Engineered wnt7a proteins and uses thereof

Abstract

The disclosure discloses an engineered Wnt7a protein, which introduces a specific single amino acid at the C-terminal of the Wnt7a protein. The disclosure overcomes the technical defect that wild-type Wnt7a lacks receptor specificity, improves the accuracy of signal pathway activation, reduces the risk of non-targeting effect, and provides a safer and more effective potential tool for the treatment of cerebrovascular diseases or blood-brain barrier damage.

Description

Technical Field

[0001] This disclosure relates to an engineered Wnt7a protein and its applications, which can specifically activate the RECK-Gpr124-Fzd-mediated classical Wnt signaling pathway, which cannot be activated when RECK and Gpr124 are absent. Background Technology

[0002] The Wnt signaling pathway plays a crucial role in many aspects, including embryonic development, organogenesis, and maintaining adult homeostasis. Nineteen Wnt ligands and ten Fzd receptors have been identified in the mammalian genome. The widespread distribution and diverse combinations of Wnt ligands and Fzd receptors, as well as the emergence of Wnt co-receptors, allow for the activation of different Wnt signaling pathways under varying tissue and physiological conditions.

[0003] Wnt7a and Wnt7b are essential for angiogenesis in the central nervous system. When the Wnt7a and Wnt7b genes are double-knocked out of mouse embryos, all double-knockout mutant mouse embryos die and exhibit severe central nervous system-specific hemorrhage phenotypes and neural tissue disorders (Stenman et al., Science, 2008). A 2018 study experimentally verified that Wnt7a and Wnt7b stimulate vascular budding in mouse choroidal explants (Lin et al., Exp EyeRes, 2018). In summary, Wnt7a and Wnt7b play important roles in inducing central nervous system cell differentiation and the formation and maintenance of blood-brain block (BBB) ​​by activating the canonical Wnt signaling pathway.

[0004] RECK and Gpr124 are co-receptors of Wnt7a and Wnt7b. When Wnt7a and Wnt7b specifically activate the RECK-Gpr124-Fzd-mediated canonical Wnt signaling pathway, they can promote angiogenesis and the formation and maintenance of the blood-brain barrier in the central nervous system (Vallon et al., Cell Reports, 2018; Eubelen et al., Science, 2018).

[0005] Public content

[0006] The technical problem this disclosure aims to solve is that wild-type Wnt7a protein lacks receptor specificity when activating signaling pathways, causing it to activate the Wnt signaling pathway in the presence or absence of RECK and Gpr124, potentially leading to off-target effects. This disclosure provides an engineered Wnt7a protein and its applications. By introducing a specific single amino acid into the C-terminus of Wnt7a, conditional activation of the signaling pathway is achieved, enabling it to activate specific signaling pathways only in the presence of RECK and Gpr124, thus providing a more precise and safer tool for signaling pathway regulation. Wild-type Wnt7a, on the other hand, activates the Wnt signaling pathway in the presence or absence of RECK and Gpr124. The engineered Wnt7a designed in this disclosure can be applied to the prevention or treatment of cerebrovascular diseases or blood-brain barrier damage in the central nervous system, while also providing new strategies and approaches for research on the Wnt signaling pathway.

[0007] In order to solve the above-mentioned technical problems, the present disclosure adopts the following technical solution.

[0008] The first aspect of this disclosure provides an engineered Wnt7a protein, wherein a specific single amino acid is introduced at the C-terminus of the Wnt7a protein, which can specifically activate RECK-Gpr124-Fzd co-mediated Wnt signaling, and wherein the engineered Wnt7a protein, under conditions of RECK and GPR124 deficiency, activates or completely disables Frizzled / LRP-mediated Wnt signaling.

[0009] In some embodiments of this disclosure, the engineered Wnt7a protein comprises an amino acid sequence as shown in any of SEQ ID NO: 8-16.

[0010] A second aspect of this disclosure provides an isolated nucleic acid that encodes the engineered Wnt7a protein as described in the first aspect of this disclosure.

[0011] A third aspect of this disclosure provides a nucleic acid expression cassette comprising isolated nucleic acids as described in the second aspect of this disclosure, the isolated nucleic acids being operatively linked to one or both of a promoter and a regulatory signal, the regulatory signal being a transcriptional regulatory signal or a translational regulatory signal.

[0012] A fourth aspect of this disclosure provides a recombinant expression vector comprising a nucleic acid as described in a second aspect of this disclosure or a nucleic acid expression cassette as described in a third aspect of this disclosure.

[0013] A fifth aspect of this disclosure provides a transformant comprising one or more of the nucleic acids described in the second aspect of this disclosure, the nucleic acid expression cassettes described in the third aspect of this disclosure, and the recombinant expression vectors described in the fourth aspect of this disclosure; the transformant is not an animal or plant species.

[0014] In some embodiments of this disclosure, the transformant is a eukaryotic cell.

[0015] In some embodiments of this disclosure, the host cell of the transformant is HEK 293T cell.

[0016] A sixth aspect of this disclosure provides a kit comprising one or more of the following: an engineered Wnt7a protein as described in the first aspect of this disclosure, a nucleic acid as described in the second aspect of this disclosure, a nucleic acid expression cassette as described in the third aspect of this disclosure, a recombinant expression vector as described in the fourth aspect of this disclosure, and a transformant as described in the fifth aspect of this disclosure.

[0017] A seventh aspect of this disclosure provides a method for preparing engineered Wnt7a protein, the method comprising culturing a transformant as described in a fifth aspect of this disclosure to obtain the engineered Wnt7a protein.

[0018] An eighth aspect of this disclosure provides a pharmaceutical composition comprising one or more of the following: an engineered Wnt7a protein as described in the first aspect of this disclosure, a nucleic acid as described in the second aspect of this disclosure, a nucleic acid expression cassette as described in the third aspect of this disclosure, a recombinant expression vector as described in the fourth aspect of this disclosure, and a transformant as described in the fifth aspect of this disclosure, and optionally a pharmaceutically acceptable carrier and / or excipients.

[0019] The ninth aspect of this disclosure provides the use of engineered Wnt7a protein as described in the first aspect of this disclosure, nucleic acid as described in the second aspect of this disclosure, nucleic acid expression cassette as described in the third aspect of this disclosure, recombinant expression vector as described in the fourth aspect of this disclosure, transformant as described in the fifth aspect of this disclosure, kit as described in the sixth aspect of this disclosure, or pharmaceutical composition as described in the eighth aspect of this disclosure in the preparation of medicaments for the prevention or treatment of neurovascular diseases or central nervous system diseases including neurovascular dysfunction.

[0020] In some embodiments of this disclosure, the neurovascular diseases are selected from ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, cerebral aneurysm, arteriovenous malformation, cavernous malformation, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformation, carotid artery stenosis, moyamoya disease, and intracranial arteriosclerosis and combinations thereof; the central nervous system diseases are selected from multiple sclerosis, ischemic stroke, brain cancer, epilepsy, dementia, vascular dementia, HIV-1 related dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, infectious encephalopathy, traumatic brain injury, migraine, and chronic traumatic encephalopathy and combinations thereof.

[0021] The tenth aspect of this disclosure provides engineered Wnt7a protein as described in the first aspect of this disclosure, nucleic acid as described in the second aspect of this disclosure, nucleic acid expression cassette as described in the third aspect of this disclosure, recombinant expression vector as described in the fourth aspect of this disclosure, transformant as described in the fifth aspect of this disclosure, kit as described in the sixth aspect of this disclosure, or pharmaceutical composition as described in the eighth aspect of this disclosure for the prevention or treatment of neurovascular diseases or central nervous system diseases including neurovascular dysfunction.

[0022] In some embodiments of this disclosure, the neurovascular diseases are selected from ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, cerebral aneurysm, arteriovenous malformation, cavernous malformation, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformation, carotid artery stenosis, moyamoya disease, and intracranial arteriosclerosis and combinations thereof; the central nervous system diseases are selected from multiple sclerosis, ischemic stroke, brain cancer, epilepsy, dementia, vascular dementia, HIV-1 related dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, infectious encephalopathy, traumatic brain injury, migraine, and chronic traumatic encephalopathy and combinations thereof.

[0023] The eleventh aspect of this disclosure provides a method for preventing or treating neurovascular diseases or central nervous system diseases including neurovascular dysfunction, the method comprising administering to a subject in need an effective amount of an engineered Wnt7a protein as described in the first aspect of this disclosure, a nucleic acid as described in the second aspect of this disclosure, a nucleic acid expression cassette as described in the third aspect of this disclosure, a recombinant expression vector as described in the fourth aspect of this disclosure, a transformant as described in the fifth aspect of this disclosure, a kit as described in the sixth aspect of this disclosure, or a pharmaceutical composition as described in the eighth aspect of this disclosure.

[0024] In some embodiments of this disclosure, the neurovascular diseases are selected from ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, cerebral aneurysm, arteriovenous malformation, cavernous malformation, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformation, carotid artery stenosis, moyamoya disease, and intracranial arteriosclerosis and combinations thereof; the central nervous system diseases are selected from multiple sclerosis, ischemic stroke, brain cancer, epilepsy, dementia, vascular dementia, HIV-1 related dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, infectious encephalopathy, traumatic brain injury, migraine, and chronic traumatic encephalopathy and combinations thereof.

[0025] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this disclosure.

[0026] All reagents and raw materials used in this disclosure are commercially available.

[0027] The positive advancements of this disclosure are as follows: By engineering the introduction of a specific single amino acid into the C-terminus of Wnt7a, an engineered Wnt7a protein with conditional activation properties has been successfully constructed. This engineered Wnt7a protein can only activate specific signaling pathways in the presence of RECK and Gpr124, while remaining inactive in their absence. This overcomes the technical deficiency of wild-type Wnt7a lacking receptor specificity, significantly improves the accuracy of signaling pathway activation, reduces the risk of off-target effects, and provides a safer and more effective potential tool for the prevention and treatment of cerebrovascular diseases or blood-brain barrier damage. It also opens up new technical pathways for the research and regulation of the Wnt signaling pathway. Attached Figure Description

[0028] Figure 1A To investigate the activation intensity of the classical Wnt signaling pathway mediated by Fzd or RECK-Gpr124-Fzd by different engineered Wnt7a with the introduction of multiple amino acids at the C-terminus of wild-type Wnt7a in the TOP Flash assay, this study was performed in HEK293T cells. WT represents wild-type Wnt7a (SEQ ID NO: 1), and WT+x represents the introduction of the corresponding amino acid (SEQ ID NO: 2-7) at the C-terminus of wild-type Wnt7a. Each group was divided into three replicates; P < 0.05 was considered statistically significant, and ns indicated no statistically significant difference. This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This means P < 0.0001.

[0029] Figure 1BTo investigate the activation intensity of different engineered Wnt7a (SEQ ID NO: 8-16) mediated by Fzd or RECK-Gpr124-Fzd in the classical Wnt signaling pathway by introducing one or more amino acids (Ile(I), Trp(W), Tyr(Y), Asn(N), Gln(Q), Asp(D), Glu(E), Leu(L), and Cys(C)) at the C-terminus of wild-type Wnt7a using the TOP Flash assay. This experiment was performed in HEK293T cells. WT represents wild-type Wnt7a (SEQ ID NO: 1), and WT+x represents the introduction of the corresponding amino acid (SEQ ID NO: 8-16) at the C-terminus of wild-type Wnt7a. Each group had three replicates; P < 0.05 was considered statistically significant, and ns indicated no significant difference. This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This means P < 0.0001.

[0030] Figure 1C To investigate the activation intensity of different engineered Wnt7a (SEQ ID NO: 17-25) mediated by Fzd or RECK-Gpr124-Fzd in the classical Wnt signaling pathway by introducing one or more amino acids (Thr(T), Val(V), Ala(A), Met(M), Phe(F), Pro(P), His(H), Gly(G), or Arg(R)) at the C-terminus of wild-type Wnt7a in the TOP Flash assay, this study aimed to evaluate the effect of different engineered Wnt7a (SEQ ID NO: 17-25) with the introduction of the corresponding amino acid (SEQ ID NO: 17-25) at the C-terminus of wild-type Wnt7a. Each group was divided into three replicates; P < 0.05 was considered statistically significant, and ns indicated no statistically significant difference. This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This means P < 0.0001.

[0031] Figure 1DTo investigate the activation intensity of the classical Wnt signaling pathway mediated by Fzd or RECK-Gpr124-Fzd by different engineered Wnt7a (SEQ ID NO: 26-27) obtained by introducing one amino acid S (Ser) or K (Lys) to the C-terminus of wild-type Wnt7a in a TOP Flash assay. This experiment was performed in HEK293T cells. WT represents wild-type Wnt7a (SEQ ID NO: 1), and WT+x represents the introduction of the corresponding amino acid (SEQ ID NO: 26-27) to the C-terminus of wild-type Wnt7a. Each group was divided into three replicates; P < 0.05 was considered statistically significant, and ns indicated no statistically significant difference. This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This means P < 0.0001. Detailed Implementation

[0032] In this disclosure, unless otherwise stated, scientific and technical terms used herein have meanings commonly understood by those skilled in the art. Furthermore, the procedures used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all standard procedures widely used in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below: In this disclosure, the letters in the amino acid sequence represent abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A (where the letter before the hyphen is the three-letter abbreviation of the amino acid, and the letter after the hyphen is the single-letter abbreviation, the same below), arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T. As will be known to those skilled in the art, in this disclosure, amino acids and amino acid residues can be used interchangeably.

[0033] In this disclosure, "nucleic acid" refers to a nucleotide chain of any length. A nucleotide can be a deoxyribonucleotide (DNA), a ribonucleotide (RNA), a modified nucleotide or base, and / or an analogue thereof, or any substrate that can be incorporated into the chain by a DNA or RNA polymerase.

[0034] In this disclosure, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of this disclosure are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this disclosure may contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring nucleotide linkages, or both. Modified nucleotide or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0035] In this disclosure, "Wnt ligands" refers to a highly conserved family of secretory lipid-modified proteins that mediate intercellular communication during animal development and adult tissue homeostasis, and exert pleiotropic functions by regulating cell proliferation, differentiation, migration, apoptosis, polarity, and genetic stability. This disclosure particularly focuses on Wnt7a ligands, which play an exclusive control role in the development of cerebral blood vessels in the mammalian forebrain and ventral spinal cord.

[0036] In this disclosure, "engineered Wnt7a protein" refers to the introduction of precisely designed specific amino acid residues into the carboxyl terminus (C-terminus) of Wnt7a protein through protein engineering technology. This modification can change the conformation or interaction interface of the protein, thereby regulating its binding specificity with the receptor. This disclosure demonstrates that this specific C-terminal modification can significantly improve the interaction specificity of engineered Wnt7a protein with the RECK-Gpr124-Fzd complex.

[0037] In this disclosure, the "Wnt signaling pathway" refers to a specific signal transduction mechanism in which RECK (a retroactively induced cysteine-rich protein with a Kazal motif) binds specifically to Wnt7a / b in a manner different from Frizzled. GPR124 (a G protein-coupled receptor) acts as a transmembrane connector between RECK and the intracellular Disheveled scaffold, bridging Frizzled and GPR124 through Disheveled to form a ligand-specific RECK / Gpr124 / Frizzled / LRP signaling complex, which participates in regulating vascular development and the formation and maintenance of the blood-brain barrier in the central nervous system.

[0038] In this disclosure, the "blood-brain barrier (BBB)" refers to an important physiological structure regulating endothelial Wnt / β-linkin signaling, which controls the expansion and maturation of the CNS vascular network during embryogenesis and helps maintain the adult BBB. The patent states that at a dysfunctional BBB, mere stimulation of Wnt / β-linkin signaling is sufficient to significantly improve ischemic stroke and brain cancer in mice.

[0039] In this disclosure, "specific activation" refers to a highly selective activation of signaling pathways. The engineered Wnt7a protein of this disclosure can recognize and preferentially activate the RECK-Gpr124-Fzd co-mediated Wnt signaling pathway, while having very low or no activation of other signaling pathways (especially the Frizzled / LRP-mediated pathway in the absence of RECK and GPR124). This specificity is crucial for precisely targeting the central nervous system of the brain while avoiding systemic side effects.

[0040] In this disclosure, "risk of off-target effects" refers to the possibility that insufficient selectivity in the use of bioactive molecules (such as Wnt signaling agonists) may lead to the activation of unintended signaling pathways, potentially causing adverse reactions or side effects. The engineered Wnt7a protein of this disclosure significantly reduces the risk of off-target effects through its specific activation mechanism, providing a safer treatment option for the prevention and treatment of neurovascular diseases or blood-brain barrier damage.

[0041] In this disclosure, "neurovascular diseases or central nervous system diseases including neurovascular dysfunction" refers to a class of diseases related to abnormal vascular function of the central nervous system, including but not limited to ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, cerebral aneurysm, arteriovenous malformation, cavernous malformation, vasculitis, cerebral hemorrhage, multiple sclerosis, brain cancer, epilepsy, and neurodegenerative diseases. The engineered Wnt7a protein of this disclosure promotes the recovery of blood-brain barrier function and the stability of neurovascular units by specifically activating the RECK-Gpr124-Fzd-mediated Wnt signaling pathway, providing a new potential tool for the prevention and treatment of these diseases.

[0042] The present disclosure is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present disclosure. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0043] Example 1: Detection of the activation intensity of engineered Wnt7a on the classical Wnt signaling pathway mediated by Fzd or RECK-Gpr124-Fzd using TOP Flash.

[0044] 1.1 Expression plasmid construct

[0045] Plasmids were constructed using the Gibson seamless cloning method with LightNing DNA Assembly Mix Plus reagent (manufacturer: Yugong Biotechnology; catalog number: EG21202S). The Wnt7a (UniProtKB #O00755) gene was fused into the pcDNA3.1 vector, and the Gpr124 (UniProtKB #Q96PE1), RECK (UniProtKB #O95980), and Fzd8 (UniProtKB #Q9H461) genes were fused into the pegBacMan vector. PCR experiments were performed using PrimeSTAR HS (Premix) reagent (manufacturer: Takara; catalog number: R040A). The required amino acids were introduced into the C-terminus of Wnt7a; the specific sequence is described below.

[0046] 1.2 TOPFlash Dual-Luciferase Assay

[0047] HEK 293T cells were induced at a concentration of 0.8 × 10⁻⁶. 5Inoculate the cells into 48-well cell culture plates at a seed volume of 1 / 2. After approximately 24 hours, transfect the cells according to the Lipofectamine 3000 Reagent instructions (manufacturer: Thermo Fisher Scientific; catalog number: L3000015), adding 200 ng of the target gene plasmid (plus 50 ng Wnt ligand, 50 ng Fzd8 receptor, 50 ng RECK receptor, and 50 ng Gpr124 receptor as specified). Adjust the total target gene amount to 200 ng using an empty pcDNA3.1 vector, and transfect 100 ng of Super TOP Flash plasmid (company: addgene; catalog number: 12456) (containing 7 TCF / LEF binding sites and the firefly luciferase reporter gene) and 2 ng of Renilla luciferase reporter gene plasmid (company: addgene; catalog number: 18016) into the 48-well plates.

[0048] After transfection, cells were cultured at 37 °C and 5% CO2 for 48 h before detection. Cells were treated with the Promega Dual-Luciferase Reporter Assay System kit (manufacturer: Promega; catalog number: E1960), and luciferase substrate was added. The fluorescence intensity of firefly luciferase and Renilla luciferase was then measured sequentially using a Tecan microplate analyzer (model: Spark; manufacturer: Tecan). Statistical analysis of the activity of engineered Wnt7a was performed using a GraphPad Prism 10. The ratio of fluorescence intensity of firefly luciferase to that of the corresponding Renilla luciferase was calculated to obtain the activation intensity of the Wnt signaling pathway by different engineered Wnt7a under different receptor-mediated conditions. The results are shown in [Figure number missing]. Figures 1A-1D . Figure 1A The results showed that when multiple amino acids were introduced at the C-terminus of wild-type Wnt7a, engineered Wnt7a could specifically activate the RECK-Gpr124-Fzd-mediated classical Wnt signaling pathway, but the signal intensity was reduced. Moreover, the signal intensity increased as the number of amino acids introduced at the C-terminus decreased (SEQ ID NO: 2-7). Figure 1BThe results showed that when one or more amino acids Ile (I), Trp (W), Tyr (Y), Asn (N), Gln (Q), Asp (D), Glu (E), Leu (L), or Cys (C) were introduced at the C-terminus of wild-type Wnt7a (SEQ ID NO: 8-16), engineered Wnt7a could specifically activate the RECK-Gpr124-Fzd-mediated Wnt signaling pathway. When RECK and Gpr124 were absent, the activation of this signaling pathway was weakened or completely absent. In contrast, wild-type Wnt7a could activate the classical Wnt signaling pathway in the presence or absence of RECK and Gpr124. Figure 1C The results showed that when one or more amino acids Thr(T), Val(V), Ala(A), Met(M), Phe(F), Pro(P), His(H), Gly(G), and Arg(R) were introduced at the C-terminus of wild-type Wnt7a (SEQ ID NO: 17-25), the activation of the Wnt signaling pathway was weakened or completely inhibited in the presence or absence of RECK and Gpr124. Figure 1D The results showed that when one amino acid S (Ser) or K (Lys) was introduced at the C-terminus of wild-type Wnt7a (SEQ ID NO: 26-27), the activation intensity of the RECK-Gpr124-Fzd-mediated signaling pathway remained unchanged between engineered Wnt7a and wild-type Wnt7a.

[0049] The sequence of wild-type Wnt7a (Uniprot ID: O00755), and all specific sequences of modified Wnt7a.

[0050] Human Wnt7a (WT) amino acid sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNA RTLMNLHNNEAGRKILEENMKLECKCHGVSSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK (SEQ ID NO: 1)

[0051] >WT+ENLYFQGSDYKDDDDK sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKENLYFQGSDYKDDDDK (SEQ ID NO: 2)

[0052] >WT+ENLYFQGSGGAGGSGG sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKENLYFQGSGGAGGSGG (SEQ ID NO: 3)

[0053] >WT+ENLYFQGS sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKENLYFQGS (SEQ ID NO: 4)

[0054] >WT + ENLYFQ Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKENLYFQ (SEQ ID NO: 5)

[0055] >WT + ENLY Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKENLY (SEQ ID NO: 6)

[0056] >WT+EN Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKEN (SEQ ID NO: 7)

[0057] >WT+I Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKI (SEQ ID NO: 8)

[0058] >WT+W Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKW (SEQ ID NO: 9)

[0059] >WT+Y Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKY (SEQ ID NO: 10)

[0060] >WT+N Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKN (SEQ ID NO: 11)

[0061] >WT+Q Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKQ (SEQ ID NO: 12)

[0062] >WT+D Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKD (SEQ ID NO: 13)

[0063] >WT+E Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKE (SEQ ID NO: 14)

[0064] >WT+L Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKL (SEQ ID NO: 15)

[0065] >WT+C Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKC (SEQ ID NO: 16)

[0066] >WT+T Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKT (SEQ ID NO: 17)

[0067] >WT+V Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKV (SEQ ID NO: 18)

[0068] >WT+A sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKA (SEQ ID NO: 19)

[0069] >WT+M sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKM (SEQ ID NO: 20)

[0070] >WT+F Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKF (SEQ ID NO: 21)

[0071] >WT+P Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKP (SEQ ID NO: 22)

[0072] >WT+H Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKH (SEQ ID NO: 23)

[0073] >WT+G Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKG (SEQ ID NO: 24)

[0074] >WT+R Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKR (SEQ ID NO: 25)

[0075] >WT+S Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKS (SEQ ID NO: 26)

[0076] >WT+K Sequence: MNRKARRCLGHLFLSLGMVYLRIGGFSSVVALGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKK (SEQ ID NO: 27)

Claims

1. An engineered Wnt7a protein, characterized in that, The engineered Wnt7a protein has a specific single amino acid introduced at the C-terminus of the Wnt7a protein, which can specifically activate the Wnt signaling co-mediated by RECK-Gpr124-Fzd. Furthermore, the engineered Wnt7a protein weakens or completely fails to activate Frizzled / LRP-mediated Wnt signaling under the condition of RECK and GPR124 deficiency.

2. The engineered Wnt7a protein as described in claim 1, characterized in that, The engineered Wnt7a protein comprises an amino acid sequence as shown in any of SEQ ID NO: 8-16.

3. An isolated nucleic acid, characterized in that, The nucleic acid encodes the engineered Wnt7a protein as described in claim 1 or 2.

4. A nucleic acid expression cassette, characterized in that, The nucleic acid expression cassette comprises the isolated nucleic acid of claim 3, wherein the isolated nucleic acid is operatively linked to one or both of a promoter and a regulatory signal, wherein the regulatory signal is a transcriptional regulatory signal or a translational regulatory signal.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 3 or the nucleic acid expression cassette as described in claim 4.

6. A transformant, characterized in that, The transformant comprises one or more of the nucleic acid as described in claim 3, the nucleic acid expression cassette as described in claim 4, and the recombinant expression vector as described in claim 5; the transformant is not an animal or plant species. Preferably, the transformant is a eukaryotic cell; More preferably, the host cell of the transformant is HEK 293T cell.

7. A reagent kit, characterized in that, The kit comprises one or more of the engineered Wnt7a protein as described in claim 1 or 2, the nucleic acid as described in claim 3, the nucleic acid expression cassette as described in claim 4, the recombinant expression vector as described in claim 5, and the transformant as described in claim 6.

8. A method for preparing engineered Wnt7a protein, characterized in that, The method comprises culturing the transformant as described in claim 6 to obtain the engineered Wnt7a protein.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises one or more of the engineered Wnt7a protein as claimed in claim 1 or 2, the nucleic acid as claimed in claim 3, the nucleic acid expression cassette as claimed in claim 4, the recombinant expression vector as claimed in claim 5, and the transformant as claimed in claim 6, and optionally a pharmaceutically acceptable carrier and / or excipient.

10. The use of one or more of the engineered Wnt7a protein as described in claim 1 or 2, the nucleic acid as described in claim 3, the nucleic acid expression cassette as described in claim 4, the recombinant expression vector as described in claim 5, the transformant as described in claim 6, the kit as described in claim 7, and the pharmaceutical composition as described in claim 9 in the preparation of a medicament for the prevention or treatment of neurovascular diseases or central nervous system diseases including neurovascular dysfunction; Preferably, the neurovascular disease is selected from ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, cerebral aneurysm, arteriovenous malformation, cavernous malformation, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformation, carotid artery stenosis, moyamoya disease, and intracranial arteriosclerosis and combinations thereof; the central nervous system disease is selected from multiple sclerosis, ischemic stroke, brain cancer, epilepsy, dementia, vascular dementia, HIV-1 related dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, infectious encephalopathy, traumatic brain injury, migraine, and chronic traumatic encephalopathy and combinations thereof.

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