is determined by dividing the number of matches by the length of the query polypeptide, followed by multiplying the resulting value by 100. The percent identity value may, but need not, be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 may be rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 may be rounded up to 78.2. It is further noted that the detailed view for each segment of alignment as outputted by BI2seq already conveniently includes the percentage of identities.
A variant of a protein, polypeptide, peptide or nucleic acid may be a homologue (e.g., orthologue or paralogue) of said protein, polypeptide, peptide or nucleic acid. As used herein, the term "homology" generally denotes structural similarity between two macromolecules from same or different taxons, wherein said similarity is due to shared ancestry.
A variant of a protein, polypeptide, or peptide may comprise one or more amino acid additions, deletions, point mutations or substitutions relative to (i.e., compared with) the corresponding protein or polypeptide. For example, a variant (substitution variant) of a protein, polypeptide, or peptide may comprise up to 70 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, 50, 60, or 70) conservative amino acid substitutions relative to (i.e., compared with) the corresponding protein or polypeptide; and/or a variant (substitution variant) of a protein, polypeptide, or peptide may comprise up to 20 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, or 19) non-conservative amino acid substitutions relative to (i.e., compared with) the corresponding protein or polypeptide.
A conservative amino acid substitution is a substitution of one amino acid for another with similar characteristics. Conservative amino acid substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (i.e., basic) amino acids include arginine, lysine and histidine. The negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.
Alternatively, or in addition, for example, a variant (deletion variant) of a protein, polypeptide, or peptide may lack up to 20 amino acid segments (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 segments) relative to (i.e., compared with) the corresponding protein or polypeptide. The deletion segment(s) may each independently consist of one amino acid, two contiguous amino acids or three contiguous amino acids. The deletion segments may be non-contiguous, or two or more or all of the deletion segments may be contiguous.
A variant of a nucleic acid may comprise one or more nucleotide additions, deletions, or substitutions relative to (i.e., compared with) the corresponding nucleic acid.
Reference to "fragment or variant" or "variant or fragment" of any peptide, polypeptide, protein or nucleic acid, also encompasses fragments of variants of such peptide, polypeptide, protein or nucleic acid, and variants of fragments of such peptide, polypeptide, protein or nucleic acid.
Particularly envisaged are biologically active fragments and/or variants of the recited peptides, polypeptides or proteins. The term "biologically active" is interchangeable with terms such as "functionally active" or "functional", denoting that the fragment and/or variant at least partly retains the biological activity or intended functionality of the respective or corresponding peptide, polypeptide or protein. Reference to the "activity" of a peptide, polypeptide or protein may generally encompass any one or more aspects of the biological activity of the peptide, polypeptide or protein, such as without limitation any one or more aspects of its biochemical activity, enzymatic activity, signaling activity, interaction activity, ligand activity, and/or structural activity, e.g., within a cell, tissue, organ or an organism.
Preferably, a functionally active fragment or variant may retain at least about 20%, e.g., at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, e.g., at least 60%, more preferably at least about 70%, e.g., at least 80%, yet more preferably at least about 85%, still more preferably at least about 90%, and most preferably at least about 95% or even about 100% of the intended biological activity or functionality compared with the corresponding peptide, polypeptide or protein. In certain embodiments, a functionally active fragment or variant may even display higher biological activity or functionality compared with the corresponding peptide, polypeptide or protein, for example may display at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% of the intended biological activity or functionality compared with the corresponding peptide, polypeptide or protein. By means of an example, where the activity of a given peptide, polypeptide or protein can be readily measured in an assay with a quantitative output, for example an enzymatic assay or a signaling assay or a binding assay producing a quantifiable signal, a functionally active fragment or variant of the peptide, polypeptide or protein may produce a signal which is at least about 20%, or at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, or at least 60%, more preferably at least about 70%, or at least 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% of the signal produced by the corresponding peptide, polypeptide or protein.
The terms "complex", "protein complex" or "polypeptide complex" are well- understood in the art. By means of further guidance and without limitation, the terms broadly denote a cluster comprising two or more proteins or polypeptides. The cluster may be stabilized by non-covalent bonds, more particularly non-covalent protein-protein interactions, wherein all or only part of the polypeptides present within the cluster physically interact. A protein complex can be comprised entirely of peptides, polypeptides or proteins, or it may include other molecules or macromolecules such as carbohydrates, lipids, glycolipids, nucleic acids, oligonucleotides, nucleoproteins, nucleosides, nucleoside phosphates, enzyme cofactors, porphyrins, metal ions and the like. The terms encompass without limitation protein complexes which may be obligate or non-obligate, transient or permanent, and/or homomultimeric or heteromultimeric. The terms encompass without limitation protein complexes which may be located at the cell membrane (plasma membrane), extracellularly, within cytoplasm, within cellular organelles, or at membranes of cellular organelles. Complexes located at the cell membrane typically contain at least one membrane-anchored or transmembrane protein. The terms also encompass membrane microdomains and membrane-associated macromolecular organelle-like structures known as "signalosome" which compartmentalize a given signaling pathway. Such higher-order protein complexes can be at least partly stabilized by intracellular scaffolds. The terms may thus also denote situations in which certain proteins or polypeptides are locally concentrated or accumulated at a given site of a cell membrane, such as to allow signal transduction through the cell membrane at said site mediated by said proteins or polypeptides.
The term "Wnt signaling" as used herein specifically refers to the mechanism by which a biologically active Wnt ligand exerts its effect upon a cell to modulate said cell's activity and/or actions. Biologically active Wnt ligands modulate cell activity and/or action by binding to Wnt receptor(s), such as the FZD/LRP receptor complex. Once activated by binding of the Wnt ligand, the Wnt receptor(s) will activate one or more intracellular signaling pathways. Three Wnt signaling pathways are classically recognized: the canonical (i.e., mediated by p-catenin activation as a transcriptional co-activator) Wnt pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt/calcium pathway. All three pathways are typically activated by binding of a Wnt ligand to a FZD receptor. However, the recruitment of the LRP receptor appears to be a prerequisite for inducing canonical (or p-catenin- dependent) Wnt signaling. As known in the art, in the absence of a Wnt/FZD/LRP- complex ("inactive canonical Wnt signaling"), p-catenin is phosphorylated in the cytoplasm by Casein Kinase and glycogen synthase kinase-3 (GSK-3). The interaction between these kinases and p-catenin is facilitated by the scaffolding proteins, Axin and adenomatous polyposis coli (APC). Together, these proteins form a 'degradation complex', which allows phosphorylated p-catenin to be recognized by beta-transducin repeat-containing protein (P-TrCP), targeted for ubiquitination, and degraded by the proteasome. Active canonical (or beta-catenin-dependent) Wnt signaling involves binding of Wnt ligands to a receptor complex of FZD and LRP on the cell surface. During signaling, FZD cooperates with LRP in such a way that binding of the Wnt protein leads to dimerization of the two receptors. It is theorized that this dimerization leads to a conformational change of the FZD and LRP receptors. As a consequence, the cytoplasmic tail of LRP recruits and binds to the scaffold protein Axin in a phosphorylation-dependent manner and leads to formation of a complex involving DVL, Axin, and GSK3. Multimers of receptor-bound DVL and Axin molecules might support the formation of the LRP-FZD dimer. As a result of the recruitment of GSK to the cell membrane, p-catenin phosphorylation is inhibited, releasing p- catenin from the degradation complex and allowing p-catenin to accumulate in the cytoplasm. The accumulation of p-catenin in the cytoplasm allows to p-catenin to enter the nucleus and to interact with TCF/LEF transcription factors.
In addition, other mechanisms are available by which Wnt signaling can be initiated (e.g. via Norrin, antibodies, and more). "Wnt7" in the present context refers to both "Wnt7a" and/or "Wnt7b", which are part of the Wnt-family of proteins.
The terms "bind", "interact", "specifically bind" or "specifically interact" as used throughout this specification mean that an agent binds to or influences one or more desired molecules or analytes substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related. The terms do not necessarily require that an agent binds exclusively to its intended target(s). For example, an agent may be said to specifically bind to target(s) of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold or more greater, such as, e.g., at least about 1000-fold or more greater, at least about lxl04-fold or more greater, or at least about lxl05-fold or more greater, than its affinity for a nontarget molecule.
By "encoding" is meant that a nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides, or to another nucleic acid sequence in a template-transcription product (e.g. RIMA or RNA analogue) relationship.
The term "nucleic acid expression cassettes" as used herein refers to nucleic acid molecules, typically DNA, to which nucleic acid fragments, preferably the recombinant nucleic acid molecule as defined herein, may be inserted to be expressed, wherein said nucleic acid molecules comprise one or more nucleic acid sequences controlling the expression of the nucleic acid fragments. Non-limiting examples of such more nucleic acid sequences controlling the expression of the nucleic acid fragments include promoter sequences, open reading frames and transcription terminators.
An "open reading frame" or "ORF" refers to a succession of coding nucleotide triplets (codons) starting with a translation initiation codon and closing with a translation termination codon known per se, and not containing any internal in-frame translation termination codon, and potentially capable of encoding a protein, polypeptide or peptide. Hence, the term may be synonymous with "coding sequence" as used in the art.
An "operable linkage" is a linkage in which regulatory sequences and sequences sought to be expressed are connected in such a way as to permit said expression. For example, sequences, such as, e.g., a promoter and an ORF, may be said to be operably linked if the nature of the linkage between said sequences does not: (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter to direct the transcription of the ORF, (3) interfere with the ability of the ORF to be transcribed from the promoter sequence. Hence, "operably linked" may mean incorporated into a genetic construct so that expression control sequences, such as a promoter, effectively control transcription I expression of a sequence of interest.
Reference to a "promoter" is to be taken in its broadest context and includes transcriptional regulatory sequences required for accurate transcription initiation and where applicable accurate spatial and/or temporal control of gene expression or its response to, e.g., internal or external (e.g., exogenous) stimuli. More particularly, "promoter" may depict a region on a nucleic acid molecule, preferably DNA molecule, to which an RNA polymerase binds and initiates transcription. A promoter is preferably, but not necessarily, positioned upstream, i.e., 5', of the sequence the transcription of which it controls. Typically, in prokaryotes a promoter region may contain both the promoter per se and sequences which, when transcribed into RNA, will signal the initiation of protein synthesis (e.g., Shine-Dalgarno sequence). A promoter sequence can also include "enhancer regions", which are one or more regions of DNA that can be bound with proteins (namely the trans-acting factors) to enhance transcription levels of genes in a gene-cluster. The enhancer, while typically at the 5' end of a coding region, can also be separate from a promoter sequence, e.g., can be within an intronic region of a gene or 3' to the coding region of the gene.
Promoters contemplated herein may be constitutive or inducible. A constitutive promoter is understood to be a promoter whose expression is constant under the standard culturing conditions. Inducible promoters are promoters that are responsive to one or more induction cues. For example, an inducible promoter can be chemically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a chemical inducing agent such as an alcohol, tetracycline, a steroid, a metal, or other small molecule) or physically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a physical inducer such as light or high or low temperatures). An inducible promoter can also be indirectly regulated by one or more transcription factors that are themselves directly regulated by chemical or physical cues. Non-limiting examples of promoters include T7, U6, Hl, retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.
The terms "terminator" or "transcription terminator" refer generally to a sequence element at the end of a transcriptional unit which signals termination of transcription. For example, a terminator is usually positioned downstream of, i.e., 3' of ORF(s) encoding a polypeptide of interest. For instance, where a recombinant nucleic acid contains two or more ORFs, e.g., successively ordered and forming together a multi- cistronic transcription unit, a transcription terminator may be advantageously positioned 3' to the most downstream ORF.
The terms "expression vector" or "vector" as used herein refers to nucleic acid molecules, typically DNA, to which nucleic acid fragments, preferably the recombinant nucleic acid molecule as defined herein, may be inserted and cloned, i.e., propagated. Hence, a vector will typically contain one or more unique restriction sites, and may be capable of autonomous replication in a defined cell or vehicle organism such that the cloned sequence is reproducible. A vector may also preferably contain a selection marker, such as, e.g., an antibiotic resistance gene, to allow selection of recipient cells that contain the vector. Vectors may include, without limitation, plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PAC, BAC, linear nucleic acids, e.g., linear DNA, transposons, viral vectors, etc., as appropriate (see, e.g., Sambrook et al., 1989; Ausubel 1992). Viral vectors may include inter alia retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno- associated viral vectors, for example, vectors based on HIV, SV40, EBV, HSV or BPV. Expression vectors are generally configured to allow for and/or effect the expression of nucleic acids or open reading frames introduced thereto in a desired expression system, e.g., in vitro, in a cell, organ and/or organism. For example, expression vectors may advantageously comprise suitable regulatory sequences.
The term "isolated" with reference to a particular component (such as for instance a nucleic acid, protein, polypeptide or peptide) generally denotes that such component exists in separation from - for example, has been separated from or prepared and/or maintained in separation from - one or more other components of its natural environment. For instance, an isolated human or animal protein or complex may exist in separation from a human or animal body where it naturally occurs. The term "isolated" as used herein may preferably also encompass the qualifier "purified". As used herein, the term "purified" with reference to peptides, polypeptides, proteins, or nucleic acids does not require absolute purity. Instead, it denotes that such peptides, polypeptides, proteins, or nucleic acids are in a discrete environment in which their abundance (conveniently expressed in terms of mass or weight or concentration) relative to other analytes is greater than in the starting composition or sample. A discrete environment denotes a single medium, such as for example a single solution, gel, precipitate, lyophilisate, etc. Purified nucleic acids, proteins, polypeptides or peptides may be obtained by known methods including, for example, laboratory or recombinant synthesis, chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification, etc. Purified peptides, polypeptides or proteins may preferably constitute by weight s 10%, more preferably > 50%, such as > 60%, yet more preferably > 70%, such as > 80%, and still more preferably > 90%, such as > 95%, > 96%, > 97%, > 98%, > 99% or even 100%, of the protein content of the discrete environment. Protein content may be determined, e.g., by the Lowry method (Lowry et al. 1951. J Biol Chem 193: 265), optionally as described by Hartree 1972 (Anal Biochem 48: 422-427). Purity of peptides, polypeptides, or proteins may be determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Quantity of nucleic acids may be determined by measuring absorbance A260. Purity of nucleic acids may be determined by measuring absorbance A260/A280, or by agarose- or polyacrylamidegel electrophoresis and ethidium bromide or similar staining.
The term "gene therapy" as used herein refers to the introduction of an exogenous polynucleotide into a host cell for therapeutic or prophylactic purposes, irrespective of the method used for the introduction. Such methods include a variety of well- known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as described elsewhere herein. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art. The terms "treat" or "treatment" encompass the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction, as well as reducing the progression of a neurovascular disorder or a CNS disorder comprising neurovascular dysfunction. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers in a patient, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. "Treatment" can also mean prolonging survival or improving well-being in a patient as compared to expected survival or well-being if not receiving treatment. The term "treatment" also refers to prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. It must be clear that the terms "treat," "treating" or "treatment", as used herein, include alleviating, abating or ameliorating disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition.
The phrases "central nervous system (CNS) disorder comprising neurovascular dysfunction" or "CNS disease comprising neurovascular dysfunction" as used herein refer to a group of neurological disorders that affect the structure and/or function of the brain and/or spinal cord, which collectively form the CNS, that are caused by, contributed to, or typified by neurovascular dysfunction, or which lead to an abnormality of the structure and/or function of the blood vessels within or supplying the brain and/or spine. Non-limiting examples of central nervous system (CNS) disorders comprising neurovascular dysfunction are multiple sclerosis, ischemic stroke, brain cancer, glioblastoma, epilepsy, dementia, vascular dementia, HIV-1- associated dementia, Alzheimer's disease, Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, infectious brain diseases, traumatic brain injuries, migraine, chronic traumatic encephalopathy and retinal vascular disorders, including but not limited to Norrie disease, familial exudative vitreoretinopathy, osteoporosis- pseudoglioma syndrome, diabetic retinopathy and macular degeneration. The terms "neurovascular disorder" or "neurovascular disease" as used herein refer to a disease or pathological alteration or condition which affects the cerebral vascular system and/or the vascular system supplying the spinal cord. The terms encompass any abnormality of the blood vessels within or supplying the brain and/or spine. Abnormalities may be without limitation (i) narrowing of arteries which reduces blood flow to the brain that may lead to hypoxia, ischemia or stroke and/or (ii) weakening of arteries that may lead to brain aneurysms and increase the risk of intracranial bleeding. Non-limiting examples of neurovascular diseases are ischemic stroke, hemorrhagic stroke, ischemia/reperfusion injury, brain aneurysms, arteriovenous malformations (AVMs), cavernous malformations, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformations, carotid artery stenosis, Moyamoya disease, intracranial atherosclerosis and retinal vascular disorders, including but not limited to Norrie disease, familial exudative vitreoretinopathy, osteoporosis-pseudoglioma syndrome, diabetic retinopathy and macular degeneration.
"Blood-brain barrier", "blood brain barrier" or "BBB" refers to highly selective semipermeable properties of endothelial cells that prevents solutes in the circulating blood from non-selectively crossing into the extracellular fluid of the central nervous system where neurons reside. The blood-brain barrier is formed by endothelial cells of the capillary wall, astrocyte end-feet ensheathing the capillary, and pericytes embedded in the capillary basement membrane. This system allows the passage of some small molecules by passive diffusion, as well as the selective and active transport of various nutrients, ions, organic anions, and macromolecules that are crucial to neural function. The blood-brain barrier, by the selectivity of tight junctions between the endothelial cells of brain capillaries, restricts the passage of pathogens, the diffusion of solutes in the blood, and large or hydrophilic molecules into the cerebrospinal fluid, while allowing the diffusion of hydrophobic molecules (O2, CO2, hormones) and small non-polar molecules. Cells of the barrier actively transport metabolic products such as glucose across the barrier using specific transport proteins. The barrier also restricts the passage of peripheral immune factors, like signalling molecules, antibodies, and immune cells, into the CNS, thus insulating the brain from damage due to peripheral immune events. The BBB is composed of endothelial cells restricting passage of substances from the blood more selectively than endothelial cells of capillaries elsewhere in the body.
"Glioblastoma", "glioblastoma multiforme", "GBM" or "grade IV astrocytoma" is the most aggressive type of cancer that begins within the brain. Initially, signs and symptoms of glioblastoma are nonspecific. They may include headaches, personality changes, nausea, and symptoms similar to those of a stroke. Symptoms often worsen rapidly and may progress to unconsciousness. Glioblastomas represent 15% of all brain tumors. They can either start from normal brain cells or develop from an existing low-grade astrocytoma. The diagnosis typically is made by a combination of a CT scan, MRI scan, and tissue biopsy. There is no known method of preventing the cancer. Treatment usually involves surgery, after which chemotherapy and radiation therapy are used. The medication temozolomide is frequently used as part of chemotherapy. High-dose steroids may be used to help reduce swelling and decrease symptoms. Surgical removal (decompression) of the tumor is linked to increased survival, but only by some months. Despite maximum treatment, the tumor always recurs. Such recurrent tumor is referred to as "recurrent glioblastoma". The typical duration of survival following diagnosis is 12-15 months, with fewer than 3-7% of people surviving longer than five years. Without treatment, survival is typically three months.
Except when noted, the terms "subject" or "patient" can be used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals and primates. Preferred subjects are human subjects. The terms "subject" or "patient" include subjects in need of treatment, more particularly subjects that would benefit from treatment of a given condition, particularly a neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction. Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and/or those in who said condition is to be prevented.
The term "therapeutically active/effective amount" as used herein, refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a surgeon, researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. The term "prophylactically active/effective amount" refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician. In present context, the "therapeutically active amount" is used to refer to both the "therapeutically active amount" and "prophylactically active amount" as described above, unless the distinction is clear from the context. Methods are known in the art for determining therapeutically and/or prophylactically effective doses of a compound, a nucleic acid encoding the compound, a nucleic acid expression cassette, or a pharmaceutical composition, as taught herein. The term "therapeutically effective dose" as used herein refers to an amount of a compound, a nucleic acid encoding the compound, a nucleic acid expression cassette, or a pharmaceutical composition, as taught herein, that when administered brings about a positive therapeutic response with respect to treatment of a patient having a specific disease or disorder.
Description
Previously the inventors characterized the first ever "Wnt decoding module" capable of discriminating Wnt ligands that are otherwise largely synonymous in their capacity to bind Frizzled, thereby helping cells interpret the complexity of Wnt signaling inputs in order to orchestrate tissue development and homeostasis. In the herein characterized Wnt decoding module, selectivity is conferred by RECK, which mediates Wnt7-specific binding in a Frizzled-independent manner. G-protein coupled receptor GPR124, a RECK binding partner, acts as a signaling-deficient transmembrane protein which is able to recruit intracellular Dishevelled (Dvl). Dvl scaffolds bridge GPR124 and Frizzled, thereby assembling Wnt7 ligand-specific RECK/GPR124/Frizzled/lipoprotein receptor-related protein (LRP) signalosomes.
The inventors further demonstrated the design of novel agonists capable of activating Wnt signaling selectively in cells expressing RECK and GPR124, such agonists being useful as therapeutics, such as particularly for the treatment of neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction. Hence, the invention allows to provide inter alia novel agonists capable of stimulating Wnt/0-catenin signaling in cerebral endothelial cells substantially without cross-reactivity with other Frizzled pathways, and useful as therapeutics, particularly for neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction.
The inventors now surprisingly discovered and provide evidence for the use of such agonists in the reduction in progression and/or treatment of glioblastoma multiforme (GBM), also referred to as glioblastoma, in a subject, without activating unwanted 'off-target' (see further) Wnt-related pathways. In an embodiment, said glioblastoma is recurrent glioblastoma. In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
Accordingly, in a first aspect, the invention provides a pharmaceutical composition comprising a therapeutically active amount of a compound chosen from Wnt7 polypeptide or a fragment thereof capable of activating G-protein coupled receptor (GPR)124/RECK/Frizzled/lipoprotein receptor-related protein (LRP)-mediated Wnt signaling, wherein said Wnt7 polypeptide or fragment thereof does not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof for use in the reduction in progression and/or treatment of glioblastoma in a subject. In an embodiment, said glioblastoma is recurrent glioblastoma.
It was shown that such pharmaceutical composition is particularly effective in preventing, reducing the progression of and in the treatment of glioblastoma, such as recurrent glioblastoma.
In an embodiment, said compound is able to influence the permeability of the bloodbrain barrier (BBB) in subjects having glioblastoma or subjects prone to develop recurrent glioblastoma.
This is particularly relevant as it was shown that such subjects were characterized by a dense vascular network exhibiting disrupted BBB properties, leading to an -at least partly- dysfunctional BBB. The pharmaceutical composition for use in preventing or reducing the progression of glioblastoma of the present invention provides for a novel way for maintaining a functional BBB in subjects prone to develop glioblastoma or having glioblastoma, while the composition for use in the treatment of glioblastoma of the present invention provides for a novel way in -at least partly- restoring the BBB function in subjects having (recurrent) glioblastoma.
In an embodiment, the compound is able to repair tight-junctional defects in glioblastoma vessels. By repairing said tight-junctional defects, the BBB impermeability is increased, thereby restoring the BBB functionality. In an embodiment, the compound is able to reduce vesicular transport in glioblastoma vessels. By reducing the transport, the BBB impermeability in increased, thereby restoring the BBB functionality.
In an embodiment, the compound is able to restore the paracellular permeability and/or the transcellular permeability of the BBB in glioblastoma vessels, in particular to reduce the paracellular permeability and/or the transcellular permeability of the BBB, thereby increasing the BBB impermeability.
In an embodiment, said compound is further able to control tumor angiogenesis in a subject suffering from glioblastoma.
In an embodiment and as will be clear from the data in the present specification, the compound is able to repair tight-junctional defects in general, thereby restoring impaired BBB function in general.
In the present specification, references to any peptides, polypeptides, proteins or nucleic acids denote the respective peptides, polypeptides, proteins or nucleic acids as commonly known under the respective designations in the art. More particularly, the references to "WNT" and in particular to "WNT7", to "G-protein coupled receptor 124" (GPR124), "Reversion-inducing cysteine-rich protein with Kazal motifs" (RECK), "Frizzled" (FZD), or "lipoprotein receptor-related protein" (LRP) denote the respective peptides, polypeptides, proteins or nucleic acids, as apparent from the context, as commonly known under said designations in the art.
The terms encompass the peptides, polypeptides, proteins or nucleic acids when forming a part of a living organism, organ, tissue or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources. The terms also encompass the peptides, polypeptides, proteins or nucleic acids when produced by recombinant or synthetic means.
Unless otherwise apparent from the context, reference herein to any peptide, polypeptide, protein or nucleic acid also encompasses modified forms of said peptide, polypeptide, protein or nucleic acid, such as forms bearing post-expression modifications including, for example, phosphorylation, glycosylation, palmitoylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, ubiquitination, oxidation of methionine to methionine sulphoxide or methionine sulphone, signal peptide removal, N-terminal Met removal, conversion of proenzymes or pre-hormones into active forms, and the like. A broader definition is given above.
The pharmaceutical composition for use of the present invention comprises in particular a therapeutically active amount of a compound chosen from Wnt7 polypeptide or a fragment thereof, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof. In an embodiment, said Wnt7 is Wnt7a or Wnt7b.
In certain embodiments, the Wnt polypeptide or fragment thereof, or nucleic acid encoding for said Wnt7 polypeptide or fragment thereof as employed herein is of animal origin, preferably warm-blooded animal origin, more preferably vertebrate origin, yet more preferably mammalian origin, including human origin and nonhuman mammalian origin, still more preferably human origin.
By means of an example, human WNT7A gene is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) Gene ID 7476. Human WNT7A mRNA (transcript variant 1) is annotated under NCBI Genbank accession number NM_004625.3. Nucleotides 306 (start codon) to 1355 (stop codon) of NM_004625.3 constitute the WNT7A coding sequence. Human WNT7A protein sequence is annotated under NCBI Genbank accession number NP_004616.2, and Uniprot accession number 000755.2, and is further reproduced below (SEQ ID NO: 9):
> NP_004616.2 protein Wnt-7a precursor [Homo sapiens]
MNRKARRCLGHLFLSLGMVYLRIGGFSSWALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGL D ECQFQFRN G RW N CSALG E RTV FG KE LKVG SREAAFTYAIIAAG VAH AITAACTQG N LS DCGCD KEKQ GQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGV SGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSP NYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSE RTEMYTCK.
By means of an example, the mature form of human Wnt7a protein (not comprising the signal peptide) comprises an amino sequence as further reproduced below (SEQ ID NO: 2):
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK. By means of an example, human WNT7B gene is annotated under NCBI Genbank Gene ID 7477. Human WNT7B mRNA (transcript variant 1) is annotated under NCBI Genbank accession number NM_058238.2. Nucleotides 375 (start codon) to 1424 (stop codon) of NM_058238.2 constitute the WNT7B coding sequence. Human WNT7B protein sequence is annotated under NCBI Genbank accession number NP_478679.1, and Uniprot accession number P56706.2, and is further reproduced below (SEQ ID NO: 10):
> NP_478679.1 protein Wnt-7b precursor [Homo sapiens]
MHRNFRKWIFYVFLCFGVLYVKLGALSSWALGANIICNKIPGLAPRQRAICQSRPDAIIVIGEGAQMGIN ECQYQFRFGRWNCSALGEKTVFGQELRVGSREAAFTYAITAAGVAHAVTAACSQGNLSNCGCDREKQG YYNQAEGWKWGGCSADVRYGIDFSRRFVDAREIKKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVS GSCTTKTCWTTLPKFREVGHLLKEKYNAAVQVEWRASRLRQPTFLRIKQLRSYQKPMETDLVYIEKSPN YCEEDAATGSVGTQGRLCNRTSPGADGCDTMCCGRGYNTHQYTKVWQCNCKFHWCCFVKCNTCSER TEVFTCK
In particular embodiments, the compound as disclosed herein is or consists essentially of a fragment of a Wnt7 polypeptide, such as a fragment of a Wnt7a or Wnt7b polypeptide, preferably a fragment of a human Wnt7 polypeptide, such as a fragment of a human Wnt7a or human Wnt7b polypeptide.
In particular embodiments, the fragment of the Wnt7 polypeptide has at least 30%, and preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, preferably at least 100% or more, of the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length Wnt7 polypeptide.
A GPR124/RECK/FZD/LRP receptor complex broadly denotes a protein complex, particularly a membrane-associated protein complex, more particularly a plasma membrane-associated protein complex comprising at least one GPR124 polypeptide, at least one RECK polypeptide, at least one FZD polypeptide and at least one LRP polypeptide. A GPR124/RECK/FZD/LRP receptor complex, when located at the plasma membrane of a cell, is capable of activating Wnt/0-catenin signaling in said cell in response to extracellularly provided Wnt7 ligand.
A FZD/LRP receptor complex broadly denotes a protein complex, particularly a membrane-associated protein complex, more particularly a plasma membrane- associated protein complex comprising at least one FZD polypeptide and at least one LRP polypeptide. A FZD/LRP receptor complex, when located at the plasma membrane of a cell, is capable of activating Wnt/0-catenin signaling in said cell in response to extracellularly provided Wnt ligand, such as, but not limited to Wnt7 ligand.
"LRP" or "lipoprotein receptor-related protein" encompasses any and all lipoprotein receptor-related proteins, also known in the art as low-density lipoprotein receptor- related proteins or prolow-density lipoprotein receptor-related proteins. In certain particularly preferred embodiments, the terms denote LR.P5, LR.P6, or LR.P5 and LR.P6 (LRP5/6).
Hence, also disclosed is a composition comprising a therapeutically active amount of a compound chosen from Wnt7 polypeptide or a fragment thereof capable of activating GPR124/RECK/Frizzled/LRP5/6-mediated Wnt signaling, wherein said Wnt7 polypeptide or fragment thereof does not activate Frizzled/LRP5/6-mediated Wnt signaling in the absence of RECK and/or GPR124, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof for use in the reduction in progression and/or treatment of glioblastoma in a subject.
A skilled person can appreciate that any sequences represented in sequence databases or in the present specification may be of precursors of the respective peptides, polypeptides, proteins or nucleic acids and may include parts which are processed away from mature molecules.
The reference to any peptides, polypeptides, proteins or nucleic acids encompasses such peptides, polypeptides, proteins or nucleic acids of any organism where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and non-human mammals, still more preferably of humans.
Hence, in certain embodiments, one or more and preferably all of WNT7, GPR124, RECK, FZD and LRP as employed herein is or are of animal origin, preferably warmblooded animal origin, more preferably vertebrate origin, yet more preferably mammalian origin, including human origin and non-human mammalian origin, still more preferably human origin.
In an embodiment, the pharmaceutical composition for use of the present invention acts by Wnt7-specific RECK/GPR124/Frizzled/LRP-mediated signaling, in which Wnt7 (Wnt7a or Wnt7b) binds specifically to Reck in a FZD-independent manner and GPR124, a RECK binding partner, bridges RECK-bound Wnt7 to the FZD/LRP complex via intracellular DVL scaffolds, thereby assembling Wnt7-ligand specific RECK/GPR124/FZD/LRP signalosomes and activating canonical Wnt signaling.
The skilled person shall further appreciate that a GPR124/RECK/FZD/LRP receptor complex as envisaged herein may include further component(s), which may or need not functionally modulate the complex. For example, the complex may include Dishevelled (Dvl), forming intracellular scaffolds capable of bridging GPR124 and Frizzled.
In particular embodiments, cells expressing GPR124, RECK, FZD and LRP polypeptides at its plasma membrane is a cell naturally expressing all GPR124, RECK, FZD and LRP polypeptides at the cell surface, such as a cerebral endothelial cell.
In particular embodiments, the capability of the compound chosen from Wnt7 polypeptide or a fragment thereof or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof to activate GPR124/RECK/Frizzled/LRP-mediated Wnt signaling, but not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124, denotes the capability of the compound to activate Wnt signaling in cells positive for GPR124, RECK, FZ and LRP, but not in cells positive for FZ and LRP and negative for GPR124 and/or RECK, wherein the cells positive GPR124, RECK, FZ and LRP and the cells positive for FZ and LRP and negative for GPR124 and/or RECK are otherwise substantially identical.
The capability of activating Wnt signaling refers to the ability of the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein to mimic, reproduce or approximate the signal transduction effect and/or activity of a natural Wnt ligand binding to FZD and LRP, such as to an FZD/LRP complex.
Activation of Wnt signaling may be suitably determined and/or quantitated by measuring the expression of one or more Wnt target genes, TCF reporter gene expression, beta-catenin stabilization, LRP phosphorylation, and/or translocation of Axin from cytoplasm to cell membrane as known in the art. For instance, activation of Wnt signaling may be suitably determined and/or quantitated by measuring the expression of TCF gene (e.g., by RT-PCR or any other transcript detection method), a primary output of Wnt signaling (Nature, 1997, vol. 385(6619), 829-33). For example, a TCF reporter assay (also known as TOP/FOP or TOPflash) may be used to assess changes in the transcription of TCF/LEF controlled genes. The TCF reporter assay may be a luciferase reporter assay. Further for example, activation of Wnt signaling may be suitably determined and/or quantitated by measuring the expression of c-myc, n-myc, LEF1, or c-jun.
Alternatively, activation of Wnt signaling may be determined by measuring the location, level and/or phosphorylation status of p-catenin. A non-limiting example of such an assay is the "P-Catenin Redistribution Assay” (Thermo Scientific) which provides recombinant U20S cells stably expressing human p-catenin fused to the C- terminus of enhanced green fluorescent protein (EGFP). The assay allows visualization and monitoring of the translocation of a GFP- p-catenin fusion protein from the membrane to the nucleus. Another way of determining activation of Wnt signaling is the visualization of Axin translocation, for example with a GFP-Axin fusion protein.
In particular embodiments, the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein may be considered capable of activating (canonical) Wnt signaling if the compound enhances Wnt/p-catenin signaling at least 10-fold more, at least 20-fold more, at least 30-fold more, at least 40-fold more, at least 50-fold more, at least 100-fold more, at least 250-fold more, at least 500-fold more, at least 750-fold more, at least 1000-fold more, at least lxl04-fold more, or at least lxl05-fold more compared to Wnt/ p-catenin signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.
In particular embodiments, the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein may be considered to not activate (canonical) Wnt signaling if the compound enhances Wnt/p-catenin signaling less than 10-fold more, such as particularly at most 5-fold more or at most 2.5-fold more, or if the compound does not enhance or even reduces (e.g., 2-fold less or 5-fold less or 10- fold less) Wnt/p-catenin signaling compared to Wnt/ p-catenin signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.
In particular embodiments, the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein may be considered to activate the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling, but not activate Frizzled/LRP- mediated Wnt in the absence of RECK and/or GPR124, if the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity induced by said compound is at least 3.5-fold more, at least 5-fold more, at least 10-fold more, at least 15-fold more, at least 20-fold more, at least 25-fold more, at least 50-fold more, at least 100-fold more, at least 500-fold more, at least 1000-fold more, at least lxl04-fold more, or at least lxl05-fold more, preferably at least 50-fold more, than the Frizzled/LRP-mediated Wnt signaling activity induced by said compound, in absence of RECK and/or GPR124. Before comparing the GPR124/RECK/Frizzled/LRP- mediated Wnt signaling activity (denoted "activity 1" in this paragraph) and the Frizzled/LRP-mediated Wnt signaling activity in the absence of RECK and/or GPR124 (denoted "activity 2" in this paragraph), activity 1 and activity 2 induced by the compound may be normalized to activity 1 and activity 2 induced by wild-type Wnt7a, respectively, the latter for example set to represent 100% activity.
By means of an example, in an in vitro or in vivo cell assay system comprising 1) cells expressing GPR124, RECK, FZ and LRP and separately 2) cells expressing FZ and LRP but not expressing GPR124 and/or RECK, wherein the cells under 1) and 2) are otherwise substantially identical, the compound chosen from Wnt7 polypeptide or a fragment thereof may be considered to activate GPR124/RECK/Frizzled/LRP- mediated Wnt signaling, but not activate Frizzled/LRP -mediated Wnt signaling in the absence of RECK and/or GPR124, when the Wnt signaling activity induced by the same quantity of said compound under substantially identical conditions is at least 3.5-fold more, 5-fold more, at least 10-fold more, at least 15-fold more, at least 20- fold more, at least 25-fold more, at least 50-fold more, at least 100-fold more, at least 500-fold more, at least 1000-fold more, at least lxl04-fold more, or at least lxl05-fold more in cells under 1) than in cells under 2). For example, the cells under 1) and 2) may be from the same primary cell source, or may be of the same cell line, and may be genetically engineered to differ in expression of GPR124 and/or RECK.
Activation of the Wnt signaling pathway may occur by promoting the close association or mutual proximity of the Frizzled and LRP polypeptides at the cell membrane, thereby forming membrane-associated hetero-oligomers comprising the Frizzled and LRP polypeptides. Upon ligand-driven formation of the Frizzled-LRP hetero-oligomer, the intracellular portion of the LRP polypeptide becomes accessible for phosphorylation, for example by CK1 and GSK-3, which greatly increases its affinity for Axin. Second, when present in a hetero-oligomer with the LRP polypeptide, the intracellular portion of the Frizzled polypeptide is able to induce the phosphorylation and recruitment of DVL. The resulting assembly of an activated LRP- FZD-DVL-Axin complex leads indirectly to the dissociation of the destruction complex of beta-catenin, thereby allowing beta-catenin to accumulate in the cytoplasm and translocate to the cell nucleus where beta-catenin may induce gene transcription.
Accordingly, in particular embodiments, the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein is capable of concurrently binding to Frizzled and LRP polypeptides at a cell membrane, in the presence of RECK and GPR124, but not in the absence of RECK and/or GPR124.
In particular embodiments, the compound as disclosed herein is capable of binding to one or more different Frizzled polypeptides, such as one or more Frizzled polypeptides selected from the group consisting of Fzd 1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and FzdlO. Preferably, the compound as disclosed herein may be capable of specifically binding to at least Fzd4, and optionally to one or more other Fzd; or may be capable of specifically binding to Fzd4 substantially to the exclusion of other Fzd. Fzd4 is believed to be the dominant Fzd family member in endothelial cells of the central nervous system. More preferably, the compound as disclosed herein is capable of specifically binding to human Fzd4. The compound as disclosed herein may be selective for the one or more preferred Frizzled polypeptides, for example having a specificity for the one or more preferred Frizzled polypeptides of at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 1000-fold, at least lxl04-fold, or at least lxl05-fold, compared to other non-preferred Frizzled polypeptides.
In particular embodiments, the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein is capable of binding to one or more different LRP polypeptides involved in Wnt signaling. Preferably, the compound as disclosed herein is capable of binding to LRP5 and/or LRP6, e.g., any one or each of LRP5 and LRP6. More preferably, the compound as disclosed herein is capable of binding to human LRP5 and/or LRP6, e.g., any one or each of human LRP5 and human LRP6. The compound as disclosed herein may be selective for the one or more preferred LRP polypeptides, for example having a specificity for the one or more preferred LRP polypeptides of at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 1000-fold, at least lxl04-fold, or at least lxl05-fold, compared to other non-preferred LRP polypeptides.
In particular embodiments, the compound as disclosed herein is capable of binding to the GPR124 and/or the RECK polypeptide. In particular embodiments, the compound as disclosed herein is capable of concurrently binding to Frizzled and LRP polypeptides, and in addition to the GPR124 and/or the RECK polypeptide.
In particular embodiments, the compound as disclosed herein is capable of binding to the RECK polypeptide.
In particular embodiments, the compound as disclosed herein is capable of concurrently binding to Frizzled, LRP and RECK polypeptides.
RECK is composed of five N-terminal cysteine-knot (CK) motifs or regions (i.e. CK1, CK2, CK3, CK4 and CK5), a cysteine-rich domain (CRD) and three Kazal motifs preceding a Glycosylphosphatidylinositol (GPI)-anchor site. The CK motifs, the CRD and the Kazal motifs are located extracellularly. Accordingly, the compound capable of binding to the RECK polypeptide as disclosed herein may bind the CK1 motif, CK2 motif, CK3 motif, CK4 motif, CK5 motif, CRD, and/or one or more of the Kazal motifs of the RECK polypeptide.
It was found that especially the binding of the compound capable of binding to the RECK polypeptide as disclosed herein to the CK4 and/or CK5 regions of the RECK polypeptide appears important for establishing activation of Wnt signaling mediated by the GPR124/RECK/Frizzled/ LRP receptor complex.
Accordingly, in particular embodiments, the compound as disclosed herein is capable of binding to the CK4 and/or CK5 regions of RECK polypeptide.
The CK4 motif spans from amino acid C216 to C263, and the CK5 motif spans from amino acid C292 to C338 of the amino acid sequence of the human RECK protein annotated under NCBI Genbank accession number NP_066934.1 as disclosed elsewhere herein. Accordingly, the CK4 motif of human RECK comprises, consists essentially of or consists of the amino acid sequence CCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQC (SEQ ID NO: 3) and the CK5 motif of human RECK comprises, consists essentially of or consists of the amino acid sequence
CCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVSMLTC (SEQ ID NO: 4).
In particular embodiments, the compound as disclosed herein is capable of binding to the amino acid sequence of SEQ ID NO: 3 and/or the amino acid sequence of SEQ ID NO: 4. The Frizzled receptor is a G protein-coupled receptor protein and ranges in length from about 500 to about 700 amino acids. The N-terminus is predicted to be extracellular and comprises a cysteine rich domain (CRD) of approximately 120 amino acids followed by a hydrophilic linker region of approximately 40-100 amino acids. The Frizzled receptor also comprises seven hydrophobic domains that are predicted to form transmembrane alpha-helices. The intracellular C-terminal domain has a variable length and, though the intracellular domain is overall not well conserved among different family members, it comprises a proximal KTXXXW amino acid motif (SEQ ID NO: 5), wherein X can be any amino acid, which is highly conserved in Frizzled polypeptides and which is required for canonical Wnt signaling. The Frizzled CRD domain comprises a motif of 10 invariantly spaced cysteines and is largely conserved between the known Frizzled family members, but also in several other proteins, such as RECK, secreted frizzled related proteins (SFRPs), receptor tyrosine kinases (RTKs), and collagen ol XVIII. The CRD domain is important for ligand (e.g. Wnt) binding to the Frizzled polypeptide, for example by recognition and/or binding of cis-unsaturated fatty acyl groups present in the ligand contact site 1 or the residues located at the Wnt ligand "index" contact site 2.
In particular embodiments, the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein is capable of binding to the cysteine-rich domain (CRD) of the Frizzled polypeptide. In certain embodiments, the compound as disclosed herein is capable of binding to the cis-unsaturated fatty acyl group- binding domain located within the CRD domain of the Frizzled polypeptide or the "index" contact site within the CRD domain.
The N-terminal extracellular domain of LRP is composed of four YWTD (SEQ ID NO: 6) repeat domains (or beta-propeller domains), four epidermal growth factor (EGF)- like domains and an LDLR-like domain (LDLRLD). A single YWTD repeat domain comprises six tandem YWTD sequences. The first two, most N-terminal YWPD repeat domains are predicted to bind to Wnt ligands, and the second two sets of YWPD repeat domains are predicted to bind to Dickkopf (DKK). The N-terminal extracellular domain is followed by a single membrane-spanning segment and a cytoplasmic tail harboring between one and three NPXY motifs (SEQ ID NO: 7), wherein X can be any amino acid (e.g. in LRP1, LRP2, LRP4, APOER2, LDLR, LRP9), or between one and five PPPSP motifs (SEQ ID NO: 8) (e.g. in LRP5 and LRP6). The term "Dickkopf" or "DKK" encompasses any and all members of the DKK family, such as without limitation the known human DKK proteins including DKK1 (RefSeq Protein: NP_036374.1; GenelD: 22943), DKK2 (RefSeq Protein: NP_055236.1; GenelD: 27123), DKK3 (RefSeq Protein: NP_001317149.1; GenelD: 27122), and DKK4 (RefSeq Protein: NP_055235.1; GenelD: 27121). In certain embodiments, the terms may particularly denote DKK1.
In particular embodiments, the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein is capable of binding to the extracellular domain of the LRP polypeptide.
In particular embodiments, the compound as disclosed herein is capable of binding to the DKK-binding site of the LRP polypeptide. In more particular embodiments, the compound as disclosed herein is capable of binding to the DKKl-binding site of the LRP5 and/or LRP6 polypeptide.
In particular embodiments, the compound as disclosed herein is capable of binding to the Wnt-binding site of the LRP polypeptide. In more particular embodiments, the compound as disclosed herein is capable of binding to the Wnt-binding site of the LRP5 and/or LRP6 polypeptide.
In further particular embodiments, the compound as disclosed herein is capable of binding to the DKK-binding site and the Wnt-binding site of the LRP polypeptide.
In particular embodiments, the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein is capable of binding to 0-propeller-EGF-like domains 1 and 2 (P1E1P2E2) and/or p-propeller-EGF-like domains 3 and 4 (P3E3P4E4) of the LRP polypeptide.
In particular embodiments, the compound chosen from Wnt7 polypeptide or a fragment thereof or nucleic acid encoding such polypeptide or fragment as disclosed herein is or consists essentially of a fragment of a Wnt7 polypeptide, such as a fragment of a Wnt7a or Wnt7b polypeptide, preferably a fragment of a human Wnt7 polypeptide, such as a fragment of a human Wnt7a or human Wnt7b polypeptide.
In particular embodiments, the fragment of the Wnt7 polypeptide has at least 30%, and preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, preferably at least 100% or more, of the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length Wnt7 polypeptide.
In particular embodiments, the fragment of the Wnt7 polypeptide has a GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity which is higher (e.g., 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold or 2-fold higher or even higher) than the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length Wnt7 polypeptide.
It was demonstrated that the N-terminal domain of Wnt7a is capable of binding to RECK and activating Wnt signaling mediated by the GPR124/RECK/Frizzled/LRP receptor complex, while not activating Wnt signaling mediated by the Frizzled/LRP receptor complex in the absence of RECK and/or GPR124.
Hence, in particular embodiments, the fragment of the Wnt7 polypeptide has Frizzled/LRP-mediated Wnt signaling activity in the absence of RECK and/or GPR124 which is at least 10-fold less, or at least 100-fold less, or at least 1000-fold less, or at least 1 x 104-fold less, or at least 1 x 105-fold less, or at least 1 x 106-fold less than the Frizzled/LRP-mediated Wnt signaling activity of the full-length Wnt7 polypeptide in the absence of RECK and/or GPR124.
In particular embodiments, said fragment of the Wnt7 polypeptide is or consists essentially of the N-terminal domain (NTD) of the Wnt7 polypeptide, such as the NTD of a Wnt7a or Wnt7b polypeptide, preferably the NTD of human Wnt7 polypeptide, such as human Wnt7a or Wnt7b. The N-terminal domain of the human Wnt7a or Wnt7b polypeptide typically ranges from the Leucine (L) residue at the position corresponding to position 1 in SEQ ID NO: 1 (Wnt7a) or SEQ ID NO: 11 (Wnt7b) to the cysteine (C) residue at the position corresponding to position 247 in SEQ ID NO: 1 (Wnt7a) or SEQ ID NO: 11 (Wnt7b).
In particular embodiments, said fragment of the Wnt7 polypeptide comprises at least the NTD of the Wnt7 polypeptide and does not comprise the C-terminal domain (CTD) of the Wnt7 polypeptide.
In particular embodiments, the fragment of Wnt7 polypeptide or nucleic acid encoding such fragment as disclosed herein comprises, consists essentially of, or consists of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, contiguous amino acids of the NTD of human Wnt7a polypeptide, more particularly, the compound as disclosed herein comprises, consists essentially of or consists of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, contiguous amino acids of the amino acid sequence: LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFG KELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSA DIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWT TLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 1) or LGANIICNKIPGLAPRQRAICQSRPDAIIVIGEGAQMGINECQYQFRFGRWNCSALGEKTVFG QELRVGSREAAFTYAITAAGVAHAVTAACSQGNLSNCGCDREKQGYYNQAEGWKWGGCSA DVRYGIDFSRRFVDAREIKKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCW TTLPKFREVGHLLKEKYNAAVQVEVVRASRLRQPTFLRIKQLRSYQKPMETDLVYIEKSPNYC (SEQ ID NO: 11), preferably SEQ ID NO: 1.
In particular embodiments, the compound chosen from Wnt7 polypeptide or a fragment thereof or nucleic acid encoding such polypeptide or fragment as disclosed herein comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 11, preferably SEQ ID NO: 1, or an amino acid sequence having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, sequence identity to the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 11, preferably SEQ ID NO: 1.
The skilled person will understand that if it is envisaged to express and secrete the compound as taught herein by a host cell, the nucleic acid encoding the compound as taught herein preferably encodes a precursor form of the compound including an N-terminal signal peptide sequence. Accordingly, the nucleic acid may encode a fragment of the precursor polypeptide of Wnt7 (i.e. including the signal peptide), such as the precursor polypeptide of human Wnt7a or Wnt7b. In particular embodiments, the nucleic acid encodes an compound comprising, consisting essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 11 or an amino acid sequence having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 11, wherein said amino acid sequence is preceded N-terminally by a signal peptide having an amino acid sequence MNRKARRCLGHLFLSLGMVYLRIGGFSSVVA (SEQ ID NO: 12) or MHRNFRKWIFYVFLCFGVLYVKLGALSSVVA (SEQ ID NO: 13), respectively. Alternatively, the nucleic acid encoding the compound as taught herein may be comprised within a vector providing for a signal peptide. The signal peptide may be a homologous or heterologous signal peptide, depending on the host cell used for production of the compound as taught herein. Furthermore, for prokaryotic expression of the compound as taught herein, a protease cleavage site motif may be present C-terminally of said signal peptide and N-terminally of the compound as taught herein.
Present inventors have demonstrated that certain variants of the human or murine Wnt7a polypeptide are as effective or more effective in activating Wnt signaling mediated by the GPR124/RECK/Frizzled/LRP receptor complex, while not activating Wnt signaling mediated by the Frizzled/LRP receptor complex in the absence of RECK and/or GPR124, compared to the wild-type human or murine Wnt7a polypeptide, respectively.
Accordingly, in particular embodiments of the pharmaceutical composition for use, the compound chosen from Wnt7 polypeptide or a fragment thereof or nucleic acid encoding such polypeptide or fragment as disclosed herein is a variant of a Wnt7 polypeptide, such as a variant of a Wnt7a or Wnt7b polypeptide. In further particular embodiments, the compound as disclosed herein is a variant of the NTD of a Wnt7a or Wnt7b polypeptide.
As indicated above, the N-terminal domain of human Wnt7a comprises an amino acid sequence referred to as SEQ ID NO: 1, while the mature form of human Wnt7a protein (not comprising the signal peptide) comprises an amino sequence referred to as SEQ ID NO: 2.
In other of further particular embodiments of the pharmaceutical composition for use, the compound chosen from Wnt7 polypeptide or a fragment thereof or nucleic acid encoding such polypeptide or fragment as disclosed herein is a mutated Wnt7 polypeptide, such as a mutated of a Wnt7a or Wnt7b polypeptide. In further particular embodiments, the compound as disclosed herein is a mutated NTD of a Wnt7a or Wnt7b polypeptide, the NTD of Wnt7a or Wnt7b polypeptide which is a fragment of the Wnt7a or Wnt7b polypeptide. In certain embodiments, in such variant or mutant, the glutamine (Q) residue at the position corresponding to position 17 in SEQ ID NO: 1 or SEQ ID NO: 2; the isoleucine (I) residue at the position corresponding to position 20 in SEQ ID NO: 1 or SEQ ID NO: 2; the proline (P) residue at the position corresponding to position 25 in SEQ ID NO: 1 or SEQ ID NO: 2; the alanine (A) residue at the position corresponding to position 27 in SEQ ID NO: 1 or SEQ ID NO: 2; the isoleucine (I) residue at position corresponding to position 28 in SEQ ID NO: 1 or SEQ ID NO: 2; the glutamate (E) residue at the position corresponding to position 33 in SEQ ID NO: 1 or SEQ ID NO: 2; the methionine (M) residue at the position corresponding to position 37 in SEQ ID NO: 1 or SEQ ID NO: 2; the leucine (L) residue at the position corresponding to position 39 in SEQ ID NO: 1 or SEQ ID NO: 2; the glutamate (E) residue at the position corresponding to position 41 in SEQ ID NO: 1 or SEQ ID NO: 2; the phenylalanine (F) residue at the position corresponding to position 44 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 50 in SEQ ID NO: 1 or SEQ ID NO: 2; the asparagine (N) residue at the position corresponding to position 52 in SEQ ID NO: 1 or SEQ ID NO: 2; the valine (V) residue at the position corresponding to position 68 in SEQ ID NO: 1 or SEQ ID NO: 2; the isoleucine (I) residue at the position corresponding to position 129 in SEQ ID NO: 1 or SEQ ID NO: 2; the phenylalanine (F) residue at the position corresponding to position 131 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 133 in SEQ ID NO: 1 or SEQ ID NO: 2; the phenylalanine (F) residue at the position corresponding to position 135 in SEQ ID NO: 1 or SEQ ID NO: 2; the isoleucine (I) residue at the position corresponding to position 141 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 146 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 158 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 181 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 191 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 198 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 200 in SEQ ID NO: 1 or SEQ ID NO: 2; the valine (V) residue at the position corresponding to position 205 in SEQ ID NO: 1 or SEQ ID NO: 2; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 1 or SEQ ID NO: 2; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 214 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 216 in SEQ ID NO: 1 or SEQ ID NO: 2; the proline (P) residue at the position corresponding to position 218 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 222 in SEQ ID NO: 1 or SEQ ID NO: 2; the isoleucine (I) residue at the position corresponding to position 223 in SEQ ID NO: 1 or SEQ ID NO: 2; the tyrosine (Y) residue at the position corresponding to position 229 in SEQ ID NO: 1 or SEQ ID NO: 2; the proline (P) residue at the position corresponding to position 232 in SEQ ID NO: 1 or SEQ ID NO: 2; the threonine (T) residue at the position corresponding to position 235 in SEQ ID NO: 1 or SEQ ID NO: 2; the glutamate (E) residue at the position corresponding to position 248 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 289 in SEQ ID NO: 1 or SEQ ID NO: 2; the tryptophan (W) residue at the position corresponding to position 291 in SEQ ID NO: 1 or SEQ ID NO: 2; the threonine (T) residue at the position corresponding to position 307 in SEQ ID NO: 1 or SEQ ID NO: 2; and/or the lysine (K) residue at the position corresponding to position 318 in SEQ ID NO: 1 or SEQ ID NO: 2, is substituted by one or more (preferably not more than three, preferably not more than two, more preferably one) other amino acid residue, such as preferably but without limitation by an alanine (A) residue, an arginine (R) residue or a glutamine (Q) residue, more preferably by an alanine (A) residue. In certain embodiments, in such variant, the glutamine (Q) residue at the position corresponding to position 17 in SEQ ID NO: 1 or SEQ ID NO: 2; the isoleucine (I) residue at the position corresponding to position 20 in SEQ ID NO: 1 or SEQ ID NO: 2; the proline (P) residue at the position corresponding to position 25 in SEQ ID NO: 1 or SEQ ID NO: 2; the isoleucine (I) residue at position corresponding to position 28 in SEQ ID NO: 1 or SEQ ID NO: 2; the glutamate (E) residue at the position corresponding to position 33 in SEQ ID NO: 1 or SEQ ID NO: 2; the methionine (M) residue at the position corresponding to position 37 in SEQ ID NO: 1 or SEQ ID NO: 2; the leucine (L) residue at the position corresponding to position 39 in SEQ ID NO: 1 or SEQ ID NO: 2; the glutamate (E) residue at the position corresponding to position 41 in SEQ ID NO: 1 or SEQ ID NO: 2; the phenylalanine (F) residue at the position corresponding to position 44 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 50 in SEQ ID NO: 1 or SEQ ID NO: 2; the valine (V) residue at the position corresponding to position 68 in SEQ ID NO: 1 or SEQ ID NO: 2; the isoleucine (I) residue at the position corresponding to position 129 in SEQ ID NO: 1 or SEQ ID NO: 2; the phenylalanine (F) residue at the position corresponding to position 131 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 133 in SEQ ID NO: 1 or SEQ ID NO: 2; the phenylalanine (F) residue at the position corresponding to position 135 in SEQ ID NO: 1 or SEQ ID NO: 2; the isoleucine (I) residue at the position corresponding to position 141 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 146 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 158 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 181 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 191 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 198 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 200 in SEQ ID NO: 1 or SEQ ID NO: 2; the valine (V) residue at the position corresponding to position 205 in SEQ ID NO: 1 or SEQ ID NO: 2; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 1 or SEQ ID NO: 2; the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 214 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 216 in SEQ ID NO: 1 or SEQ ID NO: 2; the proline (P) residue at the position corresponding to position 218 in SEQ ID NO: 1 or SEQ ID NO: 2; the lysine (K) residue at the position corresponding to position 222 in SEQ ID NO: 1 or SEQ ID NO: 2; the isoleucine (I) residue at the position corresponding to position 223 in SEQ ID NO: 1 or SEQ ID NO: 2; the tyrosine (Y) residue at the position corresponding to position 229 in SEQ ID NO: 1 or SEQ ID NO: 2; the proline (P) residue at the position corresponding to position 232 in SEQ ID NO: 1 or SEQ ID NO: 2; the threonine (T) residue at the position corresponding to position 235 in SEQ ID NO: 1 or SEQ ID NO: 2; the glutamate (E) residue at the position corresponding to position 248 in SEQ ID NO: 1 or SEQ ID NO: 2; the arginine (R) residue at the position corresponding to position 289 in SEQ ID NO: 1 or SEQ ID NO: 2; the tryptophan (W) residue at the position corresponding to position 291 in SEQ ID NO: 1 or SEQ ID NO: 2; the threonine (T) residue at the position corresponding to position 307 in SEQ ID NO: 1 or SEQ ID NO: 2; and/or the lysine (K) residue at the position corresponding to position 318 in SEQ ID NO: 1 or SEQ ID NO: 2, is substituted by an alanine (A) residue; and/or the alanine (A) residue at the position corresponding to position 27 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an arginine (R) residue; and/or the asparagine (N) residue at the position corresponding to position 52 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by a glutamine (Q) residue; and/or the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an alanine (A), a serine (S) or a leucine (L) residue. In certain embodiments, the variant or mutant of the Wnt7 polypeptide or a fragment thereof comprises two or more (e.g., preferably two, preferably three, more preferably four) of the amino acid substitutions listed above.
In more particular embodiments, the NTD of the Wnt7a or Wnt7b polypeptide comprises two or more (e.g., preferably two, preferably three, more preferably four) of the amino acid substitutions listed above.
Hence, in particular embodiments, the compound chosen from Wnt7 polypeptide or a fragment thereof as disclosed herein comprises, consists essentially of or consists of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, wherein the residue at position 17 is not glutamine, the residue at position 20 is not isoleucine, the residue at position 25 is not proline, the residue at position 27 is not alanine, the residue at position 28 is not isoleucine, the residue at position 33 is not glutamate, the residue at position 37 is not methionine, the residue at position 39 is not leucine, the residue at position 41 is not glutamate, the residue at position 44 is not phenylalanine, the residue at position 50 is not arginine, the residue at position 52 is not asparagine, the residue at position 68 is not valine, the residue at position 129 is not isoleucine, the residue at position 131 is not phenylalanine, the residue at position 133 is not lysine, the residue at position 135 is not phenylalanine, the residue at position 141 is not isoleucine, the residue at position 146 is not arginine, the residue at position 158 is not arginine, the residue at position 159 is not lysine, the residue at position 181 is not lysine, the residue at position 191 is not arginine, the residue at position 198 is not lysine, the residue at position 200 is not lysine, the residue at position 205 is not valine, the residue at position 208 is not glutamate, the residue at position 214 is not arginine, the residue at position 216 is not lysine, the residue at position 218 is not proline, the residue at position 222 is not lysine, the residue at position 223 is not isoleucine, the residue at position 229 is not tyrosine, the residue at position 232 is not proline, the residue at position 235 is not threonine, the residue at position 248 is not glutamate, the residue at position 289 is not arginine, the residue at position 291 is not tryptophan, the residue at position 307 is not threonine, and/or the residue at position 318 is not lysine; preferably wherein the residue at position 27 is arginine, the residue at position 28 is alanine, the residue at position 33 is alanine, the residue at position 41 is alanine, the residue at position 44 is alanine, the residue at position 50 is alanine, the residue at position 52 is glutamine, the residue at position 68 is alanine, the residue at position 129 is alanine, the residue at position 131 is alanine, the residue at position 133 is alanine, the residue at position 135 is alanine, the residue at position 141 is alanine, the residue at position 146 is alanine, the residue at position 158 is alanine, the residue at position 159 is alanine, the residue at position 181 is alanine, the residue at position 191 is alanine, the residue at position 198 is alanine, the residue at position 200 is alanine, the residue at position 205 is alanine, the residue at position 208 is alanine, the residue at position 214 is alanine, the residue at position 216 is alanine, the residue at position 218 is alanine, the residue at position 222 is alanine, the residue at position 223 is alanine, the residue at position 229 is alanine, the residue at position 232 is alanine, the residue at position 235 is alanine, the residue at position 248 is alanine, the residue at position 289 is alanine, the residue at position 291 is alanine, the residue at position 307 is alanine, and/or the residue at position 318 is alanine.
In preferred embodiments, the compound is a fragment of Wnt7 polypeptide as disclosed herein and is a variant of the NTD of human Wnt7a comprising, consisting essentially of or consisting of an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the residue at position 17 is not glutamine, the residue at position 20 is not isoleucine, the residue at position 25 is not proline, the residue at position 27 is not alanine, the residue at position 28 is not isoleucine, the residue at position 33 is not glutamate, the residue at position 37 is not methionine, the residue at position 39 is not leucine, the residue at position 41 is not glutamate, the residue at position 44 is not phenylalanine, the residue at position 50 is not arginine, the residue at position 52 is not asparagine, the residue at position 68 is not valine, the residue at position 129 is not isoleucine, the residue at position 131 is not phenylalanine, the residue at position 133 is not lysine, the residue at position 135 is not phenylalanine, the residue at position 141 is not isoleucine, the residue at position 146 is not arginine, the residue at position 158 is not arginine, the residue at position 159 is not lysine, the residue at position 181 is not lysine, the residue at position 191 is not arginine, the residue at position 198 is not lysine, the residue at position 200 is not lysine, the residue at position 205 is not valine, the residue at position 208 is not glutamate, the residue at position 214 is not arginine, the residue at position 216 is not lysine, the residue at position 218 is not proline, the residue at position 222 is not lysine, the residue at position 223 is not isoleucine, the residue at position 229 is not tyrosine, the residue at position 232 is not proline, and/or the residue at position 235 is not threonine. In particular embodiments, the compound as disclosed herein comprises, consists essentially of or consists of an amino acid sequence as set forth in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95, preferably SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 33, 34, 35, 38, 39,
43, 44, 45, 46, 47, 48, 50, 54, 55, 57, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 78, 79, 80, 81, 82, 83, 84, 85, 86, 88, 92, 93 or 95, more preferably SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 33, 34, 35, 38, 39, 43, 44, 45, 46, 47, 48, 50, 54, 55, or 57.
Sequences below are mature Wnt7a polypeptide variants or NTD fragments thereof, wherein one amino acid residue has been substituted by another residue (in bold). hWnt7aNTD K159A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLM N LHN N EAGRAILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 14) hWnt7aK159A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLM N LHN N EAGRAILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 59) hWnt7aNTD A27R:
LGASIICNKIPGLAPRQRAICQSRPDRIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 15) hWnt7aA27R:
LGASIICNKIPGLAPRQRAICQSRPDRIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 60) hWnt7aNTD E33A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGAGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLMN LHN N EAGRKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 16) hWnt7aE33A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGAGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 61) hWnt7aNTD E41A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDACQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 17) hWnt7aE41A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDACQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 62) hWnt7aNTD E44A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQAQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 18) hWnt7aE44A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQAQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 63) hWnt7aNTD N52Q:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWQCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 19) hWnt7aN52Q:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWQCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 64) hWnt7aNTD I129A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGAGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 20) hWnt7aI129A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGAGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 65) hWnt7aNTD F131A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGAAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 21) hWnt7aF131A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGAAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 66) hWnt7aNTD K133A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAAVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 22) hWnt7aK133A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAAVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 67) hWnt7aNTD I141A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REAKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 23) hWnt7aI141A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REAKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 68) hWnt7aNTD R158A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAGAKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 24) hWnt7aR158A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAGAKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 69) hWnt7aNTD K181A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLMNLHNN EAGRKILEENMKLECKCHGVSGSCTTATCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 25) hWnt7aK181A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLMNLHNN EAGRKILEENMKLECKCHGVSGSCTTATCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 70) hWnt7aNTD R191A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFAELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 26) hWnt7aR191A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFAELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 71) hWnt7aNTD K198A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLADKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 27) hWnt7aK198A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLADKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 72) hWnt7aNTD V205A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAAH V EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 28) hWnt7aV205A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAAH V EPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYEEDPVTGSVGTQGRACNKTAPQASGCDLMC CGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 73) hWnt7aNTD E208A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV APVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 29) hWnt7aE208A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV APVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 74) hWnt7aNTD K216A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNARPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 30) hWnt7aK216A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNARPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 75) hWnt7aNTD K222A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLAIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 31) hWnt7aK222A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLAIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 76) hWnt7aNTD I223A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKAKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 32) hWnt7aI223A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKAKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 77) hWnt7aNTD Y229A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSARKPMDTDLVYIEKSPNYC (SEQ ID NO: 33) hWnt7aY229A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSARKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 78) hWnt7aNTD P232A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKAMDTDLVYIEKSPNYC (SEQ ID NO: 34) hWnt7aP232A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKAMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 79) hWnt7aNTD T235A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKPMDADLVYIEKSPNYC (SEQ ID NO: 35) hWnt7aT235A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKPMDADLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 80) hWnt7aE248A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCAEDPVTGSVGTQGRACN KTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 36) hWnt7aR289A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYAAVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 37) hWnt7aW291A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVAQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 38) hWnt7aT307A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLM N LHN N EAGRKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNACSERTEMYTCK (SEQ ID NO: 39) hWnt7aNTD Q17A:
LGASIICNKIPGLAPRARAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 43) hWnt7aQ17A:
LGASIICNKIPGLAPRARAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 81) hWnt7aNTD I20A:
LGASIICNKIPGLAPRQRAACQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 44) hWnt7aI20A:
LGASIICNKIPGLAPRQRAACQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACN KTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 82) hWnt7aNTD P25A:
LGASIICNKIPGLAPRQRAICQSRADAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 45) hWnt7aP25A:
LGASIICNKIPGLAPRQRAICQSRADAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 83) hWnt7aNTD M37A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQAGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 46) hWnt7aM37A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQAGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 84) hWnt7aNTD L39A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGADECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 47) hWnt7aL39A:
LG ASIICN KI PG LAPRQRAICQS RPDAIIVIG EGSQM G AD ECQFQFRN G RW N CSALG ERTVFG KE LKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 85) hWnt7aNTD I28A:
LGASIICNKIPGLAPRQRAICQSRPDAAIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 48) hWnt7aI28A:
LGASIICNKIPGLAPRQRAICQSRPDAAIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 86) hWnt7aNTD R50A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGAWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 49) hWnt7aR50A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGAWNCSALGERTVFGKELKVG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LHN N EAGRKILEEN MKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 87) hWnt7aNTD V68A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKAG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 50) hWnt7aV68A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKAG SREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVD AREIKQNARTLMN LHN N EAGRKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVH VEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL MCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 88) hWnt7aNTD F135A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVAVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 51) hWnt7aF135A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVAVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 89) hWnt7aNTD R146A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNAATLM N LH N N EAGRKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 52) hWnt7aR146A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNAATLM N LH N N EAGRKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 90) hWnt7aNTD K159S:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLMNLHNNEAGRSILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 53) hWnt7aK159S:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLMNLHNNEAGRSILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 91) hWnt7aNTD K159L:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLMNLHNNEAGRLILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 54) hWnt7aK159L:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLMNLHNNEAGRLILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 92) hWnt7aNTD K200A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDAYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 55) hWnt7aK200A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDAYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 93) hWnt7aNTD R214A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASANKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 56) hWnt7aR214A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASANKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 94) hWnt7aNTD P218A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRATFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO: 57) hWnt7aP218A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQN ARTLM N LH N N EAG RKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRNKRATFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 95) hWnt7aK318A:
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGS REAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDA REIKQNARTLM N LH N N EAGRKILEEN M KLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYN EAVHV EPVRASRN KRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLM CCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCA (SEQ ID NO: 58)
Table 1 gives an overview of the position of amino acid substitutions in the amino acid sequence of the (mouse) Wnt7a precursor polypeptide and their corresponding position in the amino acid sequence of the mature (mouse) Wnt7a polypeptide:
Table 1
Figure imgf000064_0001
Figure imgf000065_0001
In particularly preferred embodiments, in such variant, the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by one or more (preferably not more than three, preferably not more than two, more preferably one) other amino acid residue, such as preferably but without limitation by an alanine (A) residue, a serine (S) or a leucine (L) residue. It will be clear to a person skilled in the art that said K residue at the position corresponding to position 159 in SEQ ID NO: 1 or SEQ ID NO: 2, relates to the K residue at position 190 in the Wnt7a precursor polypeptide, as shown in Table 1.
For consistency reasons the inventors mostly substituted to alanine, albeit with some exceptions. A person skilled in the art will however understand that also other substitutions towards other amino acids are able to reproduce the desired effect. This was indeed confirmed by further experiments that showed the desired effect with other substitutions than alanine. The inventors postulate that the mutations have an effect on the stability of the Wnt7 polypeptide.
In even more preferred embodiments, the compound as disclosed herein comprises, consists essentially of or consists of an amino acid sequence as set forth in SEQ ID NO: 14, 54, 55, 59, 92 or 93, preferably SEQ ID NO: 14 or 59, more preferably SEQ ID NO: 14.
In particular embodiments, the variant of the Wnt7 polypeptide has at least 35%, preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, preferably 100%, of the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length wildtype Wnt7 polypeptide. In preferred embodiments, the variant of the Wnt7 polypeptide has at least 70% of the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length wild-type Wnt7 polypeptide. In particular embodiments, the variant of the Wnt7 polypeptide has a GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity which is higher (e.g. 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold or 2-fold higher or even higher) than the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length wild-type Wnt7 polypeptide.
In particular embodiments, the compound as disclosed herein is soluble in water. For example, the solubility of the compound can be increased by using a Frizzled- specific antibody or fragment thereof, instead of a palmitoylated Frizzled-binding domain of a Wnt polypeptide.
The compound as disclosed herein can be a protein, polypeptide or a peptide. In another embodiment, said compound is a nucleic acid encoding the Wnt7 protein or fragment as disclosed herein. Said nucleic acid may be inserted into a nucleic acid expression cassette and/or vector, as is well-known in the art.
Accordingly, a further aspect relates to a nucleic acid expression cassette comprising the nucleic acid encoding the compound as disclosed herein, operably linked to a promoter and/or transcriptional and translational regulatory signals.
Preferably, the nucleic acid expression cassette may comprise one or more open reading frames (ORF) encoding said one or more proteins, polypeptides or peptides. The precise nature of transcriptional and translational regulatory sequences or elements required for expression may vary between expression environments, but typically include a transcription terminator, and optionally an enhancer.
In particular embodiments, the nucleic acid expression cassette comprises the nucleic acid encoding the compound as disclosed herein, operably linked to one or more promoters, enhancers, ORFs and/or transcription terminators.
A further aspect relates to vector comprising the nucleic acid encoding the compound as disclosed herein, or the nucleic acid expression cassette as disclosed herein, such as a viral vector.
Factors of importance in selecting a particular vector include inter alia: choice of recipient cell, ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in particular recipient cells; whether it is desired for the vector to integrate into the chromosome or to remain extra-chromosomal in the recipient cells; and whether it is desirable to be able to "shuttle" the vector between recipient cells of different species.
Expression vectors can be autonomous or integrative. A nucleic acid can be in introduced into a cell in the form of an expression vector such as a plasmid, phage, transposon, cosmid or virus particle. The recombinant nucleic acid can be maintained extrachromosomally or it can be integrated into the cell chromosomal DNA. Expression vectors can contain selection marker genes encoding proteins required for cell viability under selected conditions (e.g., URA3, which encodes an enzyme necessary for uracil biosynthesis, or LEU2, which encodes an enzyme required for leucine biosynthesis, or TRP1, which encodes an enzyme required for tryptophan biosynthesis) to permit detection and/or selection of those cells transformed with the desired nucleic acids. Expression vectors can also include an autonomous replication sequence (ARS). The ARS may comprise a centromere (CEN) and an origin of replication (ORI). For example, the ARS may be ARS18 or ARS68.
Integrative vectors generally include a serially arranged sequence of at least a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment. The first and second insertable DNA fragments are each about 200 (e.g., about 250, about 300, about 350, about 400, about 450, about 500, or about 1000 or more) nucleotides in length and have nucleotide sequences which are homologous to portions of the genomic DNA of the cell species to be transformed. A nucleotide sequence containing a nucleic acid of interest for expression is inserted in this vector between the first and second insertable DNA fragments, whether before or after the marker gene. Integrative vectors can be linearized prior to transformation to facilitate the integration of the nucleotide sequence of interest into the cell genome. Prior to introducing the vectors into a cell of interest, the vectors can be grown (e.g., amplified) in bacterial cells such as Escherichia coli (E. coli). The vector DNA can be isolated from bacterial cells by any of the methods known in the art, which result in the purification of vector DNA from the bacterial milieu. The purified vector DNA can be extracted extensively with phenol, chloroform, and ether, to ensure that no E. coli proteins are present in the plasmid DNA preparation, since these proteins can be toxic to mammalian cells.
As noted elsewhere, a compound may comprise a protein, polypeptide or peptide. Such may be suitably obtained through expression by host cells or host organisms, transformed with an expression construct encoding and configured for expression of said protein, polypeptide or peptide in said host cells or host organisms, followed by purification of the protein, polypeptide or peptide.
Hence, a further aspect provides a host cell comprising the nucleic acid, nucleic acid expression cassette or vector as taught herein.
In certain embodiments, the host cell may be a bacterial cell, a yeast cell, an animal cell, or a mammalian cell.
The terms "host cell" and "host organism" may suitably refer to cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals. Contemplated as host cells are inter alia unicellular organisms, such as bacteria (e.g., E. coli, Salmonella typhimurium, Serratia marcescens, or Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris), (cultured) plant cells (e.g., from Arabidopsis thaliana or Nicotiana tobaccum) and (cultured) animal cells (e.g., vertebrate animal cells, mammalian cells, primate cells, human cells or insect cells). Contemplated as host organisms are inter alia multi-cellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrate animals, still more preferably mammals, yet more preferably primates; particularly contemplated are such animals and animal categories which are non-human. Such protein, polypeptide or peptide may be suitably isolated.
Further, there are several other well-known methods of introducing nucleic acids into animal cells, any of which may be used herein. At the simplest, the nucleic acid can be directly injected into the target cell I target tissue. Other methods include fusion of the recipient cell with bacterial protoplasts containing the nucleic acid, the use of compositions like calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids or liposomes or methods like receptor- mediated endocytosis, biolistic particle bombardment ("gene gun" method), infection with viral vectors (i.e. derived from lentivirus, adeno-associated virus (AAV), adenovirus, retrovirus or antiviruses), electroporation, and the like. Other techniques or methods which are suitable for delivering nucleic acid molecules to target cells include the continuous delivery of an NA molecule from poly (lactic-Co- Glycolic Acid) polymeric microspheres or the direct injection of protected (stabilized) NA molecule(s) into micropumps delivering the product. Another possibility is the use of implantable drug-releasing biodegradable microspheres. Also envisaged is encapsulation of NA or providing NA in various types of liposomes (immunoliposomes, PEGylated (immuno) liposomes), cationic lipids and polymers, nanoparticles or dendrimers, poly (lactic-Co-Glycolic Acid) polymeric microspheres, implantable drug-releasing biodegradable microspheres, etc.; and co-injection of NA with protective agent like the nuclease inhibitor aurintricarboxylic acid. It shall be clear that also a combination of different above-mentioned delivery modes or methods may be used.
In particular embodiments, the compound is provided in a liposome or lipid nanoparticle.
The term "lipid nanoparticle" or "LNP" refers to a particle having at least one dimension in the order of nanometers (e.g., 1-1,000 nm) and comprises a plurality of lipid molecules physically associated with each other by intermolecular forces. The lipid nanoparticles may be, e.g., microspheres (including unilamellar and multilamellar vesicles, e.g. "liposomes"), a dispersed phase in an emulsion, micelles or an internal phase in a suspension. An active agent or therapeutic agent, such as a nucleic acid or polypeptide, is encapsulated or provided in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response. LNPs are commonly formulated with two or more excipients: (i) a sterol, which enhances the stability of the LNP bilayer and promotes membrane fusion; (ii) optionally a phospholipid, which fortifies the LNP bilayer structure and also aids in endosomal escape; and (iii) a lipid-polyethylene glycol (PEG) conjugate, which inserts into the LNP bilayer and provides a PEG coating that reduces LNP aggregation, reduces nonspecific binding of proteins due to sterically hindrance, and reduces nonspecific endocytosis by immune cells. A LNP may further comprise one of more buffering agents.
In other particular embodiments, the vector comprising the nucleic acid as described herein is a viral vector, preferably a viral vector specifically directed towards the central and/or peripheral nervous system (e.g., a brain-specific viral vector). In further particular embodiments, the viral vector is a central nervous system (CNS) neuron-specific adeno-associated virus serotype 9 (AAV9) mutant.
In preferred embodiments, the viral vector is a blood brain barrier endothelial cellspecific viral vector. In further preferred embodiments, the viral vector is a blood brain barrier endothelial cell-specific capsid adeno-associated virus serotype 2 (AAV2) mutant.
In another or further embodiment of the pharmaceutical composition for use, comprising a therapeutically active amount of a compound chosen from Wnt7 polypeptide or a fragment thereof or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof as described in any of the previous embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable" as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.
As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active substance, its use in the therapeutic compositions may be contemplated.
Illustrative, non-limiting carriers for use in formulating the pharmaceutical compositions include, for example, oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents suitable for intravenous (IV) use, liposomes or surfactant-containing vesicles, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to one of ordinary skill in the art.
Pharmaceutical compositions as intended herein may be formulated for essentially any route of administration, such as without limitation, oral administration (such as, e.g., oral ingestion or inhalation), intranasal administration (such as, e.g., intranasal inhalation or intranasal mucosal application), parenteral administration (such as, e.g., subcutaneous, intravenous (I.V.), intramuscular, intraperitoneal, intrathecal or intracisternal injection or infusion), transdermal or transmucosal (such as, e.g., oral, sublingual, intranasal) administration, topical administration, rectal, vaginal or intratracheal instillation, and the like. In this way, the therapeutic effects attainable by the methods and compositions can be, for example, systemic, local, tissue-specific, etc., depending of the specific needs of a given application.
For example, for oral administration, pharmaceutical compositions may be formulated in the form of pills, tablets, lacquered tablets, coated (e.g., sugar-coated) tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions. In an example, without limitation, preparation of oral dosage forms may be is suitably accomplished by uniformly and intimately blending together a suitable amount of the compound as disclosed herein in the form of a powder, optionally also including finely divided one or more solid carrier, and formulating the blend in a pill, tablet or a capsule. Exemplary but non-limiting solid carriers include calcium phosphate, magnesium stearate, talc, sugars (such as, e.g., glucose, mannose, lactose or sucrose), sugar alcohols (such as, e.g., mannitol), dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. Compressed tablets containing the pharmaceutical composition can be prepared by uniformly and intimately mixing the compound as disclosed herein with a solid carrier such as described above to provide a mixture having the necessary compression properties, and then compacting the mixture in a suitable machine to the shape and size desired. Moulded tablets maybe made by moulding in a suitable machine, a mixture of powdered compound moistened with an inert liquid diluent. Suitable carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc.
For example, for oral or nasal aerosol or inhalation administration, pharmaceutical compositions may be formulated with illustrative carriers, such as, e.g., as in solution with saline, polyethylene glycol or glycols, DPPC, methylcellulose, or in mixture with powdered dispersing agents, further employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilising or dispersing agents known in the art. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the compound as taught herein or their physiologically tolerable salts in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents. If required, the formulation can also additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant. Illustratively, delivery may be by use of a single-use delivery device, a mist nebuliser, a breath-activated powder inhaler, an aerosol metered-dose inhaler (MDI) or any other of the numerous nebuliser delivery devices available in the art. Additionally, mist tents or direct administration through endotracheal tubes may also be used.
Examples of carriers for administration via mucosal surfaces depend upon the particular route, e.g., oral, sublingual, intranasal, etc. When administered orally, illustrative examples include pharmaceutical grades of mannitol, starch, lactose, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate and the like, with mannitol being preferred. When administered intranasally, illustrative examples include polyethylene glycol, phospholipids, glycols and glycolipids, sucrose, and/or methylcellulose, powder suspensions with or without bulking agents such as lactose and preservatives such as benzalkonium chloride, EDTA. In a particularly illustrative embodiment, the phospholipid 1,2 dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) is used as an isotonic aqueous carrier at about 0.01-0.2% for intranasal administration of the compound of the subject invention at a concentration of about 0.1 to 3.0 mg/ml.
For example, for parenteral administration, pharmaceutical compositions may be advantageously formulated as solutions, suspensions or emulsions with suitable solvents, diluents, solubilisers or emulsifiers, etc. Suitable solvents are, without limitation, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose, invert sugar, sucrose or mannitol solutions, or alternatively mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. The compound and pharmaceutically acceptable salts thereof of the invention can also be lyophilised and the lyophilisates obtained used, for example, for the production of injection or infusion preparations. For example, one illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP Water for Injection (WFI). Other illustrative carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral vegetable oil-in-water emulsion. Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solution, 5% dextrose in WFI and 0.01-0.1% triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a 1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol and the balance an acceptable isotonic solution such as 5% dextrose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI and 1 to 10% squalene or parenteral vegetable oil-in-water emulsions.
Where aqueous formulations are preferred, such may comprise one or more surfactants. For example, the composition can be in the form of a micellar dispersion comprising at least one suitable surfactant, e.g., a phospholipid surfactant. Illustrative examples of phospholipids include diacyl phosphatidyl glycerols, such as dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoyl phosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines, such as dimyristoyl phosphatidylcholine (DPMC), dipalmitoyl phosphatidylcholine (DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acids, such as dimyristoyl phosphatidic acid (DPMA), dipahnitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); and diacyl phosphatidyl ethanolamines such as dimyristoyl phosphatidyl ethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine (DPPE) and distearoyl phosphatidyl ethanolamine (DSPE). Typically, a surfactant:active substance molar ratio in an aqueous formulation will be from about 10: 1 to about 1 : 10, more typically from about 5: 1 to about 1 :5, however any effective amount of surfactant may be used in an aqueous formulation to best suit the specific objectives of interest.
When rectally administered in the form of suppositories, these formulations may be prepared by mixing the compounds according to the invention with a suitable nonirritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidity and/or dissolve in the rectal cavity to release the drug.
Suitable carriers for microcapsules, implants or rods are, for example, copolymers of glycolic acid and lactic acid.
One skilled in this art will recognize that the above description is illustrative rather than exhaustive. Indeed, many additional formulations techniques and pharmaceutically-acceptable excipients and carrier solutions are well-known to those skilled in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.
In preferred embodiments, the pharmaceutical composition comprising the compound or nucleic acid encoding the compound, as taught herein is administered parenterally. More preferably, the pharmaceutical composition as taught herein is administered intravenously, for example by infusion, or intrathecally.
In particular embodiments, the pharmaceutical composition for use comprising the compound, the nucleic acid encoding the compound, or the nucleic acid expression cassette comprising the nucleic acid as taught herein is used in gene therapy, in particular in blood brain barrier endothelial cell-directed gene therapy, most in particular in glioblastoma-directed gene therapy.
Accordingly, also provided herein is a method for gene therapy, in particular central and/or peripheral nervous system-directed gene therapy, in a subject in need of said gene therapy comprising: introducing in the subject, in particular in the central and/or peripheral nervous system of the subject, a nucleic acid expression cassette or a vector as described herein; and expressing a therapeutically effective amount of the compound encoded by the nucleic acid as taught herein in the subject, in particular the central and/or peripheral nervous system of the subject. In particular embodiments, the pharmaceutical composition for use comprising the compound or the nucleic acid encoding the compound as taught herein is used in mRNA therapy, in particular in blood brain barrier endothelial cell-directed mRNA therapy, most in particular in glioblastoma-directed mRNA therapy.
Accordingly, also provided herein is a method for mRNA therapy in a subject suffering from glioblastoma, in particular central and/or peripheral nervous system-directed mRNA therapy, in a subject in need of said mRNA therapy comprising : introducing in the subject, in particular in the central and/or peripheral nervous system of the subject, a nucleic acid encoding the compound as taught therein; and expressing a therapeutically effective amount of the compound encoded by the nucleic acid as taught herein in the subject, in particular the central and/or peripheral nervous system of the subject.
An advantage of the use of mRNAs therapy, is that mRNAs do not integrate into the genome and therefore do not have the risk of insertional mutagenesis.
In particular embodiments, the pharmaceutical composition as taught herein is administered to the subject by the injection (e.g., intravenously) or transplantation of allogeneic cells transformed with the vector comprising the nucleic acid or the nucleic acid expression cassette as taught herein. When administered, the injected or transplanted allogenic cells will transcribe and translate the nucleic acid encoding the compound as taught herein in vivo. The dosage or amount of the compound as taught herein, optionally in combination with one or more other active compounds to be administered, depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect. Thus, the unit dose and regimen depend on the nature and the severity of the disorder to be treated, and also on factors such as the species of the subject, the sex, age, body weight, general health, diet, mode and time of administration, immune status, and individual responsiveness of the human or animal to be treated, efficacy, metabolic stability and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the compound described in any of the embodiments above. In order to optimize therapeutic efficacy, the compound as taught herein can be first administered at different dosing regimens. Typically, levels of the compound in a tissue can be monitored using appropriate screening assays as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. The frequency of dosing is within the skills and clinical judgement of medical practitioners (e.g., doctors, veterinarians or nurses). Typically, the administration regime is established by clinical trials which may establish optimal administration parameters. However, the practitioner may vary such administration regimes according to the one or more of the aforementioned factors, e.g., subject's age, health, weight, sex and medical status. The frequency of dosing can be varied depending on whether the treatment is prophylactic or therapeutic.
Toxicity and therapeutic efficacy of the compound as described herein or pharmaceutical composition for use of the invention as described in any of the embodiments, comprising the same can be determined by known pharmaceutical procedures in, for example, cell cultures or experimental animals. These procedures can be used, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Pharmaceutical compositions that exhibit high therapeutic indices are preferred. While pharmaceutical compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to normal cells (e.g., non-target cells) and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in appropriate subjects. The dosage of such pharmaceutical compositions lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a pharmaceutical composition used as described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the pharmaceutical composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.
Without limitation, depending on the type and severity of the disease, a typical dosage (e.g., a typical daily dosage or a typical intermittent dosage, e.g., a typical dosage for every two days, every three days, every four days, every five days, every six days, every week, every 1.5 weeks, every two weeks, every three weeks, every month, or other) of the compound as taught herein may range from about 10 pg/kg to about 500 pg/kg body weight of the subject, per dose, depending on the factors mentioned above, e.g., such as 20-400 pg/kg or 20-200 pg/kg or 20-100 pg/kg or 40-80 pg/kg body weight of the subject.
By means of example and without limitation, the compound as taught herein may be administered at about 5 pg/kg, or at about 10 pg/kg, or at about 15 pg/kg, or at about 20 pg/kg, or at about 25 pg/kg, or at about 30 pg/kg, or at about 35 pg/kg, or at about 40 pg/kg, or at about 45 pg/kg, or at about 50 mg/kg, or at about 60 pg/kg, or at about 70 pg/kg, or at about 80 pg/kg, or at about 90 pg/kg, or at about 100 pg/kg, or at about 200 pg/kg, or at about 300 pg/kg, or at about 400 pg/kg, or at about 500 pg/kg per dose.
In particular embodiments, the compound as taught herein is administered using a sustained delivery system, such as a (partly) implanted sustained delivery system. Skilled person will understand that such a sustained delivery system may comprise a reservoir for holding the compound as taught herein, a pump and infusion means (e.g., a tubing system). For example, the sustained delivery system may be a mini- osmotic pump system implanted in the brain.
In particular embodiment, the compound as disclosed herein is the main or only active ingredient of the pharmaceutical composition.
In another embodiment, the pharmaceutical composition for use as described in any of the embodiments is combined with a second therapy, preferably chosen from surgery, chemotherapy, radiotherapy or immunotherapy.
A further aspect relates to the pharmaceutical composition comprising the compound as disclosed herein, the nucleic acid encoding the compound as disclosed herein the nucleic acid expression cassette as disclosed herein or the vector as disclosed herein, for use as a medicament.
A further aspect provides a method of preventing, reducing in progression or treating glioblastoma in a subject in need thereof, comprising administering to a subject a therapeutically active amount of pharmaceutical composition comprising a compound chosen from Wnt7 polypeptide or a fragment thereof capable of activating G-protein coupled receptor (GPR)124/RECK/Frizzled/lipoprotein receptor-related protein (LRP)-mediated Wnt signaling, wherein said Wnt7 polypeptide or fragment thereof does not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof, as described in any of the embodiments throughout this specification.
It will be clear that the current invention equally provides methods for the treatment of glioblastoma, as well as is directed to the use of a therapeutically active amount of pharmaceutical composition comprising a compound chosen from Wnt7 polypeptide or a fragment thereof capable of activating G-protein coupled receptor (GPR)124/RECK/Frizzled/lipoprotein receptor-related protein (LRP)-mediated Wnt signaling, wherein said Wnt7 polypeptide or fragment thereof does not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof, as described in any of the embodiments throughout this specification for the manufacture of a medicament for the treatment or reduction of progression of glioblastoma in a subject. Further embodiments are as described above.
The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.
EXAMPLES AND DESCRIPTION OF FIGURES
Material and methods
Constructs and antibodies
For Super Top Flash (STF) assays, the coding sequence of mouse Reck, Gprl24, Fzl- 10, Lrp5, Wnt7a, and Wnt7b (and their variants), zebrafish Wnt7-aa, -ab, -ba, and -bb, Reck and Gprl24 were expressed from the CMV promoter of the pCS2+ or the pRK5 (for Fzs and Lrp5) plasmid. C-terminal V5-tagged single-residue and fusion variants of Wnt7a were obtained using In-Fusion cloning (ST0345, Takara) with tandem overlapping PCR products and were confirmed by Sanger sequencing. The NTD coding sequences correspond to amino-acids 1-278 of Wnt7a and Wnt7b, and the CTD part of the chimeric ligands to 279-360 (Wnt2), 281-351 (Wnt4), 310-380 (Wnt5a), 294-364 (Wnt6), 347-417 (WntlOa), 284-354 (Wntll), 294-364 (Wntl6) and 261-358 (XWnt8a). The collection of active human WNT-V5 ligands (Addgene #43807 to #43825 from the Xi He laboratory) was used in the Wnt/Fz pairwise STF experiments of Fig. 8. Firefly luciferase activities (derived from a genomic transgene in STF cells or ectopically expressed from the transfected M50 Super 8x TOPFIash plasmid, Addgene plasmid #12456 in other cells) were normalized to Renilla luciferase activities (pTK-Renilla vector transfection). For PLA (proximity ligation assay), IF (immunofluorescence) and IP (immunoprecipitation) experiments, pCS2+ plasmids coding for N-terminally HA-tagged mouse Reck and Fz5 were used together with the Wnt-V5 variants described above.
The following commercial primary antibodies were used in this study: mouse monoclonal antibody against V5 (R960-25, Thermo Fisher Scientific; RRID: AB_2556564) at 1 :400; rabbit polyclonal antibodies against Wnt7a (GTX128106, GenTex) at 1 :300 for IF and 1: 1000 for WB; purified rabbit polyclonal anti-HA antibodies (H6908, Sigma-Aldrich, RRID:AB_260070) at 1:400 for PLA and 1 : 1000 for WB; rabbit polyclonal anti-Glut-1 (07-1401, Millipore, RRID:AB_1587074) at 1 :200 for mouse IF; rabbit polyclonal anti-Fibrinogen (ab34269, Abeam, RRID:AB_732367) at 1 :300; rabbit polyclonal anti-Mfsd2A (a gift from D. Silver, (65)) at 1 :500, rabbit polyclonal anti-Desmin (abl5200, Abeam, RRID:AB_301744) at 1300; rabbit monoclonal antibody against LEF1 (2230, C12A5, Cell Signaling, RRID:AB_823558) at 1 :200; Alexa 647-conjugated rabbit monoclonal antibody against ERG (abl96149, Abeam) at 1 :250; rabbit monoclonal antibody against Dkkl (abl09416, Abeam, RRID:AB_10861912) at 1 : 1000 ; rabbit polyclonal antibody against Claudin-5 (Thermo Fisher Scientific 34-1600 RRID:AB_2533157) at 1 :250; rabbit monoclonal antibody against Caveolin-1 (3267, Cell Signaling Technology, RRID:AB_2275453) at 1/200; rabbit monoclonal antibody against S100[3 (ab52642, abeam, RRID:AB_882426) at 1:300); rabbit monoclonal antibody against Ibal (abl78846, Abeam, RRID:AB_2636859) at 1 :300; biotin-conjugated rat monoclonal anti-HA (3F10, 12158167001, Roche, RRID:AB_390915) at 1 ng.pl- 1; rat monoclonal anti-Laminin y-1 (sc-65643, 3E10, Santa Cruz Biotechnology, RRID: AB_1123687) at 1 :500; rat monoclonal anti-CD31 (553370, BD Pharmigen,RRID:AB_396660) at 1 :250; rat monoclonal anti-GFAP (13-0300, Thermo Fisher Scientific, RRID:AB_2532994) at 1:300; rat monoclonal anti-o- tubulin (MAI-80017, Pierce, RRID:AB_2210201) at 1:2000; chicken polyclonal antibodies against p Galactosidase/lacZ (ab9361, Abeam, RRID:AB_307210) at 1 :200; chicken anti-GFP (GFP-1010, Aves Labs, RRID:AB_2307313) at 1 :400, guinea pig polyclonal antibodies against NeuN (ABN90, Merk, RRID:AB_11205592) at 1:300. For additional detection of vessel and biotin we respectively used Isolectin GS-IB4 Alexa 594-Conjugate (121413, Invitrogen) at 1 :200 and Streptavidin Alexa 647conjugate (S21374, Invitrogen) at 1 : 1000. For immunofluorescence we used Alexa 488-, Alexa 568- and Alexa 641-conjugated goat secondary antibodies (Molecular probes) at 1 :400 and for western blotting we used HRP conjugated secondary antibodies (Promega) at 1 :5000.
Cell CRISPR/Cas9 editing and culture
HEK293T cells were obtained from ATCC (CRL-3216), and HEK293 STF cells were kindly provided by Jeremy Nathans (John Hopkins). They were grown in DMEM/F12 1 : 1 medium (Lonza) supplemented with 10% fetal bovine serum (Biowest, S1810) and maintained at 37°C in a humidified incubator equilibrated with 5% CO2. HEK293(T) cells were transfected with lipofectamine 2000 (Invitrogen) for STF assays or with Polyethylenimine (PEI max, Polyscience) for IP and IF at 70% confluency 24 h after plating. For staining experiments, HEK293(T) cells were seeded on poly-lysine (0.1 mg.ml-1) coated 12 mm coverslips. The CRISPR/Cas9- engineered GPR124-/-;RECK-/- mutant HEK293 STF line has been described before, together with the FZ1-10-/- HEK293T clone used to build the FZl-10-/-;GPR124-/- ;RECK-/- HEK293T line. In order to generate the RECK-/- HEK293T and FZ1-10-/- ;GPR124-/-;RECK-/- HEK293T cell lines, GPR124 (5'-GCATCCGCTGGTACCACAAC-3'; SEQ ID NO: 96) and RECK (5'-ATTGTTGATGGTCTCATCGA-3'; SEQ ID NO: 97) CRISPR/Cas9 guide sequences were designed using the http://crispr.mit.edu website and cloned into pSpCas9(BB)-2A-GFP. The top 1% of GFP+ cells were isolated by FACS (Arialll, BD Biosciences) 48 h after transfection and distributed in 96-well plates for clonal expansion and lesion determination by Sanger sequencing.
STF dual luciferase assay
Dual luciferase assays were performed 2 days after transfection in 96-well plates using DualLuciferase Reporter Assay system (E1960, Promega) according to the manufacturers' protocol: after extraction in passive lysis buffer, the ratio of Firefly and Renilla luciferases activities of the cell lysates were measured. The co-culture assay was performed by seeding transfected reporter cells with Wnt7 ligands expressing HEK293T cells at a 1 : 1 ratio 1 day after transfection and 1 day prior to luciferase measurements. The amount of plasmid DNA transfected per well was optimized for each expression vector as follows: Renilla luciferase (0.5 ng), Wnt ligands (20 ng), Fz receptors (5 ng), Lrp5 (2.5 ng), Gprl24 (10 ng), Reck (10 ng). The total amount of DNA was adjusted to 100 ng per well with the empty pCS2 vector. Proximity ligation assay
The Duolink PLA technology (Sigma-Aldrich) was used to infer protein-protein interaction between HA-Reck and Wnt-V5 variants in situ. Two days after transfection, cells were fixed with 4% PFA for 10 min, blocked 30 min at 37°C with the Duolink blocking solution, incubated with primary antibodies anti-HA and anti- RECK for 1 h at RT, with anti-rabbit PLUS and anti-mouse MINUS PLA probes for 1 h at 37°C, with the Duolink Ligation solution for 30 min at 37°C and finally with the Duolink Amplification solution (Red) for 100 min at 37°C. Two PBS washes were included between each step. Slides were air-dried and mounted in ProLong Gold mounting medium supplemented with DAPI (Molecular probes). PLA signal was quantified using Image! PLA-associated channel was thresholded to create a binary mask in which PLA dots were detected using the Particle Analysis plugin of ImageJ set to quantify the area and the mean signal intensity of each particle. Their Nearest Neighbor Distance (NND) was additionally computed using the ImageJ BAR plugin. To sort for membrane-localized signal, we filtered out the particles whose area was < 5pm2 or NND > 4pm. PLA signal was calculated per field of view as ( Ax/,)/ N where A is the area of the particle, I its mean intensity corrected for background and N the area fraction occupied by the nuclei signal in the field (measured on DAPI thresholded pictures), indicative of the number of cells.
Surface and intracellular immunofluorescence staining
Two days after transfection, GPR124-/-;RECK-/- HEK293 cells were blocked in PBS/BSA 2% for 20 min at 4°C, incubated with primary antibody against Wnt7a for 1 h at 4°C, fixed in 4% paraformaldehyde for 10 min at RT, permeabilized with 0.1% saponin in PBS for 5 min, blocked again in PBS/BSA 2% for 1 h and sequentially incubated with primary anti-V5 and Alexaconjugated secondary antibodies for 1 h at RT. Antibodies were diluted in PBS/BSA 2% and cells were washed three times with PBS after each step. Membrane accumulation of the ligand was quantified using ImageJ as follows: maximum intensity projection of the signal obtained after permeabilization (V5 intracellular signal) was used to detect expressing cells and to measure their mean signal intensity. For each cell, the Z plane associated with the strongest membrane-associated signal (Wnt7 extracellular signal) was used to measure its mean signal intensity along a line of circa 2 pm width (30 pixels) along the cell surface. We also included in this analysis cells that were only positive for membrane signal. The ratio between extracellular and intracellular signals after background correction was calculated per cell, normalized to the ratio corresponding to a double negative cell in the same field of view and averaged per field. Co-Immunoprecipitation, dot blot, and western blot
For co-immunoprecipitation experiments, RECK-/- HEK293T were incubated with the membrane impermeable crosslinker DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate), Sigma-Aldrich) at 0.2 mM in HBS (20 mM HEPES, pH 7.4, 150 mM NaCI, 1 mM CaCI2, 0.5 mM MgCI2) for 2 h at 4°C, two days after transfection in 6- well plates. After quenching the reaction with 50 mM Tris/HCI, pH 7.4 for 15 min, total cell extracts were prepared in RIPA buffer (10 mM Tris-HCI pH 8, 150 mM NaCI, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail (Complete - Roche)) under rocking at 4°C for 30 min. Extracts were cleared by centrifugation (15 min at 14,000 rpm) and incubated, after saving 10% as input control, for 2 h at 4°C with a biotin-conjugated antibody against HA and for 1 h at 4°C with high capacity streptavidin agarose beads (Pierce) under constant rotation. Beads were washed four times with RIPA buffer and boiled for 10 min in 2X Laemmli sample buffer containing 15% Pmercaptoethanol. Input protein extracts and bead eluates were analyzed by SDS-PAGE and Western blotting, according to standard procedures and developed by autoradiography with ECL Plus substrate (PerkinElmer) after incubation with primary and secondary antibodies respectively for 1 night and 1 h at 4°C. Dot blot analysis was performed to assess ligand secretion. Cellular supernatant was collected 2 days after transfection, cleared by centrifugation, and transferred to a nitrocellulose membrane (GE Healthcare) using a BioDot SF apparatus (Bio-Rad). The membrane was then processed as described above. For in vitro analysis of Dkkl expression in Tet-off GL261 cells, cells were cultured in the absence or presence of doxycycline (0.002 to 2 pg.ml-1) for 48 h before extraction in RIPA buffer. To analyze in vivo expression levels, tumors were dissected at 21 dpi from mice fed with control or doxycycline-containing diet (1 g.kg-1 doxycycline hyclate, Safe Diets, France).
Structural modeling
The mouse Wnt7a structure was modeled based on the crystal structure of Xenopus Wnt8a in complex with mouse Fz8 CRD (PDBID:4F0A) as described previously.
Animal procedures - zebrafish
The transgenic and mutant lines used in this study are Tg(kdrl:EGFP)s843, Tg(kdrl:HRASmCherry)s896, Tg(7xTCF-Xla.Siam:GFP)ia4, Tg(-
17.0neurogl:EGFP)w61, Tg(gatala:dsRed)sd2 and gprl24s984. Zebrafish (Danio rerio) were maintained at 28°C on a 14 h light/10 h dark cycle. Embryos were obtained and raised under standard conditions in accordance with European and national ethical and animal welfare guidelines (protocol approval number: CEBEA- IBMM-2017-22: 65). The wnt7aaulb2 allele was generated using the CRISPR/Cas9 technology. The targeting sgRNA construct was cloned into pT7-gRNA (Addgene plasmid #46759) using these primers: wnt7aa-fwd 5'-TAGGTGGAACTGCTCGGCCCTC-3' (SEQ ID NO: 98), wnt7aa-rev 5'-AAACGAGGGCCGAGCAGTTCCA-3' (SEQ ID NO: 99). sgRNA was transcribed from pT7-gRNA using the MEGAshortscript T7 transcription kit (Ambion) and injected at 50 pg into one-cell stage embryos, together with 150 pg of nls-zCas9-nls mRNA transcribed from pT3TS-nCas9n (Addgene plasmid # 46757) using the mMessage mMachine T3 Kit (Ambion).
Morpholinos, RNA, and DNA constructs microinjection
4 ng of the following wnt7 splice-blocking morpholinos (GeneTools) were injected into one-cell stage embryos: wnt7aa : TTCCATTTGACCCTACTTACCCAAT (SEQ ID NO: 100), wnt7ab: AACCCCTACAAATGACCACAGAACT (SEQ ID NO: 101), wnt7ba : AAGCATTCGCTCAAACCTACAGGTA (SEQ ID NO: 102), wnt7bb: ACAGATTGAAACACTTACAGGTAGC (SEQ ID NO: 103). Synthetic mRNAs were transcribed from pCS2 plasmids after Notl digestion using the mMessage mMachine SP6 Kit (Ambion) and injected into one-cell stage zebrafish embryos at the concentration indicated in the Figures. Transient mosaic endothelial overexpression was obtained by co-injection of 10 pg of Tol2 transposase mRNA and 10 pg of the pT2-kdrl-mWnt7a(WT, K190A or NTD)-P2A-NLS-eBFP DNA constructs.
Whole-mount in situ hybridization (WISH)
Whole-mount in situ hybridization was performed as described previously (73). Embryos were hybridized with digoxigenin-labeled riboprobes using the following primers: wnt7aa-fwd : 5'-GGGGATTATTTATTTGAAGATTGG-3' (SEQ ID NO: 104); w n t7a a - rev : 5 '-TAATACG ACTC ACTATAGGGGTGC ATCTAGG AAG ACCTTTG AG - 3 ' (SEQ ID NO: 105); wnt7ab-fwd : 5'-CATGACAATGGCTGGAAATG-3' (SEQ ID NO: 106); wnt7ab-rev: 5'-TAATACGACTCACTATAGGGGCCACACACGTGAATACTGG-3' (SEQ ID NO: 107); wnt7ba-fwd : 5'- GCATCAACGAGTGCCAGTATCAG-3' (SEQ ID NO: 108); wnt7ba-rev: 5'- TAATACGACTCACTATAGGGCATCCACGAAGCGTCTGGAGAAC-3' (SEQ ID NO: 109); wnt7bb-fwd : 5'- GAGTTCTCACGGAAGTTTGTGGATG-3' (SEQ ID NO: 110); wnt7bb-rev: 5'-TAATACGACTCACTATAGGGTTGAACCGCTTTGTTGTACTTGTCC-3'
(SEQ ID NO: 111). Probes were transcribed with T7 RIMA polymerase (Roche) and detected using anti- DIG AP (1 : 5000) and NBT/BCIP substrate (Roche). Embryos were sectioned (Vibratome, 50 pm) after embedding in 4% ultrapure low melting point agarose (Thermo Fisher Scientific).
Phenotypinq
Staging was performed. CtAs, DRG neurons and TCF-positive endothelial cells were quantified on confocal Z-stack recordings of live, anesthetized (low doses of tricaine), agarose-embedded (1% low melting point agarose) transgenic zebrafish. For hemorrhagic stroke experiments, Tg(kdrl: EGFP);Tg(gatala :dsRed) embryos were treated from 34 hpf onwards with 1 pM atorvastatin (Sigma-Aldrich). Embryos were graded based on the extent of intracerebral bleeding under a bright-field stereoscope. For that purpose, brains were arbitrary divided into 4 areas (left and right, forebrain/midbrain and hindbrain) in which the level of hemorrhages was scored in a double-blind manner as 0 (no hemorrhage), 1 (small hemorrhage) or 2 (large hemorrhage). The phenotype was considered as mild if the cumulated score of the 4 zones was < 1, moderate if it was <4 and severe if it was >4. Confocal pictures were taken for illustration. Tracer leakage assays were performed by microinjection of 1 nl of 10 mg.ml-1 Alexa Fluor 488 Dextran 10.000 MW (Invitrogen) in PBS in the common cardinal vein at 54 hpf. Embryos were mounted dorsoventrally and individually imaged at 30 min post-injection using a Zeiss LSM900 inverted laser scanning microscope. Quantification was performed using ImageJ by generating maximum intensity projection of stack volumes around 20 pm thick, including the dorsal arch of the CtAs about 40 pm above the PHBC. Projections were positioned minimally 15 pm below the dorsally located ventricle to avoid fluorescence signal contamination from tracer accumulated in this structure. Mean fluorescence intensity was calculated in the area covering the hindbrain parenchyma. The relative fluorescence intensity was determined by dividing the mean parenchymal tracer intensity by the maximal intensity of the tracer signal in the CtA lumen.
Whole-mount Immunostaininq
A custom polyclonal rabbit antibody was generated against zebrafish Glutl/Slc2ala (Eurogentec, Belgium). Two KLH-MBS-conjugated peptides (Slc2ala 45-59: NETWHNRYSEYIPPT (SEQ ID NO: 112) and 471485: ADKYNRDDLNTLGAD (SEQ ID NO: 113)), designed to minimize cross-reactivity with the paralogue Slc2alb, were used for immunization. For Glut-1 immunostaining, manually dechorionated zebrafish embryos were fixed with 4% paraformaldehyde overnight at 4°C, permeabilized with 20 pg.ml-1 Proteinase K in PBS 0.5% TritonX-100 for 30 min at 37°C, fixed again with 4% paraformaldehyde 20 min at RT, blocked 1 h at RT with 10% normal goat serum, 0.5% TritonXIOO, 1% DMSO in PBS and sequentially stained overnight at 4°C with primary and secondary antibodies diluted in blocking solution. Embryos were washed several times with PBS 0.5% TritonX-100 for 5 min after each step and for 30 min after the staining steps.
Animal procedures - Xenopus
Xenopus laevis embryos, obtained from adult frogs by hormone-induced egg-laying and in vitro fertilized using standard methods, were microinjected with 15 pg mRNA of Wnt7a variants into the ventral vegetal region of four-cell stage embryos. Experiments were conducted in accordance with European and national ethical and animal welfare guidelines (protocol approval number: CEBEA-IBMM-2017-22: 19).
Animal procedures - Mouse
WT C57BL/6 (Charles River Laboratories) and transgenic BAT-gal (0-catenin- activated transgene driving expression of nuclear p-galactosidase reporter) mice were used in this study. Animals were housed under standard conditions and fed ad libitum. All animal handling was performed to minimize suffering. Experimental protocols were conducted in accordance with European and National regulations and guidelines. Protocol approval numbers: CEBEA-IBMM-2019-24: 104 (ULB), FK10/1052 (Goethe University Frankfurt), S14044 (UCSD), CMM-2865 (Ottawa Hospital Research Institute).
AAV injection
Recombinant PHP.eB AAV particles were produced and purified from HEK293T cells at the Viral Vector Facility (VVF) of the Neuroscience Center Zurich (ZNZ) from the pAAV-CAG-EGFP and pAAV-CAG-mWnt7a (WT or K190A)-2A-EGFP vectors. Mouse neonates (2 days old) were injected retro-orbitally with 50 pl of a PBS suspension containing 4.1010 vg of AAV vector packaged into the PHP.eB capsid. 8-week old mice were injected intravenously in the tail (or retroorbitally in the case of behavioral testing) with 0.4-1.1012 vg of PHP-eB AAV capsids in 200 pl PBS.
Locomotor activity of neonatally-injected mice
Locomotor activity was recorded by videotracking at P20, 18 days after AAV injection. Pools of four mice were familiarized for 10 min in open field locomotor activity boxes (14 cm x 36 cm) before being recorded for 3 min. Horizontal ambulation was analyzed at 3 Hz using the Manual Tracking plugin from ImageJ. The cumulated travelled distance was calculated and normalized to the non-injected condition.
Glioblastoma model
Mouse GL261 glioblastoma cells (DSMZ no. ACC 802) were cultured in DMEM- GlutaMAX-I medium (Invitrogen) containing 10% fetal bovine serum (Biowest, S1810). The previously generated Dkkl Tet-Off GL261 cells were cultured in the same media supplemented or not with 1 pg.ml-1 doxycycline for 5 days before implantation.
For intracerebral tumor implantations, anesthetized mice were placed into a stereotactic device and 2 pl of PBS containing 105 living GL261 cells were injected at 0.25 pl.min-1 into the striatum using the coordinates relative to bregma: 0.5 (anterior-posterior), 2 (mediolateral), and 3.5 (dorsoventral) using a Hamilton Microliter #75N injection syringe with a 26Ga needle. The needle is left in place for at least 2 min before being slowly removed. At the end of the procedure, the incision was sutured and mice were allowed to recover two days before being injected with AAVs. Alternatively, mice were injected with AAVs 2 weeks before GL261 cell implantation. For TetOff-dependent experiments, mice were fed with control or 1 g.kg-1 doxycycline hyclate containing diet (Safe Diets, France) starting 2 weeks before implantation until the end of the experiment. Mice were individually monitored at 7, 14 and/or 21 days post implantation (dpi) using Magnetic Resonance Imaging (MRI) performed on a 9.4T Biospec or on a IT ICON operating under Paravision 5.1 software (Bruker). MR signal was obtained using a mouse head transmit/receive volume coil (23 mm inner diameter). Animals were anesthetized in a chamber with isoflurane (2.53%) vaporized in oxygen (2 L.min-1) and were transferred to a dedicated imaging cradle, in which isoflurane delivery was adapted (1.5-2% in 0.4 L.min-1 oxygen flow). Their body temperature was maintained by warm water circulating in a blanket, and their respiratory rate was monitored during the whole imaging session, in accordance with CMMI protocol number 2011- 07 (LA1500589). A T2-weighted 2D TurboRARE (Rapid Acquisition with Relaxation Enhancement) sequence was used to visualize the brain on 15 slices (0.5 mm thickness (no gap), 83 microns plane resolution, repetition time (TR): 3000 ms, effective echo time (TE): 60 ms, number of averages: 16, acquisition time: 9 min 36 sec, fat saturation). Tumors were manually contoured on each slice by drawing region of interest (ROI), from which volumes were obtained with the VivoQuant software (InviCRO, version 2.0). For histology, mice were euthanized at 25 dpi and brains were embedded into OCT before freezing at -80°C. For sulfo-NHS-biotin permeability assay, anesthetized mice were perfused intracardially at 22-24 days post implantation with 10 ml of 0.5 mg.ml-1 EZ-LinkSulfo-NHS-Biotin (Thermo Fisher Scientific, 21217) followed by 10 ml of 1%PFA/PBS at a flow rate of 2-2.5 ml.min-1. The dissected brains were fixed overnight in 4% PFA/PBS at 4 °C before being rinsed in PBS, embedded in 4% agarose and cut in 50 pm thick sections using a vibratome. Floating sections were blocked/permeabilized in PBS 0.5% TritonX-100, 10% normal goat serum for 2 h at RT, incubated with primary antibodies against CD31 overnight at 4°C and 4 h at RT, with secondary antibodies additionally containing Alexa 647-conjugated streptavidin 2 h at RT, with five PBS 0.1% TritonX-100 washes after each staining step. After PBS and distilled water washes, sections were mounted in Dako medium (Agilent).
Mouse brain histology and immunostaininqs
Serial coronal sections of 12 pm were prepared on native brain using a cryostat (Leica). Brain slices were mounted on SuperFrost slides (Thermo Fisher Scientific) and were submitted to H8iE (Hematoxylin and Eosin) staining following standard procedures to assess the anatomical tumor localization and size. For immunostainings, native brain sections were fixed in 4% paraformaldehyde for 10 min at RT, washed 2 times with PBS, blocked in PBS 0.1% TritonX-100, 10% normal goat serum for 1 h at RT, incubated with primary antibodies overnight at 4°C and with secondary antibodies 2 h at RT, with 5 PBS 0.1% TritonX-100 washes after each staining step. Antibodies were diluted in the blocking solution and DAPI (1 pg.ml-1) was added to the secondary antibodies. Slides were mounted in DAKO medium (Agilent) and imaged using a confocal microscope. LacZ and LEF-1 immunostainings were used to assess 0-catenin activity in brain from BAT-Gal mice after AAV injection. Staining was performed on similarly located sections and the number of lacZ or LEF- 1 positive nuclei (co-localizing with DAPI signal) were counted. For the analysis of lacZ in the hippocampus, a 0.25 mm2 area dorsal to the dentate gyrus was analyzed, whereas a 0.5 mm2 area centered around the parafascicular nucleus was used to quantify the intensity of lacZ signal in this region of the thalamus. The distribution of EGFP expression upon AAV gene delivery was quantified by measuring of the percentage of the area occupied by the thresholded signals of vessels (CD31 or isolectin), astrocytes (GFAP) and neurons (NeuN) that is also positive for EGFP. For pericyte co-localization assessment, three-color intensity profiles along a line were made using the 'plot profile' option in ImageJ for each channel (EGFP, Desmin, CD31). Tumor vessel phenotypes were analyzed on slides from the center of the tumor. Quantifications were performed using ImageJ on maximum intensity projections from 5 different fields of view and averaged per mouse. For analysis of vessel coverage, thresholded pictures of CD31 staining were used to measure the portion of the area occupied by vessels (area fraction measurement option). Laminin dispersion was calculated as SDi/ i in which SDi is the standard deviation of intensity and i the mean intensity of the signal. For LEF-1 expression analysis, thresholded DAPI, ERG and laminin signals were used to isolate endothelial and nonendothelial nuclei using the Particle Analysis plugin. Endothelial specific mean signal intensity of LEF-1 in nuclei was measured and normalized to the nuclear LEF-1 signal outside of the vessels after background subtraction. Quantification of Claudin-5, Mfsd2A and Caveolin-1 endothelial expressions were performed by detecting vessel structures using the Particle Analysis plugin on thresholded CD31/laminin pictures and measuring their mean respective signal intensity after background subtraction. The same procedure was used to measure GLUT1 mean endothelial intensity per surface unit and this value was multiplied by the cell area (vessel area divided by the number of nucleus) to get the total GLUT1 signal per cell. For assessing fibrinogen leakage, the detected vessel areas were scaled up by 125% before measuring fibrinogen intensity in this area. For analysis of pericyte coverage, the ratio between the area of the thresholded desmin and CD31 signals was calculated. Sulfo-NHS-Biotin leakage was quantified as the intensity of the streptavidin signal in the area around the vessel after background subtraction. This area was defined as the region corresponding to a 150% scale up of the thresholded CD31 signal from which the initial vessel area was subtracted. Only vessels whose inside streptavidin signal was bigger than the background were taken into account.
Mouse brain RNAScope in situ hybridization
Whole brains were fixed overnight with 4% PFA at 4°C before being embedded into OCT, frozen at -80°C and sliced using a cryostat (Leica) into serial coronal sections of 12 pm mounted on SuperFrost slides. Sections were submitted to RNAScope® procedure with the brown detection kit (ACD-Biotechne) according to manufacturer instructions and using axin2 probe (#400331). After post-fixation with formalin for 30 min and dehydration in ethanol, slides were baked at 60°C for 30 min using an HybEZ Oven. They were then covered with the RNAScope Hydrogen Peroxidase solution for 10 min at RT, before 5 washes in distilled water. Epitope retrieval was achieved with the RNAScope Target Retrieval Reagent for 15 min at 100°C and sections were let to dry at 60°C. Tissues were then submitted to a series of incubation at 40°C in the HybEZ Oven followed by two washes in RNAScope Wash Buffer IX: 15 min with the Protease Plus solution, 2 h with the probe, 30 min with the APM1 solution, 15 min with the APM2 solution, 30 min with the APM3 solution, 15 min with the APM4 solution. The two last amplification steps were performed at RT : 30 min with the APM5 solution and 15 min with the APM6 solution. Sections were stained with a 1 : 1 ratio of RNAScope DAB-A and DAB-B solutions for 10 min at RT and counterstained with 50% Hematoxylin for 2 min at RT before dehydration and mounting.
Mouse brain transmission electron microscopy
Mice were retro-orbitally injected with 10 mg HRP (Sigma-Aldrich) in 200 pl of PBS that was let to circulate 30 min. After cervical dislocation, brains were harvested and fixed in 2% glutaraldehyde/4%PFA in 0.1 M cacodylate buffer for 1 h at RT followed by 5 h at 4°C. Brains were embedded in 4% agarose in 0.1 M cacodylate buffer and cut in 50 pm thick sections using a vibratome agarose. DAB revelation was performed by incubating floating sections with the DAB substrate (SIGMAFAST tablets, D4293-5SET, Sigma-Aldrich) for 20 min at RT. After 2 washes in 0.1 M cacodylate buffer, samples were post-fixed in 1% OsO4/1.5% ferrocyanide, dehydrated in ethanol and embedded into epoxy-resin (Agar 100 resin, Agar Scientific Ltd, UK). Ultrathin sections (70 nm) were collected and observed using a TecnailO TEM (FEI-Thermo Fisher). Images were captured with a Veleta CCD cam.
Image acquisition and processing
Images of cells, zebrafish embryos, and mouse brain slices were collected using an inverted laser scanning confocal microscope Zeiss LSM710 equipped with spectral PMT detector using 20X/0.8 dry and 63X/1.4 oil objectives and driven by Zen Blue controlling software. H8iE and DABstained brain slices were imaged on a Leica stereoscope or on a wide-field Zeiss Axio Observer Z1 microscope. Xenopus larvae were imaged with an Olympus SZX16. We used ImageJ for image preparation (adjustments of levels and contras, maximum intensity projections and sums and removal of outliers) and Imaris software (BitPlane) for the 3D representation of brain vasculature using wire diagrams. We used CellProfiler to analyze the RNAScope images. The hematoxylinDAB stained images were deconvolved into two separate channel images (UnmixColors operation) and the objects of interest (nuclei and RNAScope spots) were separated from the background using an "A trous" wavelet filter plugin we developed in-house (available at https://github.com/zindy/libatrous). With an appropriate filter and bandpass window (Lin3x3 kernel, scales 2-5), this plugin can separate the punctiliar RNAScope marker from the residual background in the DAB deconvolved channel. Spots are then detected as objects using a Primary Object Identification (IdentifyPrimaryObjects, Global Otsu) and their area was computed (MeasureObjectSizeShape). Statistical analysis
Statistical analysis was performed using GraphPad software. Data represent mean ± SD. p-values were calculated by two-way ANOVA (fig. 20E), one-way ANOVA (with post hoc Dunnett's test, fig. IB, IE, fig. 2B, fig. 6C, fig. 9A, 9B, fig. 4B-D, fig. 19) and Student's t test (fig. IOC) for multiple and single comparisons of normally distributed data, respectively and by the Kruskal- Wallis (post hoc Dunn's test, fig. 3B-E, 3H, fig. 6B, 6E-G, fig. 15A, fig. 20A-D, fig. 25A-C) and Mann-Whitney test (fig. 4E, fig. 5A, 5C-G, fig. 6H-I) for multiple and single comparisons of non-normally distributed data, respectively. *P<0.05, **P<0.01, ***P<0.001.
Results and Discussion
Proper function of the central nervous system (CNS) requires a tightly controlled environment safeguarded from harmful blood-borne components and pathogens. This environment is maintained by the blood-brain barrier (BBB), a set of properties of CNS vascular endothelial cells (ECs). The BBB is induced and maintained by signals derived from other cells of the neurovascular unit (NVU), most notably pericytes and astrocytes. Within the healthy NVU, the endothelial monolayer limits paracellular permeability by linking adjacent cell membranes through tight junctions, represses transcellular traffic by downregulating vesicular transport, constrains immune cell trafficking, and uses ATP-fueled transporters to efflux a wide range of undesirable small molecules back into the bloodstream. This physiological barrier consequently only allows carefully selected blood constituents to enter the CNS parenchyma by engaging substrate-specific receptors and transporters at the endothelial BBB gate. BBB dysfunction, along with varying degrees of cerebrovascular hyperpermeability, neurovascular uncoupling, or blood flow dysregulation, has been linked to stroke, gliomas, epilepsy, traumatic brain injury, and neurodegenerative disorders. Upon BBB breakdown, leakage of neurotoxic plasma components, infiltration of immune cells, and CNS milieu alterations contribute to neuronal demise and worsen disease outcome. However, despite their broad therapeutic potential across a wide range of disorders, clinically-approved BBB-protective strategies are currently lacking.
Among the many pathways that control neurovascular function, endothelial Wnt/0- catenin signaling acts as a master regulator of BBB physiology in response to neural- derived Wnt7a/b ligands. Wnt/0-catenin signaling initiates the BBB differentiation cascade at the earliest steps of CNS vascular invasion, and then maintains BBB function in adults. Recent evidence suggests that inhibition of Wnt signaling by conditional deletion of 0-catenin signaling in ECs causes BBB breakdown and accelerates disease progression in stroke, glioblastoma, and multiple sclerosis in mice. Conversely, recombining a constitutively active form of 0-catenin in the CNS endothelium is protective in models of brain cancer and stroke.
Hence, identifying safe modalities to enhance Wnt/0-catenin signaling selectively at the BBB constitutes a promising therapeutic avenue for a range of neurological disorders.
As Wnt7a/b are the endogenous ligands controlling p-catenin-dependent BBB maturation, they constitute, in principle, legitimate therapeutic agents to repair the dysfunctional BBB. However, Wnt signaling exerts pleiotropic functions across a range of tissues and organs, in both health and disease. More so, the structural modalities of Wnt/Frizzled (Fz) interactions a priori disqualify natural Wnt ligands as safe therapeutics. Wnt7a/b indeed belongs to a multigenic family of 19 closely related secreted glycoproteins that fold into an unusual two-domain structure reminiscent of a human hand. The N-terminal domain (NTD, Fig. 1A) exposes a palmitoleic acid moiety at its "thumb" extension, and the C-terminal domain (CTD, Fig. 1A) "index" is composed of hydrophobic residues. Wnt ligands bind to the ten members of the Fz family of receptors by pinching the globular cysteine-rich domain (CRD) of the receptors between their "thumb" (site 1) and "index" (site 2) (pale orange areas, Fig. 1A). Notably, the interaction chemistry involves conserved residues among both the ligand's and the receptor's multigenic families.
Consequently, the 19 Wnt ligands and their 10 Fz receptors interact promiscuously, with many Wnts able to engage a single Fz receptor, and multiple Fz receptors competing for any particular Wnt ligand. Delivering Wnt7a/b ligands in vivo is thus predicted to have multiple adverse outcomes, including altered organogenesis, stem cell expansion, and tumorigenesis. Accordingly, Wnt7a overexpression is incompatible with proper vertebrate development: when expressed in Xenopus embryos, Wnt7a causes axis duplication, a classical dysmorphic outcome of exacerbated Wnt/0-catenin signaling (fig. 7A). Similarly, Wnt7a and other "canonical" Wnt ligands induce posteriorization of the anterior nervous system in zebrafish embryos, resulting in the loss of forebrain and eye structures (fig. 7B). This promiscuous signaling mode, together with the widespread Frizzled expression patterns, and the difficulty in producing active Wnt proteins at bulk levels, has contributed to hampering the clinical development of Wnt-signaling based therapies.
Interestingly, cerebral ECs cell-autonomously resort to a highly atypical Wnt7a/b- specific receptor complex to activate Wnt/p-catenin signaling for brain angiogenesis and BBB regulation. This receptor complex is composed of the GPI-anchored glycoprotein Reck and the adhesion G-protein coupled receptor Gprl24. Reck, after physically binding to the linker peptide of Wnt7a/b NTD (Fig. 1A, pink residues), stabilizes the ligand in a signaling-competent lipophilic conformation and delivers it to Fz receptors via the transmembrane tethering function of Gprl24. Thereby, Reck and Gprl24 synergistically stimulate Wnt7a/b-specific responses by assembling higher-order Gprl24/Reck/Fz/Lrp5/6 complexes.
Wnt7a/b thus activates two distinct types of membrane receptor complexes. One has broad tissue distribution and binds non-discriminately to Wnt7a/b and other Wnt ligands via two contacts sites within the Fz CRD, and a less well-known interaction with Lrp5/6 (Fig. 1A, hereafter termed systemic "off-target" complex). The other complex is enriched at the level of the BBB ECs and is highly specific for Wnt7a/b. In this case, Reck provides an additional contact point by binding at least in part to the divergent linker peptide of Wnt7a/b (Fig 1A, BBB "on-target").
Taking advantage of the differential composition of these receptor complexes, we attempted to selectively target the BBB by engineering Wnt7a/b into Gprl24/Reck- specific agonists. To that end, we first established the genetic bases of Gprl24/Reck- dependent Wnt7a/b/Fz signaling.
We compared Wnt signaling induced by 17 Wnt ligands, including Wnt7a and Wnt7b, in pairwise combination with each of the ten Fz receptors in WT, GPR124-/-;RECK- /- and Gprl24/Reck-overexpressing Wnt/p-catenin reporting super top flash (STF) HEK293 cells (fig. 8). WT and GPR124-/-;RECK-/- cells revealed a near-identical Wnt/Fz signaling landscape with multiple Wnt ligands signaling through several Fz receptors and most Fz responding to multiple Wnt ligands, as anticipated from the poorly discriminative Wnt/Fz binding mechanism. Wnt7a and Wnt7b signaled preferentially through Fz5 and Fz8 in WT and GPR124-/;RECK-/- cells (fig. 8A, B). However, as reported previously, Wnt7a/b signals were selectively and potently stimulated upon Gprl24 and Reck over-expression even in the absence of a coexpressed Fz (fig. 8C). This illustrates that the endogenous pool of Fz receptors is sufficient to support Wnt7a/b signaling in HEK cells. In order to determine which Fz isoform is competent for Gprl24/Reck-stimulated Wnt7a/b signaling, we knocked out GPR124 and RECK in previously-generated FZ1-10-/- mutant cells by CRISPR/Cas9 mutagenesis. STF-based reporter assays in this 12 loci null genetic background allowed us to unambiguously test individual components by transient re-expression. This setting revealed that, albeit at different levels, most Fzs were competent for Wnt7a and Wnt7b upon expression of Reck and Gprl24, with Fzl, 4, 5, 8, and 9 exhibiting the highest potency (Fig. 1A and fig. 9A). On a cautionary note, the relative expression level or membrane localization of different Fz receptors was not assessed. In the absence of Gprl24/Reck, Fz5 and Fz8 were confirmed as the only Wnt7a or Wnt7b signaling receptors (Fig. 1A and fig. 9B).
The near-uniform competence of Fz isoforms for Gprl24/Reck-dependent Wnt7a signaling was unexpected and reveals that Wnt7a/b loses its capacity to discriminate Fz receptors when bound by Reck within the Gprl24/Reck/Fz/Lrp5/6 complex. As Wnt ligands discriminate Fz receptors primarily via site 2, we suspected that Wnt7a/b activity at the BBB might be dominated by site 1 of the NTD. Supporting this hypothesis, Reck binds Wnt7a/b exclusively within the N-terminal domain, and chimeric Wnt ligands composed of Wnt7a NTD fused to a CTD derived from another Wnt were competent for Reck binding, which correlated with Gprl24/Reck signaling (fig. 10).
This led to the exciting possibility that, in contrast to the systemic "off-target" situation where both contact sites 1 and 2 are strictly required for Wnt signaling, Wnt7-Fz interaction at site 2 might become dispensable in the context of Gprl24/Reck-stimulated signaling. In line with this prediction, a hemisected Wnt7a variant, Wnt7aNTD, composed of the sole NTD domain, retained partial activity on Gprl24/Reck-mediated signaling while showing no stimulation of Fz5 signaling, used as a paradigm "off-target" readout (Fig. IB). Furthermore, substituting three of the five Wnt7a residues involved in site 2 contacts into alanines did not affect Gprl24/Reck-mediated signaling, while reducing Fz5 signaling by more than 50% (fig. 11). Both the reciprocal single-domain CTD variant and Wnt7aS206A with impaired site 1 were fully inactive. Assuming comparable expression and secretion levels, site 1- and 2-inactive variants likely result from improper folding, inability to interact with Fz, or both. Together, these findings reveal that when presented by appropriate co-receptors, Wnt ligands can trigger Fz signaling even in the absence of the hydrophobic contacts of site 2. The signaling properties of Wnt7aNTD further demonstrated that altering the structure of Wnt ligands can modulate their signaling specificity, and more specifically, that Wnt7a can be engineered into a Gprl24/Reckspecific ligand.
Based on this proof of concept, we implemented a large-scale screen for Wnt7a variants with increased "on-target" activity, as Wnt7aNTD is only ~30% active compared to the parental Wnt7a ligand. To that end, we generated a collection of 147 single-residue variants of murine Wnt7a, in which surface-exposed charged or hydrophobic residues were mutated into alanine (or into arginine for alanine residues). This collection, the most comprehensive of any Wnt ligand, corresponds to 46% of the residues of the secreted protein, and 51% of its exposed surface. Each variant was individually tested for its Gprl24/Reck-dependent and -independent activity (Fig. 1C, D, fig. 12A). Wnt7a appeared highly sensitive to mutations when assessed on Fz5, with 37% of variants exhibiting <10% activity (red residues), and only 31% maintaining >70% activity (green residues). Unexpectedly, the large majority of critical residues are not located in the Fz binding sites. Hyperactive variants were also readily uncovered, with 5% of variants displaying over 2-fold higher Fz5 signaling activities than the natural ligand (e.g. K255A, K95A, K296A, K273A). This illustrates the evolution of Wnt ligand morphogens within carefully selected, submaximal activity windows. Although a systematic evaluation of ligand expression and secretion was not attempted, we assessed the secretion of all 17 variants inactive on both Fz5 and Gprl24/Reck. By virtue of their complete inactivity, these variants constitute preferential candidates for mis-trafficking or misexpression and, therefore, probably overestimate the secretion failure rate. Yet, 88% (15/17) showed similar levels of extracellular accumulation as Wnt7a, ruling out improper trafficking as a leading cause for the large proportion of Fz5-inactive variants (fig. 13). Accordingly, the majority of Fz5-inactive and overactive variants displayed unaltered Gprl24/Reck-dependent signaling. Indeed, in sharp contrast to Fz5, Gprl24/Reck signaling appeared homogenous and largely insensitive to Wnt7a variations, as 76% of the variants retained >70% activity (Fig. 1C, D and fig. 12A).
Notably, the enhanced Wnt7a signaling robustness associated with the presence of the BBB specific co-receptors resulted in the identification of 25 single-residue variants displaying highly specific Gprl24/Reck activity (so-called "agonists" >70% on-target, <10% off-target), scattered over the entire Wnt7a structure (Fig. ID residues highlighted in blue, fig. 12B). Reciprocal Fz5-selective variants were not found. Among these single-residue Gprl24/Reck agonists, we selected the Wnt7aK190A variant as a prototype, optimally combining a WT-like signal on Gprl24/Reck, and no "off-target" Fz5 activity (Fig. 1C, fig. 12B). It should however be clear to a skilled person that also the other agonists listed herein behave similar to the Wnt7aK190A. All data obtained on Wnt7aK190A is confirmed for the other 24 single-residue variants.
In vitro, none of the transfected Fz receptors could be stimulated by Wnt7aNTD or Wnt7aK190A in the absence of Gprl24/Reck, an observation compatible with the strict selectivity of agonists (Fig. IE). In vivo, in sharp contrast to Wnt7a, ubiquitous expression of Wnt7aNTD or Wnt7aK190A after mRNA injections failed to trigger ectopic Wnt activation during Xenopus (Fig. IF), or zebrafish (Fig. 1G) gastrulation, as assessed by the absence of posteriorizing or gross morphological alterations. Although the expression level and secretion rate of Wnt7aNTD or Wnt7aK190A were not formally compared to Wnt7a in vivo, the agonists displayed "on-target" biological activities in zebrafish, implying that the lack of morphological defects is not a trivial consequence from a complete lack of activity (see below). Dose-response analyses following mRNA injections in the zebrafish zygote revealed that 3 pg of Wnt7a mRNA was sufficient to trigger "off-target" signaling, while Wnt7aNTD and Wnt7aK190A were remarkably well tolerated even when expressed in a 100-fold excess (Fig. 1G). Of note, Wnt7a signaling in this setting is Gprl24-independent, as revealed by the analysis of zygotic and maternal-zygotic gprl24s984 mutants (fig. 7C). The morphological alterations induced, selectively, by Wnt7a mRNA injections may result either from a mere quantitative increase in Wnt signaling, as detected in cell culture using universal TOP-flash reporters, or from a qualitative difference between Gprl24/Reck dependent and -independent signaling. To resolve this, we artificially expanded the expression domain of Gprl24/Reck by mRNA injections in zebrafish embryos. Under these conditions, both Wnt7a or Wnt7aK190A mRNA injections triggered morphological alterations at low doses (fig. 14). The developmental innocuity of Wnt7aK190A thus seemingly results predominantly, although we cannot say exclusively, from the restricted expression pattern of Gprl24/Reck.
Mechanistically, the selectivity of the uncovered agonists results from their incapacity to bind, and therefore activate, Fz receptors in the absence of Gprl24/Reck. Indeed, while Wnt7a immunolocalized to the surface of RECK-/- ;GPR124-/- cells transiently transfected with Reck or Fz5, Wnt7aNTD and Wnt7aK190A labeled the membrane in the presence of Reck, but not Fz5 (Fig. 2A, B). Accordingly, the agonists bound HA-tagged Reck as efficiently as Wnt7a in antiHA co-immunoprecipitation experiments (Fig. 2C) but lost the capacity to bind HA-Fz5 in RECK-/- cells (Fig. 2D). However, Fz5 binding was restored upon co-expression of Reck (Fig. 2D). These results imply either (i) that Reck binding influences the inherent structural modalities of Wnt/Fz interaction, as exemplified by the activity of the single-domain Wnt7aNTD lacking an essential contact site for autonomous Wnt/Fz signaling, or (ii) that Reck is required to keep Wnt7a ligands in an active configuration. Wnt ligands are indeed short-lived and rapidly lose activity by oligomerization, in a process that increases their hydrosolubility. Reck has been shown to extend Wnt7a activity half-life by maintaining it in a monomeric, hydrophobic state. The widespread distribution of the 25 Gprl24/Reck agonistic mutations over Wnt7a structure, their exposed position and the nature of introduced mutations make it unlikely that the mutations intrinsically affect the interaction chemistry with Fz.
It is thus possible that the introduction of single-residue variations into Wnt7a reduces ligand stability below a threshold required for Fz signaling, a property that the stabilizing action of Reck could counteract. In agreement, when assessed across the 147 Wnt7a variants, a clear correlation was seen at the single-residue level between Fz5 and Gprl24/Reck/Fzl activities. Variants displaying even slightly reduced activity on Gprl24/Reck (yellow, orange, or red residues in Fig. 1C, D) were generally fully inactive in the more sensitive Fz5 setting (Fig. 2E). Conversely, preservation of at least partial Fz5 activity (orange, yellow, or green residues in Fig. 1C, D) strongly correlated with full activity on Gprl24/Reck (Fig. 2E). In summary, the collection of variants seems to give a similar structure-function picture in both Wnt7a receptor settings, albeit at a dramatically different level of expressivity.
As stability of morphogens influences their range of action, with more stable ligands capable of activating more distant cells, we analyzed the capacity of Wnt7a and Wnt7aK190A to activate Wnt signaling at short distances (autocrine mono-cultures) versus longer distances (paracrine co-cultures) (Fig. 2F). While Wnt7a retained partial paracrine activity, Wnt7aK190A could not reach the receiving cell in an active form. Of note, the whole collection of agonists behaved similarly in this assay (fig. 15).
Together these results indicate that achieving Gprl24/Reck selective agonism relies not only on the ligand's intrinsic properties, but also on the spatial distribution of Wnt7a/b-releasing cells and Gprl24/Reck-positive receiving cells. Consequently, the structural window for agonist engineering is probably narrow, with even subtle mutations at risk of being detrimental for selectivity. In agreement, combining agonistic mutations invariably resulted in fully inactive ligands, even when tested on Gprl24/Reck signaling in mono-cultures (Fig. 2G). Conversely, introducing K95A, K255A, K273A, and K296A mutations into Wnt7aK190A (all of which increase Fz5 activity, Fig. 1C) was sufficient to abolish selectivity by restoring partial Fz5 signaling (Fig. 2H). Finally, mutating K190 to alternative residues (G, S, L, P, D, E, or R) had variable effects on signaling specificity. Mutations to small or hydrophobic residues (A, S, or L) conferred selectivity towards Gprl24/Reck. In contrast, mutations to helix breakers (G or P) or residues of opposed charges (D or E) abrogated activity, and the charge-preserving K to R substitution reduced Fz5 activity by over 50% (Fig. 21). In sum, by recruiting Wnt7a/b into a higher-order receptor complex, Gprl24/Reck extends Wnt7a/b signaling robustness.
To test the functionality of the agonists in vivo, we generated a Wnt7a-deficiency model in zebrafish. Although the Gprl24/Reck complex was first discovered in zebrafish by its essential role in cerebrovascular and dorsal root ganglia development, the exact nature of the Wnt7a/b ligand isoform implicated in these processes has not been explored. During mouse embryogenesis, Wnt7a and Wnt7b cooperate to control brain angiogenesis and BBB formation. Wnt7a and Wnt7b are duplicated in the zebrafish genome (wnt7aa, wnt7ab, wnt7ba, and wnt7bb), and we used morpholino-mediated gene knock-down to identify the BBB-relevant ligand(s). Only wnt7aa morphants showed the anticipated phenotypes (fig. 16A) and, therefore, we generated a wnt7aa frame-shift allele (wnt7aaulb2) through CRISPR/Cas9 mutagenesis (Fig. 3A). Homozygous wnt7aaulb2 mutants (wnt7aa-/-) displayed fully penetrant cerebrovascular defects in the 60 hours post fertilization (hpf) hindbrain (Fig. 3B), associated with down-regulated Tg(7xTCF-Xla.Siam:GFP) Wnt/0-catenin reporter expression in CNS-invading vessels (Fig. 3C). The development of ngnl :GFP-positive DRG neurons was also compromised (Fig. 3B), consistent with wnt7aa expression in the zebrafish brain and trunk region (fig. 16B). In cell cultures, all tested human, mouse, and zebrafish orthologues of the Wnt7/Gprl24/Reck signaling module were functionally interchangeable (fig. 17), authorizing the functional assessment of murine ligands in zebrafish.
The Gprl24/Reck-dependent DRG neurogenesis defects resulting from wnt7aa deficiency could be partially corrected by injecting 100 pg of Wnt7aNTD or Wnt7aK190A mRNA at the one-cell stage. In contrast, the parental Wnt7a could not restore DRG development as it triggered severe morphological defects (Fig. 1G and 3D). These results not only demonstrate that these agonists can stimulate Gprl24/Reck activity in vivo, but also that they outperform Wnt7a by the lack of off- target effects (Fig. 3D). However, Wnt7aa-/- cerebrovascular phenotypes could not be rescued by transient mRNA expression, presumably due to the late onset of angiogenic sprouting in zebrafish hindbrain (36 hpf). Therefore, to test if Wnt7aNTD and Wnt7aK190A can also function as bona fide BBB instructive ligands, we assessed their activity by mosaic transgenic endothelial expression, using a kdrl (vegfr2) promoter. In this approach, Wnt7aK190A restored the formation of central arteries (CtAs) in wnt7aa-/- embryos to a level comparable to Wnt7a, and these vessels expressed the glucose transporter-1 (Glutl), a marker of BBB maturation (Fig. 3E). Consistent with its partial activity in vitro, Wnt7aNTD was also competent for Gprl24/Reck-dependent brain angiogenesis and barriergenesis, although less potently than Wnt7aK190A. This difference is compatible with a more limited intrinsic signaling potential (Fig. IE), but we cannot exclude reduced protein expression or processing in vivo.
These results, demonstrating the capacity of the agonists (in particular Wnt7aK190A) to stimulate Wnt signaling at the BBB, led us to test their protective potential in disease models. In zebrafish larvae, pharmacological exposure to atorvastatin (ATV) is used as a non-invasive hemorrhagic stroke model, reminiscent of human cerebral cavernous malformation (CCM)-like lesions. While over 90% of ATV-exposed WT larvae displayed moderate to severe intracranial hemorrhages, transgenic endothelial expression of Wnt7aK190A profoundly decreased the extent of cerebrovascular ruptures, with over half of the injected embryos showing no or little intracerebral bleeding (Fig. 3F). ATV treatment also resulted in the accumulation of intracardially injected 10 kDa Dextran into the hindbrain (Fig. 3G). To test whether Wnt7aK190A expression could counteract these more subtle ATV- induced BBB defects, we examined Dextran-injected embryos with hemispheric transgenic endothelial expression of Wnt7aK190A, allowing to contrast CNS leakage among the two hemispheres of a single animal. Wnt7aK190A expression was sufficient to reduce the leakage to control levels (Fig. 3H).
Altogether, these results in zebrafish demonstrate the efficacy of the uncovered Gprl24/Reck agonists in vivo and led us to test their potential as BBB-protective agents in mammals. In order to deliver the agonists to the mouse CNS, we used the engineered AAV-PHP.eB capsids of adeno-associated viruses, reported to transduce over 55% of cortical and striatal neurons, as well as astrocytes and endothelial cells after a single peripheral intravenous injection. We generated AAV-PHP.eB viruses expressing either EGFP alone (AAV-EGFP), Wnt7a-P2AEGFP (AAV-Wnt7a), or Wnt7aK190A-P2A-EGFP (AAV-K190A) under the control of the constitutive CAG promoter. We validated that the Wnt7a fusions to EGFP via the cleavable P2A are active in vitro, and that upon intravenous injection of 4.1011 vg (viral genomes) in 8week old mice, CD31-positive brain vessels are surrounded by EGFP+ cells (Fig. 4A), exposing endothelial cells to local sources of Wnt7a or Wnt7aK190A. We confirmed that the AAV-PHP.eB capsid drives expression of the transgenes in ~25% of cortical and striatal ECs, in ~30% of astrocytes, and in ~45% of NeuN-i- neurons (fig. 18A). Over 95% of the examined Desmin-i- pericytes were negative for EGFP. EGFP+/Desmin+ double positive cells were seemingly detected at rare occasions, although we could not exclude endothelial contributions. Pericytes are thus, at best, marginal sources of Wnt7a in this approach (fig. 18B).
Notably, although expressed from uniformly distributed cells, Wnt7a and Wnt7aK190A showed a discrete distribution, with preferential accumulation at the level of CD31-positive brain vessels, as well as some scattered parenchymal cells (Fig. 4B, asterisks). The distribution, and hence potential activity of the ligands, likely reflects the expression patterns of their receptors, with unbound ligands presumably getting cleared through the glymphatic system. Accordingly, Wnt7a, with its broader receptor repertoire, appeared to exhibit a more significant non- vascular distribution (Fig. 4B, asterisks). More so, immunodetection of 0-catenin activity (LacZ signal) in coronal brain sections of BAT-GAL mice revealed that, in contrast to Wnt7aK190A, Wnt7a triggered ectopic Wnt/0-catenin signaling in non- endothelial cells of the hippocampus and the parafascicular nucleus of the thalamus at all examined timepoints (7, 14, and 28 dpi) (Fig. 4C). These two areas are associated with high Fz5 expression. Other brain regions did not exhibit increased LacZ signal, probably reflecting the restricted expression of Fz5 and Fz8 in the adult CNS (fig. 19). Wnt transgenic reporter systems, like BAT-GAL mice, using an artificial reporter construct with concatemerized TCF/LEF sites, do not always reliably report in vivo Wnt activities due to random variegation in transgene expression.
To verify the Wnt activity patterns revealed by the BAT-GAL model, we performed RNAScope hybridization of Axin2 mRNA, possibly the transcript most reliably correlated with Wnt activity across tissues. This approach confirmed the ectopic activation of Wnt signaling induced by Wnt7a, but not Wnt7aK190A, at both 14 dpi (Fig. 4D) and 32 dpi (fig. 20) in the hippocampus and the parafascicular nucleus of the thalamus. Gene delivery of Wnt7a, but not Wnt7aK190A, was detrimental to neonatal mouse development as revealed by impaired spontaneous locomotor activity (Fig. 4E). We next focused on the Wnt activity status at the target BBB endothelium. Endothelial Wnt activity is low in adult mice and could only rarely be detected in BAT-GAL reporter mice, irrespective of Wnt7a or Wnt7aK190A expression (fig. 19). Therefore, we stained for Lymphoid Enhancer Binding Factor 1 (LEF1), a key mediator of Wnt/0-catenin signaling, which robustly labeled ~80% endothelial cells throughout the different brain regions. AAV-based delivery of Wnt7a or Wnt7aK190A did not increase LEF1 signals in ECs (fig. 21).
The absence of "off-target" Wnt signaling activity after widespread gene delivery of Wnt7aK190A in the mouse brain prompted us to test the therapeutic potential of the Gprl24/Reck agonist in CNS pathologies associated with BBB dysfunction, starting with grade IV astrocytoma or glioblastoma multiforme (GBM). GBM, the most aggressive and frequent primary brain tumor, is characterized by a dense vascular network exhibiting disrupted BBB properties. Interestingly, endothelial Wnt signaling was reported to control vascular integrity in different brain tumor models. This led us to evaluate the impact of Gprl24/Reck-specific activators on GBM tumor growth, vascularization, and BBB integrity. Two days after orthotopic implantation of 1.105 GL261 tumor cells into the C57BL/6 mouse striatum, we performed a single "hit- and-run" intravenous AAV-PHP.eB gene delivery and monitored tumor volume by MRI at 21-24 days post-implantation (dpi) (Fig. 5A). While control tumor expansion was highly variable, with volumes ranging from 10 to 80 mm3, Wnt7aK190A expression (AAV-K190A) reduced this variability and restricted tumor volume to an average value of 20 mm3. In particular, the proportion of larger tumors (> 40 mm3) was reduced upon Wnt7aK190A gene delivery (8% in K190A cohorts versus 33% in controls). At 25 dpi, mice with the largest tumors started to exhibit disease symptoms, including faulty postural syndromes and abnormal gait. This timepoint was therefore chosen for terminal analysis and tissue harvesting. Control tumors showed more prominent intratumoral microvascular hemorrhages (asterisks) and edema than AAV-K190A tumors (Fig. 5B), suggesting cerebrovascular differences between the groups. Accordingly, the tumor-associated vasculature showed features of vessel normalization, i.e. reduced CD31+ vascular density (Fig. 5C), reduced vessel diameter (Fig. 5C), and more compact and seemingly smoother distribution of laminin (Fig. 5D). Cell densities were comparable in AAV-EGFP and AAV-K190A tumors (6,425 versus 7,141 cells. mm-2, respectively). This implies that the total number of cells is smaller in AAV-K190A tumors, most likely as a consequence of reduced tumor cell proliferation. The modest differential cellular densities reflect, however, a contribution of vasogenic edema to the larger volumes of AAV-EGFP tumors. Assessment of 0-catenin transcriptional response by immunodetection of nuclear LEF-1 confirmed previous reports of reduced endothelial Wnt signaling in GBM vessels (Fig. 5E). Wnt7aK190A successfully restored Wnt activity in the tumor endothelium, reaching a level remarkably similar to the steady-state activity of non- tumoral parenchymal vessels. In contrast, it did not significantly affect LEF1 levels in vessels of the contralateral hemisphere (Fig. 5E), confirming the findings in healthy mice (fig. 21). We postulate that homeostatic feedback regulations poise endothelial Wnt activity levels at a physiological setpoint value, insensitive to ectopic external stimuli. This observation could explain the lack of detectable phenotypes in AAV-K190A mice, with Wnt activity increased selectively at the dysfunctional BBB, leaving the healthy brain unaltered. In agreement with LEF-1 nuclear accumulation, we observed re-establishment of BBB integrity in tumor vessels, as assessed by increased GLUT1 immunostaining (Fig. 5F) and reduced endogenous fibrinogen extravasation (Fig. 5G). Interestingly, the restoration of GLUT1 signal in the tumor vasculature of AAV-K190A mice was linked to reduced tumor parenchymal GLUT1 signal, paralleling the developmental switch of GLUT1 expression from the neuroepithelium to the vessels upon CNS vascularization.
To determine the source of Wnt7aK190A within the tumor microenvironment, we analyzed the distribution of the EGFP transduction marker on coronal tumor sections. Glioblastoma cells were negative, as expected from the non-replicate nature of AAV genomes, and the numerous cell divisions of these cells upon implantation (fig. 22). Within the tumor, 30% of CD31+ endothelium was EGFP+, and blood vessels accounted for ~60% of the total intratumoral EGFP+ signal (fig. 22). In addition, the EGFP signals were particularly intense in S1OO0+/GFAP- astrocytes of the tumor glial scar. Ibal+ microglial cells were EGFP- (fig. 22). Together, if we assume that EGFP is a reliable indicator for Wnt7a expression, the seemingly most prominent sources for Wnt7aK190 are the ECs themselves and the peritumoral reactive astrocytes.
Combined with the intrinsic variability associated with in vivo delivery of AAV particles, these relatively discrete Wnt7aK190A sources resulted in surprisingly uniform effects on tumor growth (Fig. 5A). Even more so, in view of the considerable heterogeneity associated with control tumors (Fig. 5A). Given that (i) GBM tumor vessels derive by vessel co-option or angiogenesis from pre-existing Wnt-positive parenchymal vessels, (ii) endothelial Wnt activity levels drop in the GL261 vasculature, and (iii) endothelial Wnt signaling slows down GL261 tumor progression, we reasoned that the heterogeneity of GL261 tumor growth could result from varying degrees of residual endothelial Wnt signaling levels. Supporting this hypothesis, endothelial LEF1 and GLUT1 levels were somewhat heterogeneous in control tumors (Fig. 5E, F). Strikingly, both markers are inversely correlated with vessel density, fibrinogen leakage, and tumor volume (Fig. 5H). These correlations suggest that the growth rate of GL261 tumors is at least partially determined by the level of residual Wnt signaling of its perfusing vasculature, and that AAV-delivery of Wnt7akl90A is sufficient to uniformly raise the signaling level to promote neurovascular normalization and tumor growth reduction (Fig. 51).
The variable degree of residual endothelial Wnt signaling within the WT GL261 tumor endothelium complicated the analysis of the mechanism underlying endothelial Wnt- induced BBB repair. Therefore, we resorted to transgenic Tet-Off GL261 cells that conditionally express the secreted Wnt inhibitor Dkkl (dickkopf WNT signaling pathway inhibitor 1). When exposed to doxycycline, these cells potently repress Dkkl expression in vitro and in vivo, without affecting their intrinsic in vitro growth rate (fig. 23). Upon implantation, the characteristics of Dkkl+ (-dox) GL261 tumors (Wnt inhibition) were compared with Dkkl- (+dox) tumors of mice injected with AAV-EGFP (control) or AAV-K190A (Wnt activation). As expected, Dkkl- tumors grew and responded to AAV-K190A as WT GL261 cells, with 95% of the AAV-K190A- treated tumors smaller than 40 mm3 at 20-24 dpi and the control cohort exhibiting more variable volumes (40-80 mm3) (Fig. 6A). Dkkl-i- tumors grew even bigger, up to 160 mm3 (Fig 6A). Endothelial Wnt activity markers (LEF1 and GLUT1) were the highest in the Wnt stimulated Dkkl-/K190A tumors and the lowest in Wnt inhibitory Dkkl-i- tumors, with Dkkl-/EGFP tumors showing intermediate values (fig. 24A, 24B). As anticipated, hemorrhage (fig. 25), vascular density (fig. 24C), and fibrinogen leakage (Fig. 6B) followed the opposite trend, being gradually reduced by the step-wise increase in endothelial Wnt signaling.
The restoration of BBB impermeability to fibrinogen (350 kDa) demonstrates that Wnt7aK190A can prevent BBB permeability to large molecules. In contrast, small molecule 557 Da sulfoNHS-Biotin leaked within the tumor parenchyma of all examined tumors, irrespective of their endothelial Wnt activation level. Of note however, Dkkl-i- tumors exhibited slightly higher leakage values (Fig. 6C). To define the mechanism of K190A-induced BBB repair to proteinsized tracers, we examined tumor and cortical vessels by electron-microscopy following intravenous injection of 44 kDa horseradish peroxidase (HRP) (Fig. 6D). In the healthy mouse cerebral cortices, blood-borne peroxidase was detected by the electron-dense 3-3' diaminobenzidine (DAB) reaction product that only penetrated the intercellular spaces of adjacent endothelial cells over small distances. The signal sharply stopped at presumptive tight junctions (arrowheads), as typically reported in healthy BBB vessels. As expected, the electron density of the endothelial basement membranes (black asterisks) was consequently much lower as compared to the lumen (white asterisks), consistent with luminal HRP retention. This spatially restricted distribution of HRP contrasted sharply with the signal observed along the entire length of the intercellular spaces of Dkkl-i- and Dkkl-/EGFP tumor vessels. In these dysfunctional vessels, no difference was found between HRP levels within blood vessel lumens and their basement membranes. However, mice injected with AAV-K190A exhibited tumor vessels that reverted to a WT phenotype, with a clear boundary at the level of the tight junction kissing points (arrowheads, Fig. 6D). These findings are compatible with the hypothesis that AAV-K190A partially corrects the tight-junctional defects of glioblastoma vessels. In line with the electron micrographs, Claudin-5 immunostaining appeared denser in AAV-K190A tumor vessels, compatible with a direct role of Wnt7akl90A on endothelial tight junctions (Fig. 6E).
The significant intra-tumoral leak of HRP resulted in modest electron-dense contrast, making unambiguous scoring of transcytosis vesicles in glioblastoma vessels impractical. Mfsd2a, an endothelium-specific inhibitor of caveolae-mediated transcytosis, was, however, increased by endothelial Wnt activation (Fig. 6F), as previously reported at the blood-retinal barrier. Accordingly, Caveolin-1 levels were lowered by AAV-K190A (fig. 26). The pericytes loss, typically associated with glioblastoma and upregulated transcytosis, could also be partially counteracted by AAV-K190A (Fig. 6G). Altogether, in GL261 tumors, Wnt7aK190A restores endothelial Wnt signaling, reduces vascular density and normalizes the BBB pleiotropically, affecting both the transcellular and paracellular permeability routes, thereby slowing tumor progression.
Discussion
Altogether, we discovered that Wnt7a ligands can be engineered into highly-specific Gprl24/Reck agonists, thereby disclosing a novel class of BBB therapeutics. This level of specificity was deemed unreachable for Wnt-derived proteins by virtue of their promiscuous interaction mode with widely expressed Fz receptors.
In contrast to Wnt7a, the uncovered Gprl24/Reck agonists were remarkably well tolerated in vivo despite the broad expression strategies adopted in this study. In Xenopus and zebrafish, their ubiquitous expression during the Wnt-sensitive steps of cleavage, gastrulation, somitogenesis, and early organogenesis failed to yield detectable morphological alterations, even when expressed in a 100-fold excess to the highest developmentally tolerated dose of the parental Wnt7a. In mice, CNS- wide expression of Gprl24/Reck agonists via AAV-PHP.eB gene delivery did not trigger ectopic Wnt activation. Accordingly, injected mice did not show any detectable adverse phenotype, even after several months. In addition to the strict signaling specificity of the Gprl24/Reck agonists, we suspect that homeostatic feedback loops maintain Wnt signaling activities within carefully controlled physiological activity windows. Accordingly, we detected significant reinforcement of endothelial Wnt signaling only in dysfunctional glioma vessels, while the signaling levels of healthy parenchyma vessels remained unaffected. The Gprl24/Reck agonists described in this study constitute BBB therapeutic molecules, in particular for use in the treatment of glioblastoma. In glioblastoma, intra-tumoral BBB leakage leads to morbid vasogenic brain edema. GBM is therefore well-positioned for targeted vascular therapies, and systemic administration of bevacizumab, an anti-VEGF antibody, is used as second-line treatment for recurrent or non-responsive glioblastomas.
Finally, our findings also exemplify that the BBB-inductive Wnt7a signals can override multiple deleterious effects of the BBB-disruptive agents typically associated with glioblastoma, most notably angiogenic growth and permeability factors like VEGF, pro-inflammatory cytokines and reactive oxygen species.
In summary, we have defined a novel modality to treat CNS disorders and more particular glioblastoma by healing the BBB. It is supposed that the present invention is not restricted to any form of realization described previously and that some modifications can be added to the presented example of fabrication without reappraisal of the appended claims.

Claims

1. A pharmaceutical composition comprising a therapeutically active amount of a Wnt 7 polypeptide or fragment thereof, or a nucleic acid encoding for said Wnt7 polypeptide or fragment thereof, wherein said Wnt7 polypeptide or fragment thereof is capable of activating G-protein coupled receptor (GPR)124/RECK/Frizzled/lipoprotein receptor- related protein (LRP)-mediated Wnt signaling, and does not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124, for use in the reduction in progression and/or treatment of glioblastoma in a subject, said Wnt7 polypeptide or fragment thereof is selected from a Wnt7 polypeptide or fragment wherein
- the glutamine (Q) residue at the position corresponding to position 17 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than glutamine (Q), preferably by an alanine (A) residue;
- the isoleucine (1) residue at the position corresponding to position 20 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than isoleucine (1), preferably by an alanine (A) residue;
- the proline (P) residue at the position corresponding to position 25 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than proline (P), preferably by an alanine (A) residue;
- the alanine (A) residue at the position corresponding to position 27 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than alanine (A), preferably by an arginine (R) residue;
- the isoleucine (I) residue at the position corresponding to position 28 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue;
- the glutamate (E) residue at the position corresponding to position 33 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue;
- the methionine (M) residue at the position corresponding to position 37 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than methionine (M), preferably by an alanine (A) residue;
- the leucine (L) residue at the position corresponding to position 39 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than leucine (L), preferably by an alanine (A) residue; - the glutamate (E) residue at the position corresponding to position 41 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue;
- the phenylalanine (F) residue at the position corresponding to position 44 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than phenylalanine (F), preferably by an alanine (A) residue;
- the arginine (R) residue at the position corresponding to position 50 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue;
- the asparagine (N) residue at the position corresponding to position 52 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than asparagine (N), preferably by a glutamine (Q) residue;
- the valine (V) residue at the position corresponding to position 68 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than valine (V), preferably by an alanine (A) residue;
- the isoleucine (I) residue at the position corresponding to position 129 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue;
- the phenylalanine (F) residue at the position corresponding to position 131 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than phenylalanine (F), preferably by an alanine (A) residue;
- the lysine (K) residue at the position corresponding to position 133 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;
- the phenylalanine (F) residue at the position corresponding to position 135 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than phenylalanine (F), preferably by an alanine (A) residue;
- the isoleucine (I) residue at the position corresponding to position 141 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue;
- the arginine (R) residue at the position corresponding to position 146 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue;
- the arginine (R) residue at the position corresponding to position 158 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue; 106
- the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an acid residue other than lysine (K), preferably by an alanine (A), serine (S) or leucine (L) residue;
- the lysine (K) residue at the position corresponding to position 181 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;
- the arginine (R) residue at the position corresponding to position 191 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue;
- the lysine (K) residue at the position corresponding to position 198 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;
- the lysine (K) residue at the position corresponding to position 200 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;
- the valine (V) residue at the position corresponding to position 205 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than valine (V), preferably by an alanine (A) residue;
- the glutamate (E) residue at the position corresponding to position 208 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue;
- the arginine (R) residue at the position corresponding to position 214 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue;
- the lysine (K) residue at the position corresponding to position 216 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;
- the proline (P) residue at the position corresponding to position 218 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than proline (P), preferably by an alanine (A) residue;
- the lysine (K) residue at the position corresponding to position 222 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue;
- the isoleucine (I) residue at the position corresponding to position 223 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than isoleucine (I), preferably by an alanine (A) residue; 107
- the tyrosine (Y) residue at the position corresponding to position 229 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than tyrosine (Y), preferably by an alanine (A) residue;
- the proline (P) residue at the position corresponding to position 232 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than proline (P), preferably by an alanine (A) residue;
- the threonine (T) residue at the position corresponding to position 235 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than threonine (T), preferably by an alanine (A) residue;
- the glutamate (E) residue at the position corresponding to position 248 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than glutamate (E), preferably by an alanine (A) residue;
- the arginine (R) residue at the position corresponding to position 289 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than arginine (R), preferably by an alanine (A) residue;
- the tryptophan (W) residue at the position corresponding to position 291 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than tryptophan (W), preferably by an alanine (A) residue;
- the threonine (T) residue at the position corresponding to position 307 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than threonine (T), preferably by an alanine (A) residue; and/or
- the lysine (K) residue at the position corresponding to position 318 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an amino acid residue other than lysine (K), preferably by an alanine (A) residue.
2. Pharmaceutical composition for use according to any of the previous claims, wherein said compound is a Wnt7 polypeptide or a fragment thereof wherein the lysine (K) residue at the position corresponding to position 159 in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted by an alanine (A) residue, an serine (S) residue or a leucine (L) residue, more preferably by an alanine (A) residue; or wherein said compound is a nucleic acid encoding for such polypeptide.
3. Pharmaceutical composition for use according to any of the previous claims wherein said compound is able to influence the permeability of the bloodbrain barrier (BBB) in subjects having glioblastoma or prone to develop glioblastoma.
4. Pharmaceutical composition for use according to any of the previous claims, wherein said compound is able to repair tight-junctional defects in glioblastoma vessels. 108 Pharmaceutical composition for use according to any of the previous claims, wherein said compound is able to reduce vesicular transport in glioblastoma vessels. Pharmaceutical composition for use according to any of the previous claims, wherein said compound is able to control tumor angiogenesis in a subject suffering from glioblastoma. Pharmaceutical composition for use according to any of the previous claims wherein said subject is human or an animal. Pharmaceutical composition for use according to any of the previous claims, wherein said compound is a nucleic acid, preferably RIMA or DNA. Pharmaceutical composition according to any of the previous claims, wherein said compound is provided in a liposome or lipid nanoparticle. Pharmaceutical composition for use according to any of the previous claims, wherein said compound is formulated in a viral vector. Pharmaceutical composition for use according to any of claims 1 to 10, wherein said compound is administered at a dosage of between 10 pg/kg to about 500 pg/kg body weight of the subject. Pharmaceutical composition for use according to any of the previous claims, wherein said composition is combined with a second therapy, preferably chosen from surgery, chemotherapy, radiotherapy or immunotherapy. Pharmaceutical composition for use according to any of the previous claims, wherein said composition is administered parenterally, preferably intravenously or intrathecally.
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