Tissue-specific Wnt signal enhancing molecule and use thereof

Abstract

To provide tissue-specific Wnt signal enhancing molecules, and related methods of using these molecules to increase Wnt signaling in targeted tissues.SOLUTION: In one embodiment, the present invention provides a tissue-specific Wnt signal enhancing molecule, or a pharmaceutically acceptable salt thereof, comprising a first domain that specifically binds one or more transmembrane E3 ubiquitin ligases selected from ZNRF3 and RNF43, and a second domain that specifically binds a tissue-specific cell surface molecule, wherein the molecule increases Wnt signaling in a tissue comprising the tissue-specific cell surface molecule.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 450,804, filed January 26, 2017, and U.S. Provisional Patent Application No. 62 / 487,135, filed April 19, 2017, each of which is incorporated by reference in its entirety. Description of sequence listing The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is SRZN_003_02WO_ST25.txt. The text file is 454KB, was created on January 26, 2018, and has been submitted electronically via EFS-Web.

[0002] The present disclosure relates to tissue-specific Wnt signal enhancing molecules, e.g., fusion proteins, comprising a domain that binds to an E3 ubiquitin ligase, ZNRF3 or RNF43, and a tissue-specific cell surface receptor binding domain, and related methods of using the tissue-specific Wnt signal enhancing molecules to mediate tissue-specific internalization or sequestration of the E3 ligase, ZNRF3 / RNF43, thus stabilizing the Wnt receptor, enhancing Wnt signaling in a tissue-specific manner, and treating and preventing various diseases and disorders. [Background technology]

[0003] Wnt ("Wingless-associated integration site" or "Wingless and Int-1" or "Wingless-Int") ligands and their signals play important roles in controlling the development, homeostasis and regeneration of many important organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste buds, ovary, cochlea and many other tissues (e.g., reviewed by Clevers, Loh and Nusse, 2014;346:1248012). Modulation of the Wnt signaling pathway has potential for the treatment of degenerative diseases and tissue injury. To this end, it is desirable to develop strategies to modulate Wnt signaling activity in a tissue-specific or cell type-specific manner to avoid undesirable effects. One of the challenges for modulating Wnt signaling as a therapeutic agent is the existence of multiple Wnt ligands and Wnt receptors, Frizzled 1-10 (Fzd1-10), with many tissues expressing multiple overlapping Fzds. Canonical Wnt signaling also involves low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) or low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) as co-receptors, in addition to Fzds, which are widely expressed in various tissues.

[0004] R-spondins 1-4 are a family of ligands that amplify Wnt signals. Each R-spondin acts through a receptor complex that contains zinc ring finger 3 (ZNRF3) or ring finger protein 43 (RNF43) at one end and leucine-rich repeat-containing G protein-coupled receptor 4-6 (LGR4-6) at the other (e.g., reviewed by Knight and Hankenson 2014, Matrix Biology;37:157-161). R-spondins may also act through additional mechanisms of action. ZNRF3 and RNF43 are two membrane-bound E3 ligases that specifically target Wnt receptors (Fzd1-10 and LRP5 or LRP6) for degradation. Binding of R-spondin to ZNRF3 / RNF43 and LGR4-6 causes clearance or sequestration of the ternary complex, which removes the E3 ligase from the Wnt receptor and stabilizes the Wnt receptor, resulting in enhanced Wnt signaling. Each R-spondin contains two furin domains (1 and 2), with furin domain 1 binding to ZNRF3 / RNF43, and furin domain 2 binding to LGR4-6. A fragment of R-spondin containing furin domains 1 and 2 is sufficient to amplify Wnt signaling. R-spondin effects are dependent on Wnt signaling, but because both LGR4-6 and ZNRF3 / RNF43 are widely expressed in various tissues, the effects of R-spondin are not tissue specific.

[0005] There is a clear need in the art for tissue-specific Wnt signal enhancing molecules for the treatment and prevention of certain diseases and disorders.The present invention addresses this need by providing compositions and methods useful for enhancing Wnt activity in a tissue-specific manner. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Knight and Hankenson 2014,Matrix Biology;37:157-161 Summary of the Invention [Means for solving the problem]

[0007] The present invention relates to tissue-specific Wnt signal enhancing molecules and their use, for example, in increasing Wnt signaling in target tissues and treating diseases and conditions that would benefit from increased Wnt signaling.

[0008] In one embodiment, the present invention provides a tissue-specific Wnt signal enhancing molecule, or a pharma- ceutically acceptable salt thereof, comprising a first domain that specifically binds to one or more transmembrane E3 ubiquitin ligases selected from ZNRF3 and RNF43, and a second domain that specifically binds to a tissue-specific cell surface molecule, wherein the molecule increases Wnt signaling in the tissue that contains the tissue-specific cell surface molecule, or a pharma- ceutically acceptable salt thereof.In certain embodiments, the tissue is selected from the group consisting of bone tissue, liver tissue, skin tissue, stomach tissue, intestinal tissue, oral mucosa tissue, kidney tissue, central nervous system tissue, mammary tissue, taste bud tissue, ovarian tissue, inner ear tissue (including cochlea and vestibular tissue), hair follicle, pancreatic tissue, retina tissue, vascular tissue, cornea tissue, heart tissue, and lung tissue.In various embodiments, either or both of the first domain and the second domain are a polypeptide, an antibody, a small molecule, a natural ligand, a non-natural ligand, or a variant thereof.

[0009] In certain embodiments of the Wnt signal enhancing molecule, the first domain comprises a first polypeptide sequence and / or the second domain comprises a second polypeptide sequence. In certain embodiments, the molecule is a fusion protein comprising the first polypeptide sequence and the second polypeptide sequence. In certain embodiments, the first polypeptide sequence comprises an R-spondin sequence or a fragment or variant thereof. In certain embodiments, the R-spondin is R-spondin-1, R-spondin-2, R-spondin-3, or R-spondin-4, such as human R-spondin 1-4. In certain embodiments, the first polypeptide sequence comprises R-spondin furin domain 1 or a fragment or variant thereof. In certain embodiments, the first polypeptide sequence is a wild-type sequence or a modified sequence. In addition, the first polypeptide sequence may have increased, equivalent, or decreased binding to LGR4-6 compared to the corresponding native full-length R-spondin. In some embodiments, the R-spondin or R-spondin furin domain 1 has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identity with any of the R-spondin or R-spondin furin 1 domains present in SEQ ID NOs: 1 to 4. In certain embodiments, the second polypeptide sequence is a polypeptide, an antibody or a fragment or variant thereof, or a ligand or a fragment or variant thereof.

[0010] In certain exemplary embodiments of the tissue-specific Wnt signal enhancing molecules disclosed herein, the tissue is bone tissue and the cell surface receptor is parathyroid hormone receptor 1 (PTH1R); the tissue is liver tissue and the cell surface receptor is asialoglycoprotein receptor 1 (ASGR1), asialoglycoprotein receptor 2 (ASGR2), transferrin receptor 2 (TFR2), or solute carrier family 10 member 1 (SLC10A1); or the tissue is oral mucosa tissue and the cell surface receptor is LY6 / PLAUR domain containing 3 (LYPD3) or desmoglein 3 (DSG3).

[0011] In certain exemplary embodiments of the tissue-specific Wnt signal enhancing molecules disclosed herein, the cell surface molecule is PTH1 and the second polypeptide sequence specifically binds to PTH1R, the cell surface molecule is ASGR1 and the second polypeptide sequence specifically binds to ASGR1, the cell surface molecule is ASGR2 and the second polypeptide sequence specifically binds to ASGR2, the cell surface molecule is SLC10A1 and the second polypeptide sequence specifically binds to SLC10A1, the cell surface molecule is TFR2 and the second polypeptide sequence specifically binds to TFR2, the cell surface molecule is LYPD3 and the second polypeptide sequence specifically binds to LYPD3, or the cell surface molecule is DSG3 and the second polypeptide sequence specifically binds to DSG3, wherein the second polypeptide is an antibody or fragment thereof, a small molecule, or a ligand of the cell surface molecule or a fragment or variant thereof.

[0012] In certain embodiments of the tissue-specific Wnt signal enhancing molecule described herein, the first domain and the second domain are linked by a linker moiety.In certain embodiments, the linker moiety is a peptidyl linker sequence.In certain embodiments, the peptidyl linker sequence comprises one or more amino acids selected from the group consisting of glycine, asparagine, serine, threonine and alanine.

[0013] In certain embodiments, the tissue-specific Wnt signal enhancer molecule described herein consists of a single polypeptide, for example, a fusion protein comprising a first domain and a second domain. In certain embodiments, the tissue-specific Wnt signal enhancer molecule described herein comprises two or more polypeptides, such as a dimer or multimer comprising two or more fusion proteins each comprising a first domain and a second domain, where the two or more polypeptides are linked, for example, through a linker moiety or via a bond between amino acid residues in each of the two or more polypeptides, for example, an intermolecular disulfide bond between cysteine ​​residues. In certain embodiments, the tissue-specific Wnt signal enhancer molecule described herein comprises two or more polypeptide sequences. For example, the tissue-specific Wnt signal enhancer molecule may comprise an antibody heavy chain and a light chain (or an antigen-binding fragment thereof) constituting either the first domain or the second domain, where the other domain (i.e., the second domain or the first domain) is linked to the antibody heavy chain or light chain as a fusion protein or via a linker moiety. In certain embodiments, the other domain is attached to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, or the C-terminus of the light chain. Such structures may be referred to herein as adduct IgG scaffolds or formats.

[0014] In a related embodiment, the invention includes a nucleic acid sequence encoding the tissue-specific Wnt signal enhancement fusion protein or subunit thereof disclosed herein, for example, an antibody heavy or light chain with a first domain or a second domain added or fused thereto. In a further related embodiment, the invention includes a vector comprising the nucleic acid sequence. In some embodiments, the vector is an expression vector comprising a promoter sequence operably linked to the nucleic acid sequence in a manner suitable for expression in, for example, a bacterial or eukaryotic cell. In another embodiment, the vector is engineered for in vitro translation and modification of functional mRNA. In a further related embodiment, the invention includes a host cell comprising the vector. In yet another related embodiment, the invention includes a method for producing a tissue-specific Wnt signal enhancement fusion protein as described herein, comprising culturing a host cell under conditions in which the fusion polypeptide is expressed by the expression vector. In some embodiments, the method further comprises isolating the fusion polypeptide produced.

[0015] In another embodiment, the present invention provides a pharmaceutical composition comprising a tissue-specific Wnt signal enhancing molecule described herein or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable diluent, adjuvant or carrier.

[0016] In another embodiment, the present invention provides a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding a tissue-specific Wnt signal enhancing molecule or a pharma-ceutically acceptable salt thereof described herein, and a pharma-ceutically acceptable diluent, adjuvant or carrier. In certain embodiments, the nucleic acid sequence comprises DNA or mRNA, optionally modified mRNA.

[0017] In another embodiment, the present invention provides a pharmaceutical composition comprising a vector comprising a nucleic acid sequence encoding a tissue-specific Wnt signal enhancing molecule or a pharma-ceutically acceptable salt thereof, and a pharma-ceutically acceptable diluent, adjuvant or carrier. In certain embodiments, the vector comprises a promoter operably linked to the nucleic acid sequence, which drives the expression of the tissue-specific Wnt signal enhancing molecule. In certain embodiments, the vector is an expression vector or a viral vector.

[0018] In another embodiment, the invention provides a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist (e.g., natural or engineered), or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable diluent, adjuvant, or carrier. In certain embodiments, the nucleic acid sequence comprises DNA or mRNA, optionally modified mRNA.

[0019] In another embodiment, the invention provides a pharmaceutical composition comprising a vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist (e.g., natural or engineered), or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable diluent, adjuvant, or carrier. In certain embodiments, the vector comprises a promoter operably linked to the nucleic acid sequence that drives expression of the Wnt polypeptide, Norrin polypeptide, or Wnt signaling agonist. In certain embodiments, the vector is an expression vector or a viral vector.

[0020] In another embodiment, the present invention provides a pharmaceutical composition comprising a tissue-specific Wnt signal enhancing molecule described herein, or a pharma- ceutically acceptable salt thereof, a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable diluent, adjuvant, or carrier.

[0021] In another embodiment, the present invention provides a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding a tissue-specific Wnt signal enhancing molecule described herein or a pharma- ceutically acceptable salt thereof, a polynucleotide comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable diluent, adjuvant, or carrier. In certain embodiments, the nucleic acid sequence comprises DNA or mRNA, optionally modified mRNA.

[0022] In another embodiment, the present invention provides a pharmaceutical composition comprising a vector comprising a nucleic acid sequence encoding a tissue-specific Wnt signal enhancing molecule or a pharma- ceutically acceptable salt thereof, a vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable diluent, adjuvant, or carrier. In certain embodiments, the vector comprises a promoter operably linked to the nucleic acid sequence that drives the expression of the tissue-specific Wnt signal enhancing molecule. In certain embodiments, the vector is an expression vector or a viral vector.

[0023] In a further embodiment, the present invention includes a method for increasing Wnt signaling in a target tissue, comprising contacting the target tissue with a tissue-specific Wnt signal enhancing molecule described herein, wherein the second domain specifically binds to a cell-specific surface molecule on the target tissue, and the tissue-specific Wnt signal enhancing molecule binds to the target tissue and sequesters or increases the endocytosis of one or more transmembrane E3 ubiquitin ligases selected from ZNRF3 and RNF43 in the target tissue.

[0024] In certain embodiments of any of the methods described herein, the tissue is bone tissue and the cell surface molecule is PTH1R, the tissue is liver tissue and the cell surface molecule is ASGR1, ASGR2, TFR2, or SLC10A1, or the tissue is oral mucosa tissue and the cell surface receptor is LYPD3 or DSG3. In certain embodiments, the target tissue or cell is contacted with a polynucleotide comprising a nucleic acid sequence encoding a tissue-specific Wnt signal enhancer molecule, or a vector, such as an expression vector or a viral vector, comprising a nucleic acid sequence encoding a tissue-specific Wnt signal enhancer molecule.

[0025] In further embodiments, the invention includes a method for increasing Wnt signaling in a target tissue, comprising contacting the target tissue with a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist, or a pharma- ceutically acceptable salt thereof. In certain embodiments, the target tissue or cell is contacted with a polynucleotide comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist, or a pharma- ceutically acceptable salt thereof, or a vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist, or a pharma- ceutically acceptable salt thereof.

[0026] In a further embodiment, the present invention includes a method for increasing Wnt signaling in a target tissue, comprising contacting the target tissue with a tissue-specific Wnt signaling enhancer molecule described herein, wherein the second domain specifically binds to a cell-specific surface molecule on the target tissue, and the tissue-specific Wnt signaling enhancer molecule binds to the target tissue and sequesters or increases the endocytosis of one or more transmembrane E3 ubiquitin ligases selected from ZNRF3 and RNF43 in the target tissue, and a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist, or a pharma- ceutically acceptable salt thereof. In certain embodiments, the target tissue or cell is contacted with a polynucleotide comprising a nucleic acid sequence encoding the tissue-specific Wnt signaling enhancer molecule and a nucleic acid encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist. In other embodiments, the target tissue or cell is contacted with a vector comprising a nucleic acid sequence encoding the tissue-specific Wnt signaling enhancer molecule and a vector encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist.

[0027] In yet another related embodiment, the invention includes a method for treating or preventing a disease or condition in a subject in need thereof, the disease or condition being associated with decreased Wnt signaling or would benefit from increased Wnt signaling, comprising providing to the subject an effective amount of a pharmaceutical composition comprising a tissue-specific Wnt signaling enhancer molecule or a pharma- ceutically acceptable salt thereof, either alone or in combination with a Wnt, Norrin, or Wnt activating / mimetic molecule. In certain embodiments, the method is carried out using a pharmaceutical composition comprising a polynucleotide (e.g., DNA or mRNA) comprising a nucleic acid sequence encoding a tissue-specific Wnt signaling enhancer molecule, or a vector (e.g., expression vector or viral vector) comprising a nucleic acid sequence encoding a tissue-specific Wnt signaling enhancer molecule, alone or in combination with a pharmaceutical composition comprising a polynucleotide (e.g., DNA or mRNA) comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule, or a vector (e.g., expression vector or viral vector) comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule.

[0028] In yet another related embodiment, the invention includes a method for treating or preventing a disease or condition in a subject in need thereof, where the disease or condition is associated with decreased Wnt signaling or would benefit from increased Wnt signaling, comprising providing to the subject an effective amount of a pharmaceutical composition comprising a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule. In certain embodiments, the method is carried out using a pharmaceutical composition comprising a polynucleotide (e.g., DNA or mRNA) comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule, or a vector (e.g., an expression vector or viral vector) comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule.

[0029] In certain embodiments of any of the methods of treatment described herein, the disease or disorder is a disease or disorder of a tissue selected from the group consisting of bone tissue, liver tissue, skin tissue, stomach tissue, intestinal tissue, oral mucosa tissue, kidney tissue, central nervous system tissue, mammary tissue, taste bud tissue, ovarian tissue, inner ear tissue (including cochlea and vestibular tissue), hair follicle, pancreatic tissue, retinal tissue, vascular tissue, corneal tissue, cardiac tissue, and lung tissue. In certain exemplary embodiments, the disease or disorder is a disease or disorder of bone tissue and the cell surface receptor is PTH1R, a disease or disorder of liver tissue and the cell surface receptor is ASGR1, ASGR2, TFR2, or SLC10A1, or a disease or disorder of oral mucosa tissue and the cell surface receptor is LYPD3 or DSG3. In certain exemplary embodiments, the disease or condition is bone fracture, osteoporosis, osteoporotic fracture, spinal fusion, osseointegration of orthopedic devices, tendon-bone integration, tooth growth and regeneration, dental implant surgery, periodontal disease, maxillofacial reconstruction, osteonecrosis of the jaw, alopecia, hearing loss, vestibular hypofunction, macular degeneration, vitreous retinopathy, retinal degeneration, diabetic retinopathy, Fuchs' dystrophy, stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, spinal cord injury, oral mucositis, intestinal mucositis, short bowel syndrome, inflammatory bowel disease (IBD), metabolic syndrome, diabetes, pancreatitis, exocrine pancreatic function. The present invention relates to a method for treating chronic liver failure, wound healing, diabetic foot ulcer, coronary artery disease, acute kidney disease, chronic kidney disease, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, acute liver failure, acute alcoholic liver disease, chronic liver disease caused by hepatitis C virus (HCV), HCV patients post-antiviral drug treatment, chronic liver disease caused by hepatitis B virus (HBV), HBV patients post-antiviral drug treatment, chronic alcoholic liver disease, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NASH), liver cirrhosis, and chronic liver failure of any cause. In certain embodiments of any of the methods for treating or preventing described herein, the pharmaceutical composition is provided systemically, parenterally, orally, intramuscularly, topically, or locally. In certain embodiments, the subject is a mammal, optionally a human. The present invention provides, for example, the following items. (Item 1) A tissue-specific Wnt ("Wingless-associated integration site" or "Wingless and Int-1" or "Wingless-Int") signal enhancing molecule, or a pharma- ceutically acceptable salt thereof, comprising a first domain that specifically binds to one or more transmembrane E3 ubiquitin ligases selected from zinc ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43), and a second domain that specifically binds to a tissue-specific cell surface molecule, wherein the compound increases Wnt signaling in the tissue containing the tissue-specific cell surface molecule, or a pharma-ceutically acceptable salt thereof. (Item 2) The molecule according to item 1, wherein the tissue is selected from the group consisting of bone tissue, liver tissue, skin tissue, stomach tissue, intestinal tissue, oral mucosa tissue, kidney tissue, central nervous system tissue, mammary tissue, taste bud tissue, ovarian tissue, inner ear tissue (including cochlear and vestibular tissue), hair follicle, pancreatic tissue, retinal tissue, corneal tissue, cardiac tissue and lung tissue. (Item 3) 3. The molecule of claim 1 or 2, wherein the first domain comprises a first polypeptide sequence and / or the second domain comprises a second polypeptide sequence. (Item 4) The molecule according to item 3, wherein the compound is a fusion protein or an antibody comprising the first polypeptide sequence and the second polypeptide sequence. (Item 5) The first polypeptide sequence is a) an R-spondin polypeptide sequence, wherein said R-spondin is optionally R-spondin-1, R-spondin-2, R-spondin-3 or R-spondin-4, or a fragment or variant thereof; or b) The molecule according to item 3 or 4, comprising an R-spondin furin domain 1 sequence and, optionally, a wild-type or mutant furin domain 2 or a fragment or variant thereof, wherein said first polypeptide sequence optionally has reduced binding to leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-6) compared to full-length R-spondin. (Item 6) 6. The molecule according to item 5, wherein the R-spondin furin domain 1 has at least 90% identity with any of the furin 1 domains present in SEQ ID NOs: 1 to 4. (Item 7) 7. The molecule according to any of items 3 to 6, wherein the second polypeptide sequence is a peptide, a polypeptide, an antibody or a fragment thereof, or a ligand or a fragment or variant thereof. (Item 8) a) the tissue is bone tissue and the cell surface molecule is parathyroid hormone receptor 1 (PTH1R); b) the tissue is liver tissue and the cell surface molecule is asialoglycoprotein receptor 1 (ASGR1), asialoglycoprotein receptor 2 (ASGR2), transferrin receptor 2 (TFR2) or solute carrier family 10 member 1 (SLC10A1); or c) The molecule according to any one of items 1 to 7, wherein the tissue is oral mucosa and the cell surface molecule is Ly6 / PLAUR domain-containing 3 (LYPD3) or desmoglein (DSG3). (Item 9) a) the cell surface molecule is parathyroid hormone receptor 1 (PTH1R) and the second polypeptide sequence specifically binds to PTH1R; b) the cell surface molecule is asialoglycoprotein receptor 1 (ASGR1) and the second polypeptide sequence specifically binds to ASGR1; c) the cell surface molecule is asialoglycoprotein receptor 2 (ASGR2) and the second polypeptide sequence specifically binds to ASGR2; or d) the cell surface molecule is solute carrier family 10 member 1 (SLC10A1) and the second polypeptide sequence specifically binds to solute carrier family 10 member 1 (SLC10A1); e) the cell surface molecule is transferrin receptor 2 (TFR2) and the second polypeptide sequence specifically binds to TFR2; or f) the cell surface molecule is Ly6 / PLAUR domain-containing 3 (LYPD3) and the second polypeptide sequence specifically binds to LYPD3; or g) the cell surface molecule is desmoglein (DSG3) and the second polypeptide sequence specifically binds to DSG3; 9. The molecule according to any of items 3 to 8, wherein the second polypeptide is an antibody or a fragment thereof or a peptide or polypeptide different from an antibody, a small molecule, or a ligand of the cell surface molecule or a fragment or variant thereof. (Item 10) 10. The molecule according to any of items 1 to 9, wherein the first domain and the second domain are linked by a linker moiety. (Item 11) 11. The molecule according to item 10, wherein the linker moiety is a peptidyl linker sequence. (Item 12) 12. The molecule of item 11, wherein the linker sequence comprises one or more amino acids selected from the group consisting of glycine, asparagine, serine, threonine and alanine. (Item 13) 13. A nucleic acid sequence encoding the fusion protein according to any one of items 4 to 12. (Item 14) 14. The nucleic acid sequence according to item 13, wherein the nucleic acid sequence is DNA or mRNA. (Item 15) A vector comprising the nucleic acid sequence according to item 13. (Item 16) 16. The vector according to item 15, wherein the vector is an expression vector comprising a promoter sequence operably linked to the nucleic acid sequence. (Item 17) 16. The vector of item 15, wherein the vector is a virus comprising a promoter sequence operably linked to the nucleic acid sequence. (Item 18) A host cell comprising the vector according to item 16. (Item 19) A method for producing a fusion polypeptide according to any one of items 4 to 12, comprising culturing a host cell according to item 18 under conditions in which the fusion polypeptide is expressed by the expression vector. (Item 20) 20. The method of claim 19, further comprising the step of isolating the fusion polypeptide produced. (Item 21) (i) an effective amount of the molecule according to any one of items 1 to 12, the nucleic acid sequence according to any one of items 13 to 14, the vector according to any one of items 15 to 17, or the host cell according to item 18; (ii) A pharmaceutical composition comprising a pharma- ceutically acceptable diluent, adjuvant or carrier. (Item 22) 22. The pharmaceutical composition according to item 21, comprising an effective amount of the molecule according to any one of items 1 to 12 and an effective amount of a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule. (Item 23) 22. The pharmaceutical composition according to item 21, comprising an effective amount of the nucleic acid sequence according to any one of items 13 to 14 and an effective amount of a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule, wherein the nucleic acid sequence encoding the Wnt polypeptide, the Norrin polypeptide, or the Wnt signaling agonist molecule is optionally DNA or mRNA. (Item 24) 24. The pharmaceutical composition of claim 23, wherein the nucleic acid sequence of any one of items 13 to 14 and / or the nucleic acid sequence encoding the Wnt polypeptide, the Norrin polypeptide, or the Wnt signaling agonist molecule is a modified mRNA. (Item 25) 23. The pharmaceutical composition according to item 22, comprising an effective amount of the vector according to any one of items 15 to 17 and an effective amount of a vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule, wherein the vector comprising the nucleic acid sequence encoding the Wnt polypeptide, the Norrin polypeptide, or the Wnt signaling agonist molecule is optionally an expression vector. (Item 26) (i) an effective amount of a nucleic acid encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule; (ii) A pharmaceutical composition comprising a pharma- ceutically acceptable diluent, adjuvant or carrier. (Item 27) 27. The pharmaceutical composition according to item 26, wherein the nucleic acid is DNA or mRNA, optionally modified mRNA. (Item 28) 27. The pharmaceutical composition according to item 26, wherein the nucleic acid is present in an expression vector, optionally a viral vector. (Item 29) 29. The pharmaceutical composition according to any of items 22 to 28, wherein the Wnt polypeptide is a mammalian Wnt polypeptide selected from Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Ant10A, Wnt10B, Wnt11, and Wnt16, and functional variants and fragments of any of these. (Item 30) 29. The pharmaceutical composition according to any of items 22 to 28, wherein the Norrin polypeptide is a mammalian Norrin polypeptide or a functional variant or fragment thereof. (Item 31) 29. The pharmaceutical composition according to any of items 22 to 28, wherein the Wnt signaling agonist molecule is a water-soluble Wnt signaling agonist that dimerizes the Frizzled (Fzd) receptor with Lrp5 / 6. (Item 32) 32. The pharmaceutical composition of claim 31, wherein the Wnt signaling agonist molecule comprises a polypeptide. (Item 33) 33. The pharmaceutical composition of claim 32, wherein the polypeptide comprises a binding domain having high affinity for one or more Fzd proteins and a binding domain having high affinity for an Lrp5 / Lrp6 protein, the binding domains being optionally directly linked or linked via a linker. (Item 34) 1. A method for increasing Wnt ("Wingless associated integration site" or "Wingless and Int-1" or "Wingless-Int") signaling in a target tissue, comprising: a) a molecule according to any one of items 1 to 12, a nucleic acid according to any one of items 13 to 14, a vector according to any one of items 15 to 17, or a host cell according to item 18, wherein the second domain specifically binds to a cell-specific surface molecule on the target tissue, and the molecule according to any one of items 1 to 12 binds to the target tissue and sequesters or increases the endocytosis of one or more transmembrane E3 ubiquitin ligases selected from zinc ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43) in the target tissue, and / or b) a host cell comprising a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule; a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule; a vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule; or an expression vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule, wherein the nucleic acid sequence encoding the Wnt polypeptide, the Norrin polypeptide, or the Wnt signaling agonist molecule is optionally DNA or mRNA, optionally modified mRNA. (Item 35) 35. The method according to item 34, comprising contacting the target tissue with a molecule according to any one of items 1 to 12. (Item 36) 35. The method according to item 34, comprising contacting the target tissue with the nucleic acid according to any one of items 13 to 14. (Item 37) 35. The method according to item 34, comprising contacting the target tissue with the vector according to any one of items 15 to 17. (Item 38) 35. The method of claim 34, comprising contacting the target tissue with the nucleic acid sequence encoding the Wnt polypeptide, the Norrin polypeptide, or the Wnt signaling agonist molecule. (Item 39) a) the tissue is bone tissue and the cell surface molecule is parathyroid hormone receptor 1 (PTH1R); b) the tissue is liver tissue and the cell surface molecule is asialoglycoprotein receptor 1 (ASGR1), asialoglycoprotein receptor 2 (ASGR2), transferrin receptor 2 (TFR2), or solute carrier family 10 member 1 (SLC10A1); or c) The method according to any one of items 34 to 39, wherein the tissue is an oral mucosal tissue and the cell surface molecule is Ly6 / PLAUR domain-containing 3 (LYPD3) or desmoglein (DSG3). (Item 40) 34. A method for treating or preventing a disease or condition in a subject in need thereof, said disease or disorder being associated with decreased Wnt ("Wingless-associated integration site" or "Wingless and Int-1" or "Wingless-Int") signaling or which would benefit from increased Wnt signaling, comprising providing to said subject an effective amount of a pharmaceutical composition according to any of items 21 to 33. (Item 41) A pharma- ceutically acceptable diluent, adjuvant or carrier and an effective amount of (a) a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule; (b) a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule, the nucleic acid sequence encoding the Wnt polypeptide, the Norrin polypeptide, or the Wnt signaling agonist molecule being optionally DNA or mRNA; (c) a vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule, the vector being optionally an expression vector; or (d) a host cell comprising an expression vector comprising a nucleic acid encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule. (Item 42) 42. The method of claim 41, comprising providing to the subject a pharmaceutical composition comprising an effective amount of the molecule of any of claims 1 to 12 and a pharmaceutical composition comprising an effective amount of a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule. (Item 43) 42. The method of claim 41, comprising providing to the subject a pharmaceutical composition comprising an effective amount of a nucleic acid sequence according to any one of claims 13-14 and an effective amount of a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule, wherein one or both of the nucleic acid sequences is optionally DNA or mRNA, optionally modified mRNA. (Item 44) 42. The method according to claim 41, comprising providing to the subject a pharmaceutical composition comprising an effective amount of the vector according to any one of claims 15 to 17 and an effective amount of a vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule, wherein one or both of the vectors is optionally an expression vector or a viral vector. (Item 45) A method for treating or preventing a disease or condition in a subject in need thereof, said disease or disorder being associated with decreased Wnt ("Wingless associated integration site" or "Wingless and Int-1" or "Wingless-Int") signaling or which would benefit from increased Wnt signaling, comprising administering to a subject in need thereof a pharmacopoeia comprising ... a) a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule, said nucleic acid sequence being optionally DNA or mRNA, optionally modified mRNA; b) a vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule, optionally an expression vector; or and c) a host cell comprising an expression vector comprising a nucleic acid encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule. (Item 46) Item 47: The method according to any one of items 41 to 45, wherein the Wnt polypeptide is a mammalian Wnt polypeptide selected from Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Ant10A, Wnt10B, Wnt11, and Wnt16, and functional variants and fragments of any of these. 46. ​​The method of any of items 41 to 45, wherein the Norrin polypeptide is a mammalian Norrin polypeptide or a functional variant or fragment thereof. (Item 48) 46. ​​The method according to any of items 41 to 45, wherein the Wnt signaling agonist molecule is a water-soluble Wnt signaling agonist that dimerizes the Frizzled (Fzd) receptor with Lrp5 / 6. (Item 49) 42. The method of claim 41, wherein the Wnt signaling agonist molecule comprises a polypeptide. (Item 50) 43. The method of claim 42, wherein the polypeptide comprises a binding domain having high affinity for one or more Fzd proteins and a binding domain having high affinity for an Lrp5 / Lrp6 protein, the binding domains being optionally directly linked or linked via a linker. (Item 51) 51. The method according to any of items 40 to 50, wherein the disease or disorder is a disease or disorder of a tissue selected from the group consisting of bone tissue, liver tissue, skin tissue, stomach tissue, intestinal tissue, oral mucosa tissue, kidney tissue, central nervous system tissue, mammary gland tissue, taste bud tissue, ovarian tissue, inner ear tissue (including cochlear and vestibular tissue), hair follicle, pancreatic tissue, retinal tissue, corneal tissue, cardiac tissue and lung tissue. (Item 52) The disease or disorder is a) a disease or disorder of bone tissue, in which the cell surface receptor is parathyroid hormone receptor 1 (PTH1R); b) a disease or disorder of liver tissue, wherein the cell surface receptor is asialoglycoprotein receptor 1 (ASGR1), asialoglycoprotein receptor 2 (ASGR2), transferrin receptor 2 (TFR2), or solute carrier family 10 member 1 (SLC10A1); or c) The method according to item 51, wherein the disease or disorder is a disease or disorder of the oral mucosal tissue, and the cell surface receptor is Ly6 / PLAUR domain-containing 3 (LYPD3) or desmoglein (DSG3). (Item 53) The disease or condition may be bone fracture, osteoporosis, osteoporotic fracture, spinal fusion, osseointegration of orthopedic devices, tendon-bone integration, tooth growth and regeneration, dental implant surgery, periodontal disease, maxillofacial reconstruction, osteonecrosis of the jaw, alopecia, hearing loss, vestibular dysfunction, macular degeneration, vitreous retinopathy, retinal degeneration, Fuchs' dystrophy, stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, spinal cord injury, oral mucositis, short bowel syndrome, inflammatory bowel disease (IBD), metabolic syndrome, diabetes, pancreatitis, exocrine pancreatic insufficiency, wound healing, diabetic foot ulcers, 51. The method according to any one of items 40 to 50, wherein the patient is selected from the group consisting of coronary artery disease, acute kidney disease, chronic kidney disease, chronic obstructive pulmonary disease (COPD), acute liver failure, acute alcoholic liver disease, chronic liver disease due to hepatitis C virus (HCV), HCV patients after antiviral drug treatment, chronic liver disease due to hepatitis B virus (HBV), HBV patients after antiviral drug treatment, chronic alcoholic liver disease, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH), cirrhosis, and chronic liver failure due to any cause. (Item 54) 54. The method according to any of items 40 to 53, wherein the pharmaceutical composition is provided parenterally, orally, intramuscularly, topically, or locally. (Item 55) 55. The method according to any one of items 40 to 54, wherein the subject is a mammal, optionally a human. [Brief description of the drawings]

[0030] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0031] [Figure 1] Diagrams representing the binding of R-spondin to ZNRF3 / RNF43 and LGR4-6 are provided. The diagram on the left shows wild-type R-spondin binding to both ZNRF3 / RNF43 and LGR4-6, while the diagram on the right shows an inactive R-spondin mutant lacking furin domain 2 (or with furin domain 2 mutated) that can bind to ZNRF3 / RNF43 but is unable or compromised in binding to LGR4-6. [Diagram 2] Provided herein is a schematic diagram of one embodiment of tissue-specific Wnt signal enhancement molecule disclosed herein.The molecule is a composite molecule that includes a fusion protein that includes, but is not limited to, a "targeting domain" that binds to tissue-specific cell surface protein and a "action domain" that can bind to ZNRF3 and / or RNF43. [Diagram 3] The figures illustrate the effect of tissue-specific Wnt signal enhancer molecules described herein and show that the molecules preferentially bind to target tissues. In non-target tissues that lack specific target cell surface receptors, tissue-specific Wnt signal enhancer molecules may or may not bind to ZNRF3 / RNF43, do not internalize or remove E3 ligase, and are essentially inactive in non-target tissues (upper figure). In target tissues that have specific target cell surface receptors, tissue-specific Wnt signal enhancer molecules bind to target tissues via their target domains, and action domains bind to ZNRF3 / RNF43 on target tissues, inducing the sequestration or endocytosis of ZNRF3 / RNF43 in target tissues (lower figure). [Figure 4]An alignment of all four human R-spondin proteins (Rspo1 (SEQ ID NO:1); Rspo2 (SEQ ID NO:2); Rspo3 (SEQ ID NO:3); and Rspo4 (SEQ ID NO:4)) is shown, with furin domains 1 (Fu1) and 2 (Fu2) shaded light and dark, respectively. The Fu1 domain generally corresponds to about amino acid residues 38-94 of SEQ ID NO:1, about amino acid residues 37-93 of SEQ ID NO:2, about amino acid residues 39-95 of SEQ ID NO:3, and about amino acid residues 32-88 of SEQ ID NO:4. The Fu2 domain generally corresponds to about amino acid residues 97-144 of SEQ ID NO:1, about amino acid residues 96-143 of SEQ ID NO:2, about amino acid residues 98-144 of SEQ ID NO:3, and about amino acid residues 91-137 of SEQ ID NO:4. [Diagram 5]We demonstrate cell-specific upregulation of Wnt signaling by an exemplary tissue-specific Wnt signal enhancing fusion protein. Figure 5A provides a scheme of the three constructs tested. From top to bottom: (1) anti-GFP fused to a functional human Rspo2 fragment (SEQ ID NO:6; encoding DNA provided in SEQ ID NO:5) containing wild-type Fu1 and Fu2 domains (amino acid residues 37-143), (2) anti-GFP fused to human Rspo2 with point mutations in the Fu2 domain (F105A / F109A) that preclude binding to LGR proteins (SEQ ID NO:8; encoding DNA provided in SEQ ID NO:7), (3) one antibody that binds to the human liver / hepatocyte specific surface receptor ASGR1 (SEQ ID NO:10; encoding DNA provided in SEQ ID NO:9) fused to the same Rspo2 mutant construct. Figure 5B shows quantitative PCR analysis of ASGR1 / 2 and ZNRF3 / RNF43 expression in human hepatoma Huh-7 cells and human epidermoid carcinoma A431 cells, representing hepatic and non-hepatic cells, respectively. The top graph shows the relevant expression levels compared to GAPDH control in the same cell line. The bottom graph shows the comparison with the relative levels of Huh-7, which were set as 1. Figure 5C shows the results of a reporter assay monitoring Wnt-enhancing activity in Huh-7 cells. The graph shows the results of a Super Top Flash (STF) reporter assay monitoring Wnt signaling activity. The cells contained a luciferase gene controlled by a Wnt-responsive promoter. The cells were transiently transfected with plasmids expressing fusion proteins designed as indicated. Three hours after transfection, Wnt3a-conditioned medium was added to constitute 10% of the total medium volume. Forty hours after transfection, the cells were assayed for luciferase activity. Luciferase activity was normalized to mock transfection (no DNA). The bottom of the figure shows a Western blot of the fusion proteins. All fusion proteins contained a signal peptide at the N-terminus for secretion (cleaved during protein maturation) and a FLAG tag at the C-terminus for detection. Antibodies were in the form of single-chain variable fragments (scFv).10 μl of culture supernatant was analyzed by Western blot using anti-FLAG monoclonal antibody M2, with the culture supernatant collected just before the luciferase assay. Figure 5D shows the results of a reporter assay monitoring Wnt-enhancing activity in A431 cells. The experimental setup was the same as that described in Figure 5C, except that the cells were co-transfected with vectors expressing either human TFR2 or human ASGR1. [Figure 6] Graphs are provided showing the effect of additional combinations of Rspo2 mutations on basal and targeted activity. Figure 6A focuses on several indicated residues in the Fu1 domain that are important for Rspo2 interaction with E3 ligases and shows the effect of these mutations in combination with the F105A / F109A double mutation. Figure 6B shows the effect of alleviating the double mutation in the LGR-binding Fu2 domain to the indicated single point mutation. Figure 6C provides an example of an additional mutation in the phenylamine 105 (F105R) residue of the Fu2 domain as an alternative to the F105A / F109A double mutation as a way to decrease interaction with the LGR protein. As shown in Figures 6A-6C, the F105R mutation, together with the F109A mutation, can reduce basal levels without significantly compromising targeted activity. In Figure 6C, the targeted domain is anti-human TFR1, which is further described in Figures 7A and 7B. Huh-7 cells were transfected with the specified constructs and assayed as described in the legends to Figures 5A-5D. [Figure 7] We provide another example by targeting a second receptor, human TFR1. Figure 7A shows a reporter assay based on transient transfection of Huh-7 cells. Figure 7B shows the same assay in A431 cells. The Western blot is an image from the same membrane detecting the FLAG tag of the fusion protein. Further procedural details can be found in the legends of Figures 5A-5D. [Figure 8-1]Enhancement of Wnt signaling activity by purified proteins fused to mutant Rspo2 targeting (anti-ASGR1 or anti-TFR1) or non-targeting (control, anti-GFP) domains in scFv form is shown. Figure 8A shows Coomassie stained gel images of proteins consisting of mutant Rspo2 as specific targeting and action domains. On the left are the kD of protein standards. Figure 8B compares the STF activity of ASGR1-targeted Rspo2 (F105A / F109A) or (F109) mutant fusion proteins with a negative control (anti-GFP construct; left) and a positive control (Rspo2, right) in Huh-7 cells, which corresponds to the human Rspo2 Fu1 and Fu2 domains (S36-E143) with a His-tag at the C-terminus. Figure 8C provides a comparison of the three target proteins with the Rspo2 positive control in three different cell lines: human hepatoma Huh-7, human colorectal adenocarcinoma HT29, and mouse normal liver FL83B. The TFR1 antibody is human specific, whereas the ASGR1 antibody can cross-react with mouse ASGR1. All three cell lines were engineered with a reporter gene and treated with specific concentrations of proteins for approximately 18 hours in the presence of 10% Wnt3a conditioned medium. For each dose tested, data from Huh-7 cells are on the left, data from HT29 cells are in the middle, and data from FL83B cells are on the right. [Figure 8-2]Enhancement of Wnt signaling activity by purified proteins fused to mutant Rspo2 targeting (anti-ASGR1 or anti-TFR1) or non-targeting (control, anti-GFP) domains in scFv form is shown. Figure 8A shows Coomassie stained gel images of proteins consisting of mutant Rspo2 as specific targeting and action domains. On the left are the kD of protein standards. Figure 8B compares the STF activity of ASGR1-targeted Rspo2 (F105A / F109A) or (F109) mutant fusion proteins with a negative control (anti-GFP construct; left) and a positive control (Rspo2, right) in Huh-7 cells, which corresponds to the human Rspo2 Fu1 and Fu2 domains (S36-E143) with a His-tag at the C-terminus. Figure 8C provides a comparison of the three target proteins with the Rspo2 positive control in three different cell lines: human hepatoma Huh-7, human colorectal adenocarcinoma HT29, and mouse normal liver FL83B. The TFR1 antibody is human specific, whereas the ASGR1 antibody can cross-react with mouse ASGR1. All three cell lines were engineered with a reporter gene and treated with specific concentrations of proteins for approximately 18 hours in the presence of 10% Wnt3a conditioned medium. For each dose tested, data from Huh-7 cells are on the left, data from HT29 cells are in the middle, and data from FL83B cells are on the right. [Figure 9]The target Wnt signal enhancement activity of constructs with targeting domains in the form of full IgG is shown. At the top is a diagram of the appended IgG constructs. The top diagram shows the Rspo2 F105R / F109A mutant appended to the N-terminus of a human IgG2 heavy chain directed against either GFP or the human TFR1 receptor. The bottom diagram shows the Rspo2 F105R / F109A mutant appended to the N-terminus of a human IgG2 light chain directed against either GFP or the human TFR1 receptor. These constructs (each with light and heavy chains) together with the TFR1 single chain variable fragment (scFv) fused to the Rspo2 F105R / F109A mutant were transiently transfected into HEK293T cells (expressing the TFR1 receptor) with a luciferase reporter. At the bottom is a schematic overview of the STF assay 40 hours after transfection. After transfection, 10% Wnt3a conditioned medium was added to the cultures. STF assays were performed as described in the legends of Figures 5A-5D. [Figure 10] We provide graphs showing results obtained with additional combinations of Rspo2 mutations as a way to reduce interaction with LGR proteins and support targeted Wnt enhancing activity when fused to a targeting domain. Specific constructs were introduced into Huh-7 cells by transient transfection. Further procedural details can be found in the legends of Figures 5A-5D. [Figure 11] STF activity of Rspo2(F105R / F109A) mutant fusion protein targeting TFR1 is compared with a negative control (anti-GFP construct) and a positive control (Rspo2) in Huh-7 cells. Luciferase activity is in arbitrary units. Further procedural details can be found in the description of Figures 8A-8C. [Figure 12]A comparison is provided of the non-ASGR1 expressing cell line A431 in response to anti-ASGR1-Rspo2 (F105A / F109A) fusion protein upon transfection with plasmids overexpressing either ASGR1 (left) or TFR2 (right, used as a control). All cell lines incorporate a luciferase reporter gene and were first transfected with the specific receptor plasmid. After 24 hours, cells were treated with the specified concentrations of protein for approximately 18 hours in the presence of 10% Wnt3a conditioned medium and then assayed for luciferase activity. [Figure 13] Wnt signal stimulatory activity of purified loaded IgG proteins targeting human TFR1 receptor. F105R / F109A Rspo2 mutant fused to the N-terminus of the heavy chain of IgG2 against TFR1 was compared to fusion with anti-GFP with Rspo2 positive control. STF assays were performed in two cell lines, Huh-7 on the left and HEK293T on the right, containing the STF reporter. Both cell lines express the target receptor TFR1. Luciferase activity is in arbitrary units. [Figure 14-1] Results obtained with an exemplary scaffold of adjunct IgG are shown. Mutant Rspo2 was covalently linked to the N-terminus of the heavy chain (N-HC), the N-terminus of the light chain (N-LC), or the C-terminus of the light chain (C-LC) as in the right diagram of FIG. 14A. Two types of IgG were analyzed: IgG2 (wild type, FIG. 14A-C) or IgG1 (with the N297G "effectorless" mutation, FIG. 14D-E). Targeting antibodies were anti-human ASGR1 and TFR1, and control antibody was anti-GFP. The STF activity of the purified proteins was tested in Huh-7 (hASGR1+ / hTFR1+; Figures 14A and 14D), 293 (hASGR1- / hTFR1+; Figures 14B and 14E), and FL83B (mouse cell line, hASGR1- / hTFR1-; Figure 14C) cell lines in the presence of 30% Wnt3a conditioned medium. Rspo2 was used as a positive control. The negative control was treatment with 30% Wnt3a conditioned medium alone ("Wnt only"). [Figure 14-2]Results obtained with an exemplary scaffold of adjunct IgG are shown. Mutant Rspo2 was covalently linked to the N-terminus of the heavy chain (N-HC), the N-terminus of the light chain (N-LC), or the C-terminus of the light chain (C-LC) as in the right diagram of FIG. 14A. Two types of IgG were analyzed: IgG2 (wild type, FIG. 14A-C) or IgG1 (with the N297G "effectorless" mutation, FIG. 14D-E). Targeting antibodies were anti-human ASGR1 and TFR1, and control antibody was anti-GFP. The STF activity of the purified proteins was tested in Huh-7 (hASGR1+ / hTFR1+; Figures 14A and 14D), 293 (hASGR1- / hTFR1+; Figures 14B and 14E), and FL83B (mouse cell line, hASGR1- / hTFR1-; Figure 14C) cell lines in the presence of 30% Wnt3a conditioned medium. Rspo2 was used as a positive control. The negative control was treatment with 30% Wnt3a conditioned medium alone ("Wnt only"). [Figure 15] We provide results from a comparison of all four human R-spondins showing targeted Wnt signal enhancement activity in selected cell lines. Figure 15A compares the Wnt signal enhancement activity of the Fu1-Fu2 domains of wild-type human Rspo1-4 fused to anti-GFP (scFv format) in human Huh-7 (left) or HEK293T (right) cells. Figure 15B shows the enhanced (targeted) activity of human Rspo2 mutants (F105A / F109A, "AA", or F105R / F109A, "RA") when fused to anti-ASGR1 or anti-TFR1 in Huh-7 (hASGR1+ / hTFR1+) or HEK293T (hASGR1- / hTFR1+) cells, using fusion to anti-GFP as a control for basal non-targeted activity. Figure 15C shows the enhanced (targeting) activity of human Rspo3 mutants when fused to anti-ASGR1 or anti-TFR1 in Huh-7 or HEK293T cells, in contrast to anti-GFP fusion controls. 30% Wnt3a conditioned medium was supplied to the STF assay. Rspo2 was used as a positive control. The negative control was treatment with 30% Wnt3a conditioned medium alone ("Wnt only"). [Figure 16]We provide non-limiting examples of Rspo3 mutations that can be used to construct tissue-specific Wnt signaling enhancers. Figure 16A lists the mutations that were tested. These mutant Rspo3 (Fu1-Fu2) domains were fused to anti-ASGR1 or anti-GFP scFv, and the activity of purified proteins was tested in Huh-7 cells by STF assay in the presence of 30% Wnt3a conditioned medium. The results are summarized in Figure 16B (for anti-GFP control) and Figure 16C (for anti-ASGR1 targeting construct). Rspo2 fused to anti-GFP and wild-type Rspo3 (Fu1-Fu2 domain) were used as positive controls in the assay. [Figure 17] Figure 17 shows an example of a Wnt signal enhancing molecule design consisting of an E3 ligase binder whose action domain is structurally independent of the Rspo scaffold. Figure 17A shows the structure of the functional molecule tested, which consists of a Fab against human ZNRF3 (or anti-GFP as a negative control) covalently linked to the N-terminus of the heavy chain of IgG2 against human ASGR1 or TFR1. Figure 17B shows the results of STF using human liver Huh-7 cells. The assay was performed in the presence of 30% Wnt conditioned medium, and Rspo2 was used as a positive control. [Figure 18]We provide an example of applying the tissue-specific Wnt signal enhancement design to other target tissues, oral mucosa via specific cell surface receptors. Figure 18A shows Q-PCR analysis of LYPD3, DSG3, ZNRF3 and RNF43 gene expression in three different cell lines. CAL27 and SCC25 are squamous cell carcinoma cell lines originally derived from human tongue, and A431 is an epidermoid carcinoma cell line derived from human skin. The top graph shows the relative expression level of each gene compared to the ACTB gene control, and the bottom graph shows a comparison with the relative level of each gene in CAL27, which was set as 1. Figure 18B shows the results of an STF reporter assay monitoring the Wnt signal enhancement activity of specific proteins in CAL27, SCC25 and A431 cells after treatment for 16-18 hours in the presence of 30% Wnt3a conditioned medium. Proteins were constructed by fusing the Rspo2(F105R / F109A) mutant to the N-terminus of the heavy chains of monoclonal antibodies against human LYPD3, human DSG3, and GFP. Rspo2 was used as a control. [Figure 19] Figure 19A shows the in vivo function of an exemplary liver-specific Wnt signal enhancer molecule. Figure 19A shows the experimental scheme. To introduce ectopic expression of hASGR1 in liver, 8-week-old mice are first injected with AAV vectors that contain hASGR1 coding sequence. After 7 days, mice are divided into 8 groups and treated with test and control proteins. After 8 hours, mice are euthanized and liver samples are taken for quantitative PCR analysis of gene expression. Figure 19B shows the expression level of ectopic hASGR1 in mouse liver. The doses of treatment were anti-GFP (1 mg / kg), Rspo2 (0.46 mg / kg), anti-GFP-Rspo2(F105R / F109A)(1 mg / kg), or anti-ASGR1-Rspo2(F105R / F109A)(1 mg / kg), either alone (four left groups) or in combination with 3 mg / kg of Wnt agonist protein (18R5-Dkk1c, Janda et al., 2017 Nature) (four right groups). Figure 19C shows the induction of the Wnt signaling target gene Axin2 in response to treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present disclosure provides tissue-specific Wnt signal enhancing molecules, in certain embodiments, that 1) selectively bind to tissue-specific or cell-specific cell surface receptors, 2) mediate internalization or sequestration of ZNRF3 / RNF43 in target tissues or cell types, and 3) enhance Wnt signaling in a tissue-specific manner. In certain embodiments, the molecules are fusion proteins. In certain embodiments, the molecules are antibodies with additional additional binding domains. Also provided are pharmaceutical compositions and methods for the use of any of the compositions disclosed herein to enhance, i.e., increase, Wnt signaling in target tissues or cell types, for example, for the treatment or prevention of a disease or disorder. These and other objects, advantages, and features of the present invention will become apparent to those skilled in the art upon reading the details of the compositions and methods as more fully described below.

[0033] definition As used herein, a "vector" refers to a macromolecule or an association of macromolecules that contains or associates with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell. Exemplary vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles.

[0034] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute a double-stranded form.

[0035] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, which means that when aligned, the percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs including BLAST, available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package from Oxford Molecular Group, Inc., a wholly owned subsidiary of Madison, Wisconsin, USA. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in sequences. Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70:173-187 (1997). Also, sequences can be aligned using the GAP program using the Needleman and Wunsch alignment method. See J. Mol. Biol. 48:443-453 (1970).

[0036] Of interest is the program BestFit (Advances in Genetics), which uses the local homology algorithm of Smith and Waterman to determine sequence identity. Applied Mathematics 2:482-489(1981)). Gap creation penalties generally range from 1 to 5, usually 2 to 4, and in many embodiments are 3. Gap extension penalties generally range from about 0.01 to 0.20, and often are 0.10. The program has default parameters determined by the sequences entered to be compared. Preferably, sequence identity is determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package, Madison, Wisconsin, USA.

[0037] Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based on the following parameters: mismatch penalty: 1.00, gap penalty: 1.00, gap size penalty: 0.33, and joining penalty: 30.0.

[0038] "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in constructs that differ from polynucleotides found in nature.

[0039] A "control element" or "control sequence" is a nucleotide sequence involved in molecular interactions that contribute to the functional regulation of a polynucleotide, including the duplication, replication, transcription, splicing, translation, or degradation of the polynucleotide. Regulation can affect the frequency, rate, or specificity of the process and can be enhancing or repressive in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region that can bind RNA polymerase under certain conditions and initiate transcription of a coding region that is usually located downstream (3' direction) of the promoter.

[0040] "Functionally linked" or "operably linked" refers to the juxtaposition of genetic elements, where the elements are in a relationship that allows them to function in an expected manner. For example, a promoter is operably linked to a coding region if it helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and the coding region, so long as this functional relationship is maintained.

[0041] An "expression vector" is a vector that contains a region that codes for a gene product of interest and is used to effect expression of the gene product in the intended target cell. An expression vector also contains a control element operably linked to the coding region to facilitate expression of the gene product in the target. The combination of a control element and a gene or genes that are operably linked for expression may be referred to as an "expression cassette", many of which are known and available in the art or can be readily constructed from elements available in the art.

[0042] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. The terms also encompass modified amino acid polymers, including, for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0043] As used herein, the term "antibody" refers to an isolated or recombinant binding agent that contains the necessary variable region sequence to specifically bind to an antigenic epitope. Thus, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, such as binding to a specific target antigen. Thus, it is used in the broadest sense, and includes, in particular, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including, but not limited to, scFv, Fab, and Fab2, so long as they exhibit the desired biological activity.

[0044] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies (e.g., Zapata et al., Protein Eng. 8(10):1057-1062(1995)), single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and an "Fc" fragment, a name reflecting its ability to crystallize easily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0045] "Comprising" means that the recited elements are required, e.g., for a composition, method, kit, etc., but that other elements may be included, within the scope of the claims, to form, e.g., a composition, method, kit, etc. For example, an expression cassette that "comprises" a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements, in addition to the gene and promoter, e.g., poly-adenylation sequences, enhancer elements, other genes, linker domains, etc.

[0046] "Consisting essentially of" means that the scope of the described, e.g., composition, method, kit, etc., is limited to the specified materials or steps that do not substantially affect the basic and novel properties of the, e.g., composition, method, kit, etc. For example, an expression cassette "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and a polyadenylation sequence may include additional sequences, e.g., linker sequences, so long as they do not substantially affect the transcription or translation of the gene. As another example, a variant, or mutant, polypeptide fragment "consisting essentially of" a recited sequence has the amino acid sequence of the recited sequence plus or minus about 10 amino acid residues, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue less than the recited binding amino acid residues, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues more than the recited binding amino acid residues, at the sequence boundaries based on the full-length native polypeptide from which it is derived.

[0047] "Consisting of" means that a composition, method, or kit excludes any element, step, or ingredient not specified in the claim. For example, a polypeptide or polypeptide domain "consisting of" a recited sequence includes only the recited sequence.

[0048] "Expression vector" as used herein includes vectors described above or known in the art, such as plasmids, minicircles, viral vectors, liposomes, etc., that contain a polynucleotide encoding a gene product of interest and are used to effect expression of the gene product in the intended target cell. Expression vectors also contain control elements operably linked to the coding region to facilitate expression of the gene product in the target. The combination of control elements, such as promoters, enhancers, UTRs, miRNA target sequences, etc., and operably linked gene(s) for expression is sometimes referred to as an "expression cassette." Many such control elements are known and available in the art or can be readily constructed from components available in the art.

[0049] "Promoter" as used herein encompasses a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and the expression of the corresponding proteins or polypeptides may be ubiquitous, i.e., strongly active in a wide range of cells, tissues and species, or may be cell type-specific, tissue-specific, or species-specific. Promoters may be "constitutive," i.e., continuously active, or "inducible," i.e., activated or deactivated by the presence or absence of a biotic or abiotic factor. Enhancer sequences, which may or may not be contiguous with the promoter sequence, are also included in the nucleic acid construct or vector of the present invention. Enhancer sequences affect promoter-dependent gene expression and may be located in the 5' or 3' region of the native gene.

[0050] The term "natural" or "wild type" as used herein refers to a nucleotide sequence, e.g., a gene, or a gene product, e.g., an RNA or protein, present in a wild-type cell, tissue, organ, or organism. The term "variant" as used herein refers to a variant of a reference polynucleotide or polypeptide sequence, e.g., a natural polynucleotide or polypeptide sequence, i.e., one that has less than 100% sequence identity to the reference polynucleotide or polypeptide sequence. In other words, a variant comprises at least one amino acid difference (e.g., an amino acid substitution, an amino acid insertion, an amino acid deletion) to a reference polynucleotide sequence, e.g., a natural polynucleotide or polypeptide sequence. For example, a variant may be a polynucleotide that has 50% or more, 60% or more, or 70% or more sequence identity to a full-length natural polynucleotide sequence, e.g., 75% or 80% or more identity, e.g., 85%, 90%, or 95% or more, e.g., 98% or 99% identity to a full-length natural polynucleotide sequence. As another example, a variant may be a polypeptide having 70% or more sequence identity to a full-length native polypeptide sequence, e.g., 75% or 80% or more, e.g., 85%, 90%, or 95% or more identity, e.g., 98% or 99% identity to a full-length native polypeptide sequence. Variants may also include variant fragments of a reference, e.g., native sequence, that share 70% or more sequence identity with a reference, e.g., a fragment of a native sequence, e.g., 75% or 80% or more, e.g., 85%, 90%, or 95% or more, e.g., 98% or 99% identity with a native sequence.

[0051] As used herein, the terms "biological activity" and "biologically active" refer to the activity attributed to a particular biological element in a cell. For example, the "biological activity" of R-spondin, or its fragment or variant, refers to its ability to enhance Wnt signal. As another example, the biological activity of a polypeptide, or its functional fragment or variant, refers to the ability of the polypeptide, or its functional fragment or variant, to perform its native function, such as, for example, binding, enzymatic activity, etc. As a third example, the biological activity of a gene regulatory element, such as a promoter, enhancer, Kozak sequence, etc., refers to the ability of the regulatory element, or its functional fragment or variant, to regulate, i.e., promote, enhance, or activate, the translation of the expression of the gene operably linked thereto, respectively.

[0052] As used herein, the terms "administering" or "introducing" or "providing" refer to delivering a composition to a cell, to a cell, tissue and / or organ of a subject, or to a subject. Such administration or introduction can occur in vivo, in vitro or ex vivo.

[0053] The terms "treatment", "treating" and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, e.g., reducing the likelihood of a disease or its symptoms occurring in a subject, and / or may be therapeutic, in terms of a partial or complete cure for a disease and / or adverse effects resulting from the disease. "Treatment", as used herein, encompasses any treatment of a disease in a mammal, including (a) preventing the disease from arising in a subject who may be predisposed to the disease, but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its onset; or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents can be administered before, during, or after the onset of the disease or injury. Of particular interest is the treatment of ongoing disease, where the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is desirably performed before complete loss of function of affected tissues. The subject therapies are desirably administered at, and in some cases after, the symptomatic stage of the disease.

[0054] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates, including monkeys and humans; mammalian sports animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pet animals (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0055] Various compositions and methods of the present invention are described below. Although certain compositions and methods are exemplified herein, it is understood that a number of alternative compositions and methods are applicable and suitable for use in practicing the present invention. It will also be understood that evaluation of the expression constructs and methods of the present invention can be performed using standard procedures in the art.

[0056] The practice of the present invention employs, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of one of ordinary skill in the art. Such techniques may be found in such publications as may be found in "Molecular Cloning: A Laboratory Manual", 2nd Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology” (DMWeir & CC Blackwell (eds.); "Gene Transfer Vectors for Mammalian Cells" (JM Miller & MP Calos (eds., 1987); "Current Protocols in Molecular Biology" (FMA Usubel et al. (eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al. (eds., 1994); and "Current Protocols in Immunology" (JE Coligan et al. (eds., 1991).

[0057] Some aspects of the present invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the present invention. However, it will be readily apparent to one skilled in the relevant art that the present invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited to the illustrated order of acts or events, and some acts may occur in a different order and / or simultaneously with other acts or events. Moreover, not all illustrated acts or events are necessary to perform the methodology according to the present invention.

[0058] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the present invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent the terms "including", "includes", "having", "has", "with" or variations thereof are used in any of the description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising".

[0059] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, the acceptable error range depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within one or more standard deviations, according to practice in the art. Alternatively, "about" may mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold. When a particular value is described in the present application and claims, the term "about" should be assumed to mean within an acceptable error range for the particular value, unless otherwise indicated.

[0060] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited, and it is understood that the present disclosure supersedes any disclosure of the incorporated publications to the extent of any conflict.

[0061] It is further noted that the claims may be drafted to exclude any element, and thus, this statement is intended to serve as a precedent for the use of exclusive terminology such as "solely," "solely," and the like or the use of "negative" limitations in connection with the recitation of claim elements.

[0062] Unless otherwise specified, all terms used herein have the same meaning as they would to one of ordinary skill in the art and the practice of the present invention employs conventional techniques of microbiology and recombinant DNA technology within the knowledge of those skilled in the art.

[0063] Tissue-specific Wnt signal enhancer In certain aspects, the present disclosure provides novel tissue-specific Wnt signal enhancing molecules that can enhance Wnt activity in a tissue-specific or cell-specific manner. In certain embodiments, the tissue-specific Wnt signal enhancing molecules are bifunctional molecules that include a first domain that binds to one or more ZNRF3 and / or RNF43 ligases, and a second domain that binds to one or more target tissues or cell types in a tissue-specific or cell-specific manner. Each of the first domain and the second domain can be any moiety that can bind to a ligase complex or a target tissue or cell, respectively. For example, each of the first domain and the second domain can be a moiety selected from, but not limited to, a polypeptide (e.g., an antibody or an antigen-binding fragment thereof, or a peptide or polypeptide different from an antibody), a small molecule, and a natural ligand or a variant, fragment, or derivative thereof. In certain embodiments, the natural ligand is a polypeptide, a small molecule, an ion, an amino acid, a lipid, or a sugar molecule. The first domain and the second domain can be the same type of moiety as each other, or can be different types of moieties. In certain embodiments, the tissue-specific Wnt signal enhancer molecule binds to a tissue-specific or cell-specific cell surface receptor. In certain embodiments, the tissue-specific Wnt signal enhancer molecule increases or enhances Wnt signaling by at least 50%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, or at least 50-fold, for example, compared to a negative control.

[0064] In certain embodiments, the tissue-specific Wnt signal enhancer molecule is a fusion protein comprising a first polypeptide sequence that binds to ZNRF3 / RNF43 and a second polypeptide sequence that binds to one or more target tissues or cell types in a tissue- or cell-specific manner.In certain embodiments, the tissue-specific Wnt signal enhancer molecule comprises two or more polypeptides, such as a dimer or multimer comprising two or more fusion proteins each comprising a first domain and a second domain, where the two or more polypeptides are linked together, for example, through a linker moiety or via a bond between amino acid residues in each of the two or more polypeptides, for example, an intermolecular disulfide bond between cysteine ​​residues.In certain embodiments, the tissue-specific Wnt signal enhancer molecule is an antibody (or an antigen-binding fragment thereof) comprising an antibody heavy chain and a light chain that constitute either the first domain or the second domain, where the other domain (i.e., the second domain or the first domain) is linked to the antibody heavy chain or light chain, either as a fusion protein or via a linker moiety. In certain embodiments, the other domain is attached to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, or the C-terminus of the light chain. Such structures may be referred to herein as IgG-added scaffolds or formats. For example, the tissue-specific Wnt signal enhancer molecule may be an antibody that binds to ZNRF3 / RNF43, where a binding domain that binds to a tissue- or cell-specific receptor is fused or added to either the heavy or light chain of the antibody that binds to ZNRF3 / RNF43. In another example, the tissue-specific Wnt signal enhancer molecule may be an antibody that binds to a tissue- or cell-specific receptor, where a binding domain that binds to ZNRF3 / RNF43 is fused or added to either the heavy or light chain of the antibody that binds to a tissue- or cell-specific receptor.

[0065] In certain embodiments, tissue-specific Wnt signal enhancing molecule comprises a first domain ("action domain") that binds to ZNRF3 / RNF43 and a second domain ("targeting domain") that binds to tissue-specific or cell-specific receptor, e.g., with high affinity. In certain embodiments, each of these two domains has substantially low activity or is inactive in enhancing Wnt signal by itself. However, when tissue-specific Wnt signal enhancing molecule binds to target tissue that expresses tissue-specific receptor, E3 ligase ZNRF3 / RNF43 is recruited to ternary complex with tissue-specific receptor, and they are sequestered and / or removed from cell surface by receptor-mediated endocytosis. As a result, Wnt signal is enhanced in a tissue-specific manner.

[0066] In certain embodiments, the action domain is a binder to ZNRF3 / RNF43E3 ligase, which can be designed based on R-spondin, for example, R-spondin1-4, including but not limited to human R-spondin1-4. In certain embodiments, the action domain is an R-spondin, for example, wild-type R-spondin1-4, optionally human R-spondin1-4, or a variant or fragment thereof. In certain embodiments, it is a variant of any of R-spondin1-4 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the corresponding wild-type R-spondin1-4 sequence. In certain embodiments, the action domain comprises or consists of the furin domain 1 of any of R-spondin, for example, R-spondin1-4, which binds to ZNRF3 / RNF43. Extended versions of furin domain 1 (including but not limited to those with mutant furin domain 2 that no longer binds to LGR4-6 or has reduced binding to LGR4-6), or engineered antibodies or any other derivative or engineered polypeptide different from the antibody, that can specifically bind to ZNRF3 / RNF43, can also be used. In certain embodiments, the action domain comprises one or more furin domains 1 of R-spondin. In certain embodiments, it does not comprise the furin domain 2 of R-spondin, or it comprises a modified or variant furin domain 2 of R-spondin, such as a furin domain 2 that has reduced activity compared to the wild-type furin domain 2. In certain embodiments, the action domain comprises a furin domain 1 but not the furin domain 2 of R-spondin. In certain embodiments, the action domain comprises two or more furin domains 1 or a multimer of furin domain 1. The action domain may comprise one or more wild-type furin domains 1 of R-spondin. In certain embodiments, the effect domain comprises a modified or variant furin domain 1 of R-spondin that has increased activity, e.g., increased binding to ZNRF3 / RNF43, compared to wild-type furin domain 1.Variants with increased binding to ZNRF3 / RNF43 can be identified, for example, by screening phage or yeast display libraries that contain variants of R-spondin furin domain 1. Peptides or polypeptides that are unrelated to R-spondin furin domain 1 but have increased binding to ZNRF3 / RNF43 can also be identified by screening. The action domain can further comprise additional moieties or polypeptide sequences, such as additional amino acid residues to stabilize the structure of the action domain or tissue-specific Wnt signal enhancement molecule in which it is present.

[0067] In certain embodiments, the targeting domain specifically binds to a cell-specific surface molecule, e.g., a cell-specific surface receptor, and may be, e.g., a natural ligand, an antibody, or a synthetic chemical. In certain embodiments, the cell-specific surface molecule is preferentially expressed in a target organ, tissue, or cell type, e.g., an organ, tissue, or cell type for which it is desirable to enhance Wnt signaling. In certain embodiments, the cell-specific surface molecule has increased or enhanced expression in a target organ, tissue, or cell type, e.g., an organ, tissue, or cell type for which it is desirable to enhance Wnt signaling, e.g., to treat or prevent a disease or disorder, when compared to one or more other non-target organs, tissues, or cell types. In certain embodiments, the cell-specific surface molecule is preferentially expressed on the surface of a target organ, tissue, or cell type, respectively, compared to one or more other organs, tissues, or cell types. For example, in certain embodiments, a cell surface receptor is considered to be a tissue- or cell-specific cell surface molecule if it is expressed at a level at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold higher in a target organ, tissue, or cell than it is expressed in one or more, five or more, all other organs, tissues, or cells, or the average of all other organs, tissues, or cells, respectively. In certain embodiments, a tissue- or cell-specific cell surface molecule is a cell surface receptor, e.g., a polypeptide receptor that includes a region located within the cell surface membrane and an extracellular region to which a targeting domain can bind. In various embodiments, the methods described herein may be performed by specifically targeting a cell surface molecule that is expressed only on the target tissue or a subset of tissues that includes the target tissue, or by specifically targeting a cell surface molecule that has a higher level of expression on the target tissue when compared to all, most, or a significant number of other tissues, e.g., higher expression in the target tissue than at least 2, at least 5, at least 10, or at least 20 other tissues.

[0068] Tissue-specific and cell-specific cell surface receptors are known in the art.Examples of tissue-specific and cell-specific surface receptors include, but are not limited to, ASGR1 (liver-specific), ASGR2 (liver-specific), TFR2 (liver-specific), SLC10A1 (liver-specific), PTH1R (bone and kidney-specific), LYPD3 (oral mucosa-specific), DSG3 (oral mucosa-specific) and the like (see Figure 2).Additional receptors for liver delivery are described, for example, in Yan et al., Tumor Biology, 2015; 36: 55-67.

[0069] An acting or targeting domain, e.g., an antibody or antigen-binding fragment thereof, that "specifically binds" or is "specific" for a particular cell surface polypeptide or receptor is one that binds to that particular polypeptide or receptor without substantially binding to other polypeptides or polypeptide epitopes. In some embodiments, the acting and targeting domains of the disclosure bind to ZNRF3 / RNF43 or a tissue-specific cell surface molecule (e.g., a receptor) with a dissociation constant (K) of 1000 nM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, 0.01 nM or less, 0.005 nM or less, 0.001 nM or less, or 0.0005 nM or less when measured at temperatures of about 4° C., 25° C., 37° C., or 42° C., respectively. d ) specifically binds to the antigen. The affinity of a binding agent, e.g., an antibody, can be readily determined using conventional techniques, such as those described by Scatchard et al. (Ann. NY Acad. Sci. USA 51:660 (1949), ELISA assays, biolayer interferometry (BLI) assays, and surface plasmon resonance (SPR) assays). The binding characteristics of an antibody to an antigen, its cells or tissues can generally be determined and evaluated using immunodetection methods, including, for example, immunofluorescence-based assays such as immunohistochemistry (IHC) and / or fluorescence-activated cell sorting (FACS).

[0070] In certain embodiments, the action domain and / or target domain of the tissue-specific Wnt signal enhancer molecule are polypeptides, and in other embodiments, the action domain and / or target domain of the tissue-specific Wnt signal enhancer molecule are small organic molecules. In certain embodiments, the action domain and target domain of the tissue-specific Wnt signal enhancer molecule are covalently linked to each other. In certain embodiments, the action domain and target domain of the tissue-specific Wnt signal enhancer fusion molecule are non-covalently linked to each other. In certain embodiments, the action domain and target domain of the tissue-specific Wnt signal enhancer molecule are present in the same fusion protein. In other embodiments, the action domain is present in a first polypeptide further comprising a first binding domain, and the target domain is present in a second polypeptide further comprising a second binding domain, where the first and second binding domains bind to each other. In some embodiments, the first and second binding domains are the same or variants thereof, e.g., Fc polypeptides. In some embodiments, the first and second binding domains are different from each other. In certain embodiments, the invention includes the use of any target domain or action domain fragment or variant described herein, including functional fragments or variants of the reference molecule.

[0071] In certain embodiments, the tissue-specific Wnt signal enhancing molecule (e.g., fusion protein) has a formula selected from R1-L-R2 and R2-L-R1, where R1 is an action domain that binds to ZNRF3 / RNF43, R2 is a target domain that binds to a tissue-specific cell surface receptor, L is a linker, and L may be present or absent. Each of R1 and R2 can be any of the various action domains and target domains described herein. Each of R1 and R2 can be one or more E3 ligases (ZNRF3 or RNF43), or any moiety that can bind to a target tissue or cell, respectively. For example, each of R1 and R2 can be a moiety selected from, but not limited to, a polypeptide (e.g., an antibody or an antigen-binding fragment thereof, or a peptide or polypeptide different from an antibody), a small molecule, and a natural ligand or a variant, fragment, or derivative thereof. In certain embodiments, the natural ligand is a polypeptide, a small molecule, an ion, an amino acid, a lipid, or a sugar molecule. The effector domain and the targeting domain (i.e., R1 and R2) can be the same type of moiety as each other, or they can be different types of moieties. In certain embodiments, R2 is an antibody of an antigen-binding fragment thereof, and in certain embodiments, R2 comprises an Fc protein or an analog thereof.

[0072] In certain embodiments, the tissue-specific Wnt signal enhancing molecule comprises a single molecule (e.g., a polypeptide), while in other embodiments, the Wnt signal enhancing fusion molecule comprises two or more molecules (e.g., polypeptides) linked together, e.g., non-covalently linked together. For example, in one embodiment, the tissue-specific Wnt signal enhancing fusion comprises two molecules having the formulas R3-L1 and R4-L2, respectively, where R3 is an action domain, R4 is a target domain, and the L1 and L2 groups are linked together, e.g., to form a dimer. In various embodiments, the L1 and L2 groups are identical to each other or different from each other. An example of an L1 or L2 group is an Fc sequence, e.g., mouse Fc2b or human Fc1, each of which is known in the art. Each of R3 and R4 can be any of the various action domains and target domains, respectively, described herein. Each of R3 and R4 can be one or more E3 ligases (ZNRF3 and / or RNF43), or any moiety that can bind to a target tissue or cell, respectively. In certain embodiments, the tissue-specific Wnt signal enhancer molecule comprises an antibody or a binding fragment thereof that binds to one or more E3 ligases (ZNRF3 and / or RNF43), wherein the antibody heavy chain and / or the antibody light chain comprises an additional binding domain that binds to a target tissue or cell. In certain embodiments, the tissue-specific Wnt signal enhancer molecule comprises an antibody or a binding fragment thereof that binds to a target tissue or cell, wherein the antibody heavy chain and / or the antibody light chain comprises an additional binding domain that binds to one or more E3 ligases (ZNRF3 and / or RNF43). The additional binding domain may be fused directly to the N-terminus or C-terminus of the antibody, e.g., as a heavy or light chain fusion protein, or may be added to the heavy or light chain via a linker moiety, e.g., to the N-terminus, C-terminus, or internal amino acid of the heavy or light chain. In some embodiments, the antibody is an IgG.

[0073] In certain embodiments, the tissue-specific Wnt signal enhancing molecule (e.g., a fusion protein) increases Wnt signaling in a target tissue or cell type contacted with the fusion protein, hi certain embodiments, Wnt signaling in the target tissue or cell type is increased by at least 50%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold.

[0074] Tissue-specific Wnt signal enhancing molecules can be produced by standard methods of organic synthesis and molecular biology known and available in the art. For example, tissue-specific Wnt signal enhancing fusion proteins can be generated by fusing a target domain (e.g., an antibody that binds to ASGR1) to an action domain (e.g., human R-spondin2 furin domain 1 alone) that corresponds to amino acids. The interaction of furin domain 2 with LGR protein is abolished or impaired by point mutations, e.g., F105A and F109A, alone or in combination). In certain embodiments, the target domain and the action domain are fused to any domain located at the N-terminus of the tissue-specific Wnt signal enhancing molecule by a linker, e.g., a glycine-serine linker. In certain embodiments, the target domain and the action domain are fused by a protein linker (e.g., albumin). Additional methods of "fusing" the target domain with the action domain include, but are not limited to, for example, "knob-in-hole" or leucine zipper-mediated dimerization. DNA sequences encoding the targeting domain, the acting domain (and optionally a linker) can be engineered to encode the desired fusion protein.

[0075] For tissue-specific Wnt signal enhancing fusion molecules comprising antibody heavy and light chains, DNA sequences encoding different portions of the fusion protein are inserted into bacterial or eukaryotic expression vectors using standard molecular cloning techniques and expressed in suitable host cells. The expressed fusion protein can be purified to homogeneity using standard techniques in protein science, such as affinity chromatography, ion exchange chromatography, and size exclusion chromatography. The present disclosure also includes functional fragments and variants of any of the polypeptide action domains, targeting domains, and fusion proteins described herein, including variants having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% polypeptide sequence identity with the action domains, targeting domains, or fusion proteins described herein. Such variants may include one or more amino acid modifications, e.g., one or more amino acid deletions, insertions or substitutions, compared to any of the sequences disclosed herein. In certain embodiments, functional fragments and variants of tissue-specific Wnt signal enhancement fusion proteins have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more Wnt signal enhancement activity compared to the tissue-specific Wnt signal enhancement fusion protein from which they are derived. In certain embodiments, functional fragments and variants of a polypeptide action domain have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more Wnt signal enhancement activity compared to the action domain from which they are derived (as measured in the context of the entire tissue-specific Wnt signal enhancement molecule).In certain embodiments, functional fragments and variants of a targeting domain have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more binding activity compared to the targeting domain from which it is derived.

[0076] The present disclosure also includes polynucleotides or nucleic acid sequences encoding one or more tissue-specific Wnt signal enhancing molecules or components thereof, such as fusion proteins or variants thereof described herein, as well as vectors including expression vectors, and cells including these vectors. In certain embodiments, the polynucleotides or nucleic acid sequences are DNA or RNA. In certain embodiments, the RNA is messenger RNA (mRNA). In certain embodiments, the RNA is modified mRNA comprising one or more modified nucleosides. Modified mRNA comprising one or more modified nucleosides has been described to have advantages over unmodified mRNA, including improved stability, higher expression levels, and reduced immunogenicity. Non-limiting examples of modified mRNAs that may be used in accordance with the present invention are described, for example, in PCT Patent Application Publication Nos. WO2011 / 130624, WO2012 / 138453, WO2013151666, WO2013 / 071047, WO2013 / 078199, WO2012045075, WO2014081507, WO2014093924, WO2014164253, U.S. Pat. Nos. 8,278,036 (describes modified mRNAs containing pseudouridine), 8,691,966 (describes modified mRNAs containing pseudouridine and / or N1-methylpsudolysine), 8,835,108 (describes modified mRNAs containing 5-methylcytidine), and 8,748,089 (describes modified mRNAs containing pseudouridine or 1-methylpsudolysine). In certain embodiments, the modified mRNA sequence encoding a tissue-specific Wnt signal enhancing polypeptide comprises at least one modification compared to unmodified A, G, U, or C ribonucleoside. In certain embodiments, the at least one modified nucleoside comprises N1-methylpsudolysine and / or 5-methylcytidine. In certain embodiments, the modified mRNA comprises a 5'-end cap sequence followed by a sequence encoding a tissue-specific Wnt signal enhancing polypeptide followed by a 3'-tailing sequence, such as a polyA or polyAG sequence.

[0077] In certain embodiments, the polynucleotide is a vector, e.g., an expression vector, and the expression vector comprises a polynucleotide sequence encoding a tissue-specific Wnt signal-enhancing fusion molecule (e.g., a fusion protein or one or both chains of an additional antibody). It is operably linked to a promoter sequence as described herein, e.g., a promoter sequence that drives expression of the polynucleotide in a cell. In certain embodiments, the vector is a viral vector, e.g., a virus that comprises a polynucleotide comprising an expression cassette that comprises a promoter operably linked to a DNA or RNA sequence encoding a tissue-specific Wnt signal-enhancing polypeptide. In certain embodiments, the expression cassette comprises 5' and / or 3' cellular or viral UTRs or derivatives thereof.

[0078] The present disclosure also includes functional fragments and variants of the polynucleotides described herein, including variants having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% polynucleotide sequence identity with the polynucleotides described herein. Such variants can include one or more nucleotide or nucleoside modifications, such as one or more nucleotide deletions, insertions or substitutions, compared to any sequence disclosed herein. In certain embodiments, the polynucleotides described herein are codon-optimized, for example, to enhance expression of the encoded polypeptide in a host cell. In certain embodiments, the polynucleotide variants include one or more modified nucleotides or nucleosides.

[0079] The present disclosure also includes cells comprising a polynucleotide or vector encoding the tissue-specific Wnt signal enhancing molecule, e.g., a fusion protein, described herein. In certain embodiments, the cell is a host cell, e.g., HEK293 cell, that can be used to produce tissue-specific Wnt signal enhancing fusion protein. Any host cell can be used in preparing the composition, including, but not limited to, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. In certain embodiments, the cell is xenogeneic or autologous to the subject treated with the tissue-specific Wnt signal enhancing polypeptide described herein. In certain embodiments, the cell is obtained from the subject and transduced with a viral vector described herein. In certain embodiments, the transduced cell is delivered to the subject for treatment.

[0080] The present disclosure also includes pharmaceutical compositions comprising one or more tissue-specific Wnt signal enhancing molecules (e.g., fusion proteins), or one or more polynucleotides or vectors comprising sequences encoding tissue-specific Wnt signal enhancing molecules.

[0081] Wnt signaling can be measured using techniques and assays known and available in the art. In certain embodiments, the increase in Wnt signaling is determined using a cell line corresponding to the target tissue or cell type. In certain embodiments, the cell line contains a reporter plasmid that contains a marker gene (e.g., luciferase gene) under the control of a Wnt signal-responsive promoter. In response to Wnt3a, Wnt3a-conditioned medium, or a recombinant source of Wnt3a, the reporter activity of cells is enhanced by adding furin domain 1 alone (or together with furin domain 2, with F105A and / or F109A point mutations). A negative control or functional R-spondin (full length or furin domain 1 and 2) as a positive control can be determined. Reporter activity in response to tissue-specific Wnt signal enhancing molecules can also be determined by contacting the reporter cell line with tissue-specific Wnt signal enhancing molecules. The negative control may be substantially, significantly, or completely negative for reporter activity, and the tissue-specific Wnt signal enhancer molecule and the positive control should show an increase in Wnt signaling response as an increase in reporter activity. Additional controls may include anti-ASGR1 antibody alone (negative), a fusion protein in which anti-GFP antibody is used instead of anti-ASGR1 antibody (negative), and an intact furin domain 1-furin domain 2 protein (positive). The tissue specificity of the tissue-specific Wnt signal enhancer molecule may be determined by similarly measuring reporter activity in response to treatment with the tissue-specific Wnt signal enhancer molecule in cell types or tissues other than the one targeted. In certain embodiments, reporter activity is higher, e.g., at least 50%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold higher, in the target tissue bound by the tissue-specific Wnt signal enhancer molecule compared to non-target tissue.

[0082] In certain embodiments, tissue-specific Wnt signal enhancing polypeptide comprises any combination of action domain and target domain, including any combination of action domain and target domain described herein.In certain embodiments, they are linked by a linker, such as albumin (e.g. human serum albumin), a peptidyl linker, or a non-peptidyl linker, where the target domain and action domain are at their N-terminus and C-terminus.Linker, such as Fc or albumin, a peptidyl linker, or a non-peptidyl linker.

[0083] The tissue-specific Wnt signal-enhancing molecule can also be conjugated to moieties such as polyethylene glycol (PEG), Fc, albumin, and the like, as known in the art, to enhance stability in vivo.

[0084] Illustrative, non-limiting examples of tissue-specific Wnt signal enhancing molecules include the following:

[0085] a) a bone tissue-specific Wnt signal enhancing polypeptide comprising an action domain comprising a variant or fragment of R-spondin (e.g., human R-spondin2) with reduced ability to enhance Wnt signaling and a targeting domain that specifically binds to PTH1R, wherein the tissue-specific Wnt signal enhancing polypeptide increases Wnt signaling in bone tissue and can be used to treat a disease or condition of bone tissue;

[0086] b) a liver tissue-specific Wnt signal enhancing polypeptide comprising an action domain comprising a variant or fragment of R-spondin (e.g., human R-spondin2) with reduced ability to enhance Wnt signaling and a targeting domain that specifically binds to ASGR1, ASGR2, TFR2 or SLC10A1; wherein the tissue-specific Wnt signal enhancing polypeptide increases Wnt signaling in liver tissue and can be used to treat a disease or condition of liver tissue; or

[0087] c) oral mucosal tissue-specific Wnt signal enhancing polypeptide comprising an action domain comprising a variant or fragment of R-spondin (e.g., human R-spondin2) with reduced ability to enhance Wnt signaling and a targeting domain that specifically binds to LYPDS3 or DSG3, wherein the tissue-specific Wnt signal enhancing polypeptide increases Wnt signaling in oral mucosal tissue and can be used to treat diseases or conditions of oral mucosal tissue.

[0088] Illustrative, non-limiting examples of tissue-specific Wnt signal enhancing molecules include sequences including, but not limited to, those described in the accompanying Examples and in Table 1. In certain embodiments, tissue-specific Wnt signal enhancing molecules comprise two or more polypeptide sequences disclosed herein, for example in the accompanying IgG or antibody format. The polypeptides disclosed herein are selected from the group consisting of SEQ ID NOs: 1-4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, , 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, or 158, and fragments thereof. In certain embodiments, the polypeptide has activity as a functional domain and / or targeting domain.

[0089] Exemplary, non-limiting examples of polynucleotides disclosed herein include those that encode any of the polypeptides, variants and fragments described herein, including those described above. The polynucleotides disclosed herein are set forth in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 11 9, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 15, 155, and 157, as well as fragments thereof. In certain embodiments, the polynucleotide encodes a polypeptide having activity as a functional domain and / or targeting domain.

[0090] Domain of action R-spondins can amplify Wnt signals. The minimal functional unit of R-spondin consists of two furin domains, furin domain 1, which binds ZNRF3 / RNF43 E3 ligase, and furin domain 2, which binds LGR4-6, bringing together a ternary complex of R-spondin, LGR, and E3 ligase. This leads to internalization of the entire complex and removes ZNRF3 / RNF43 away from their targets for destruction. Furin domain 1 alone is not functional, but it can bind both ZNRF3 and RNF43 (see Figure 1).

[0091] The action domain of the tissue-specific Wnt signal enhancing molecule described herein can be any functional moiety, such as, but not limited to, a polypeptide or organic chemical that can bind to ZNRF3 / RNF43 ligase (see FIG. 2). In certain embodiments, the action domain, such as a polypeptide comprising the furin domain 1 of R-spondin, either alone or together with a targeting domain, is substantially inactive in non-target tissues to minimize potential off-target effects. The action domain is fused or bound to the targeting domain in the context of the tissue-specific Wnt signal enhancing molecule, and when the tissue-specific Wnt signal enhancing molecule engages with the target tissue expressing the tissue-specific receptor, the E3 ligase ZNRF3 / RNF43 is recruited to the tissue-specific receptor and the ternary complex, which are relocated to, sequestered, and / or removed from the cell surface (see FIG. 3).

[0092] In certain embodiments, the action domain comprises a fragment or variant of an R-spondin polypeptide (e.g., any of R-spondins 1-4), or a functional fragment or variant thereof. In certain embodiments, the action domain comprises a fragment of wild-type R-spondin, and in other embodiments, the action domain comprises a fragment of R-spondin comprising one or more amino acid modifications. The R-spondin can be any R-spondin known in the art, or a homolog thereof, including R-spondin from any animal species, including but not limited to mammalian species, such as human R-spondin. R-spondins have been identified and described, and their polypeptides and polynucleotide sequences encoding them are known and available in the art. In certain embodiments, the R-spondin polypeptide is human R-spondin, or a homolog found in other vertebrates or invertebrates, such as mouse R-spondin. The amino acid sequences of human R-spondin1, human R-spondin2, human R-spondin3, and human R-spondin4, as well as their furin domain 1, are provided in FIG. 4 and SEQ ID NOs: 1-4, respectively. Their homologs and variants can be obtained from general database searches, such as https: / / www.dot.ncbi.dot.nlm.dot.nih.dot.gov / protein / . The present invention includes (but is not limited to) action domains that comprise or consist of any fragments and variants of these or other R-spondins. In various embodiments, any R-spondin polypeptide and fragment variants thereof comprise one or more amino acid modifications, such as deletions, additions, or substitutions, compared to wild-type R-spondin polypeptides. Modifications can be present in any region of the R-spondin variant or its fragments, including but not limited to furin domain 1 and / or furin domain 2. It is understood that amino acid modifications outside of furin domain 1 or furin domain 2 can modify the resulting variant such that the resulting variant has reduced LGR4-6 binding activity compared to wild-type R-spondin or its fragments.

[0093] In certain embodiments, the action domain comprises or consists of an R-spondin sequence, such as full-length or wild-type R-spondin-1, -2, -3, or -4, optionally human R-spondin-1, -2, -3, or -4, or a variant or fragment thereof. In certain embodiments, it is a variant of any of R-spondin 1-4 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the corresponding wild-type R-spondin 1-4 sequence. In certain embodiments, the action domain comprises or consists of a full-length R-spondin (e.g., any of R-spondin-1-4) containing one or more amino acid modifications, including but not limited to any of those disclosed herein. In certain embodiments, the action domain comprises or consists of a fragment of a wild-type or modified R-spondin (e.g., any of R-spondin-1-4). In certain embodiments, the fragment is capable of binding to ZNRF3 and / or RNF43. In certain embodiments, the action domain comprises a furin domain 1 of an R-spondin protein, or a fragment or variant of an R-spondin protein. In certain embodiments, the action domain comprises or consists of one or more (e.g., one, two or more) furin domains 1 of an R-spondin protein (e.g., R-spondin 1-4), or variants thereof having at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the R-spondin furin domain 1. In certain embodiments, the action domain comprises an R-spondin furin 1 domain or a variant or fragment thereof, and an R-spondin furin 2 domain or a variant or fragment thereof. In certain embodiments, the action domain comprises a ZNRF3 / RNF43. In certain embodiments, the action domain specifically binds to either ZNRF3 or RNF43.

[0094] In certain embodiments, the action domain comprises one or more furin domains 1 of R-spondin, such as human R-spondin 1 or human R-spondin 2, or variants thereof. In certain embodiments, the action domain comprises one or more furin domains 1 of R-spondin, but it does not comprise the furin domain 2 of R-spondin. In certain embodiments, the action domain comprises one or more furin domains 1 of R-spondin, which comprises a modified or variant furin domain 2 of R-spondin, such as a furin domain 2 with reduced activity compared to the wild-type furin domain 2. In certain embodiments, the action domain comprises an R-spondin protein with a modified or variant furin domain 2 of R-spondin, such as a furin domain 2 with reduced activity compared to the wild-type furin domain 2. In certain embodiments, the action domain comprises two or more furin domains 1 or variants thereof, or a multimer of furin domain 1 or variants thereof. In certain embodiments, the action domain comprises a variant R-spondin furin1 domain that comprises one or more point mutations, for example at amino acid residues corresponding to K58, H76, S77, R86, N91 of human R-spondin2. In certain embodiments, the action domain comprises a modified or variant furin domain1 of R-spondin that has increased activity, for example binding to ZNRF3 / RNF43, compared to wild-type furin domain1. The action domain may further comprise additional moieties or polypeptide sequences, for example additional amino acid residues to stabilize the structure of the action domain or tissue-specific Wnt signal enhancing molecule in which it is present. In certain embodiments, the action domain comprises a peptide or polypeptide that does not have obvious / strong sequence homology to R-spondin, but has binding affinity to ZNRF3 / RNF43 that is equal to or greater than that of R-spondin.

[0095] In certain embodiments, the working domain comprises a furin domain 1, or a functional fragment or variant thereof, of an R-spondin polypeptide (e.g., human R-spondin), and a modified or variant furin domain 2 of an R-spondin polypeptide, where the modified furin domain 2 has a lower binding affinity to LGR4-6 compared to the corresponding wild-type furin domain 2 (see Figures 5-7). In certain embodiments, the furin domain 2 comprises one or more point mutations, for example, at amino acid residues corresponding to F105 and / or F109 of human R-spondin2. Those skilled in the art can easily determine the corresponding amino acid residues in other R-spondin polypeptides by comparing their amino acid sequences with human R-spondin2. In certain embodiments, the working domain comprises a furin domain 1 or a variant thereof and a furin domain 2 or a variant thereof, where the furin domain 1 and / or the furin domain 2 comprise one or more point mutations. The one or more point mutations in the action domain (compared to the corresponding wild-type R-spondin sequence) can occur at any amino acid residue in the furin domain 1 and / or furin domain 2, including, but not limited to, amino acid residues K58, H76, S77, R86, N91, F105, F109, or K121, and other residues that can be modified to reduce binding affinity to LGR4-6. Regions of the furin domain 1 and furin domain 2 of human R-spondin1 that are important for their functional activity have been identified, including the conserved hydrophilic residues S48, N51, R66, R70, and Q71, and the less conserved hydrophobic residues L46, L54, I62, and L64, which are important for binding to E3 ligases. Furthermore, in human R-spondin1 furin domain 1, amino acid residues K59, S78, D85, R87, N88 and N92 form a hydrophilic interaction surface with LGR5, and FSHNF amino acid sequences have been identified as important loops for the hydrophobic surface.In certain embodiments, the working domain comprising R-spondin furin domain 1 and / or furin domain 2 may contain one or more mutations within any of these regions, surfaces or amino acid residues. In certain embodiments, the working domain comprising R-spondin furin domain 1 and / or furin domain 2 may contain one or more mutations or other changes over these regions, surfaces or amino acid residues that indirectly impair LGR4-6 binding by affecting the structure and / or stability of the binding surface. In certain embodiments, the working domain comprising R-spondin furin domain 1 and / or furin domain 2 may contain one or more mutations at any amino acid residue, including but not limited to any of those shown in the accompanying examples. In certain embodiments, the modified furin domain 2 has a binding affinity to LGR4-6 that is less than 80%, less than 50%, less than 20%, or less than 10% of the binding of the corresponding wild-type furin domain 2, for example in the context of the full-length R-spondin protein.

[0096] In certain embodiments, the action domain comprises the furin domain 1 of R-spondin polypeptide (e.g., human R-spondin), or its functional fragment or variant, and the unmodified furin domain 2 of R-spondin polypeptide (e.g., human R-spondin). In certain embodiments, the modified furin domain 2, which has a lower binding affinity to LGR4-6 compared with the corresponding wild-type furin domain 2, is more desirable for increasing the specificity of tissue targeting, but in certain embodiments, the unmodified furin domain 2 is combined. The use of the targeting domain improves tissue targeting compared to wild-type R-spondin without the targeting domain, and has utility in certain situations.

[0097] In certain embodiments, the action domain comprises a wild-type or modified R-spondinfurin domain 1, for example from any of R-spondin-1, -2, -3, -4, optionally human R-spondin-1, -2, -3 or -4. In certain embodiments, the action domain comprises an R-spondinfurin 1 domain and a wild-type or modified R-spondinfurin 2 domain, for example from any of R-spondin-1, -2, -3, -4, optionally human R-spondin-1, -2, -3 or -4. In certain embodiments, the action domain comprises a first R-spondinfurin 1 domain and a second wild-type or modified R-spondinfurin 1 domain, for example from any of R-spondin-1, -2, -3, -4, optionally human R-spondin-1, -2, -3 or -4. In certain embodiments, the modified furin domain 2 has a binding affinity to LGR4-6 that is equivalent to, or less than 80%, less than 50%, less than 20%, or less than 10% of, the binding affinity to LGR4-6 of the corresponding wild-type furin domain 2, e.g., in the context of a full-length R-spondin protein.

[0098] Target Domains Cells of a particular cell type and in a particular tissue may contain one or more cell- or tissue-specific surface molecules, such as cell surface receptors (see FIG. 2). As used herein, a molecule is said to be cell- or tissue-specific if more of the molecule is present in a particular cell or tissue type compared to one or more other cell or tissue types, or any other cell or tissue type. In certain embodiments, the more is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold compared to the amount in one or more other cell or tissue types, or any other cell or tissue type. In certain embodiments, the cell-specific surface molecule has increased or enhanced expression in a target organ, tissue, or cell type, e.g., an organ, tissue, or cell type in which it is desirable to enhance Wnt signaling, e.g., to treat or prevent a disease or disorder, when compared to one or more other non-target organs, tissues, or cell types. In certain embodiments, the cell-specific surface molecule is preferentially expressed on the surface of a target organ, tissue, or cell type compared to one or more other organs, tissues, or cell types, respectively. For example, in certain embodiments, a cell surface receptor is considered to be a tissue- or cell-specific cell surface molecule if it is expressed at a level at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold higher in a target organ, tissue, or cell than it is expressed in one or more, five or more, all other organs, tissues, or cells, or the average of all other organs, tissues, or cells, respectively. In certain embodiments, a tissue- or cell-specific cell surface molecule is a cell surface receptor, e.g., a polypeptide receptor that includes a region located within the cell surface membrane and an extracellular region to which a targeting domain can bind.In various embodiments, the methods described herein may be performed by specifically targeting a cell surface molecule that is expressed only on the target tissue or a subset of tissues that includes the target tissue, or by specifically targeting a cell surface molecule that has a higher level of expression on the target tissue when compared to all, most, or a significant number of other tissues, e.g., higher expression in the target tissue than at least 2, at least 5, at least 10, or at least 20 other tissues.

[0099] In certain embodiments, the targeting domain binds to tissue-specific surface molecules expressed in the target cell or tissue type of interest, i.e., the cell or tissue type that is desired to enhance or increase Wnt signaling activity.The targeting domain that binds to each tissue-specific surface molecule can be, but is not limited to, an antibody or its antigen-binding fragment, a peptide, the natural ligand of tissue-specific or cell-specific receptor, or their derivatives, and synthetic small molecules.

[0100] The target tissue bound by the targeting domain can be any tissue, for example, any mammalian tissue or cell type. In certain embodiments, the target tissue can be present in any organ. In certain embodiments, the target tissue is bone tissue, liver tissue, skin tissue, stomach tissue, intestinal tissue, oral mucosa tissue, kidney tissue, central nervous system tissue, mammary tissue, taste bud tissue, ovarian tissue, inner ear tissue (including cochlea and vestibular tissue), hair follicle, pancreatic tissue, retinal tissue, corneal tissue, heart tissue or lung tissue, and the targeting domain binds to tissue-specific cell surface molecules (e.g., cell surface receptors) that are preferentially expressed in bone tissue, liver tissue, skin tissue, stomach tissue, intestinal tissue, oral mucosa tissue, kidney tissue, central nervous system tissue, mammary tissue, taste bud tissue, ovarian tissue, inner ear tissue (including cochlea and vestibular tissue), hair follicle, pancreatic tissue, retinal tissue, corneal tissue, heart tissue or lung tissue, respectively.

[0101] The targeting domain can bind to any cell type, for example, any cell in any tissue, organ, or animal, including, but not limited to, mammals, such as humans. In certain embodiments, the tissue-specific Wnt signal enhancing molecule binds to a specific cell type, for example, a specific cell type associated with the target tissue. For example, in liver tissue, the targeting domain can bind to hepatocytes, hepatocyte precursors and stem cells, biliary tract cells, and / or endothelial cells or other vascular cells. For example, in bone tissue, the targeting domain can bind to osteoblasts, osteoblast precursors, mesenchymal stem cells, stem cells, and progenitor cells that give rise to bone, cartilage, and / or other cells present in bone tissue. Cell types present in various tissues, including but not limited to those described herein, are known in the art, and in various embodiments, the tissue-specific Wnt signal enhancing molecule described herein can bind to any of them.

[0102] In various embodiments, the tissue-specific surface molecule is a tissue-specific cell surface receptor. For the liver, these include, but are not limited to, ASGR1, ASGR2, TFR2, SLC10A1, etc. In certain embodiments, the targeting domain is a natural ligand, or a functional variant or fragment thereof, or an antibody or an antigen-binding fragment thereof. They bind to ASGR1, ASGR2, TFR2, SLC10A1, LYPD3, or DSG3. For bone or kidney, such tissue-specific cell surface receptors include, but are not limited to, parathyroid hormone receptor 1 (PTH1R), etc. In certain embodiments, the targeting domain is a natural ligand, or a functional variant or fragment thereof, or an antibody or an antigen-binding fragment thereof, that binds to PTH1R. For the oral mucosa, such tissue-specific cell surface receptors include, but are not limited to, LYPD 3 and DSG 3. In certain embodiments, the targeting domain is a natural ligand, or a functional variant or fragment thereof, or an antibody or an antigen-binding fragment thereof, that binds to LYPD3 or DSG3.

[0103] The asialoglycoprotein receptor (ASGPR) consists of ASGR1 and ASGR2 (reviewed, for example, by Stockert, Morell, and Ashwell, 1991, Targeted Diagnostics and Therapy 4:41-64). This receptor is a transmembrane protein that plays a key role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine residues. Thus, natural and synthetic ligands of AGPR include, but are not limited to, galactosylated cholinesterases, galactose (Gal) and N-acetylgalactosamine (GalNAc), GalNAc-containing molecules such as GalNAc-terminated glycoproteins, as well as monosaccharide-, oligosaccharide-, or polysaccharide-containing molecules or nanoparticles (reviewed, for example, by D'Souza and Devarajan 2015, Journal of Controlled Release, 203:126-139).

[0104] The SLC10 family transports bile acids, sulfated solutes, and other xenobiotics in a sodium-dependent manner. The founding members, SLC10A1 (NTCP) and SLC10A2 (ASBT), function to maintain the enterohepatic circulation of bile acids. Examples of natural and synthetic ligands for SLC10A include, but are not limited to, cholic acid, Na(+) / bile acids, Na(+) / taurocholic acid, and the preS1 domain of Hepatitis B virus and their fragments or variants (e.g., as reported in Yan et al., 2012 eLife, 1:e00049).

[0105] Transferrin receptor 2 (TFR2) is a homolog of transferrin receptor 1 (TFR1), a protein that delivers iron into cells via receptor-mediated endocytosis of dimeric transferrin (Fe2TF). TFR2 also binds Fe2TF, but it appears to function primarily in regulating systemic iron homeostasis (e.g., Worthen and (Reviewed by Enns, 2014, Frontiers in Pharmacology 5:34). Examples of natural and synthetic ligands and binding partners of TFR2 include, but are not limited to, transferrin, such as dimeric transferrin, and hemochromatosis (HFE) proteins and fragments and variants thereof.

[0106] The type 1 receptor (PTH1R) of parathyroid hormone (PTH) and PTH-related peptide (PTHrP) is highly expressed in bone and kidney (reviewed, for example, by Mannstadt, Juppner, and Gardella, 1999, American Journal of Physiology 277:F665-F675). Natural and synthetic ligands of parathyroid hormone receptor 1 (PTH1R) include, but are not limited to, PTH, PTHrP, and fragments and variants thereof.

[0107] Ly6 / PLAUR domain-containing protein 3 (LYPD3) is a GPI-anchored protein that shows highly specific expression in stratified squamous epithelia found in tissues such as the oral mucosa, skin, and esophagus. LYPD3 expression has also been shown to be upregulated in migrating keratinocytes during wound healing and in various cancers (e.g., Jacobsen, Kriegbaum, Santoni-Rugiu, and Ploug, 2014, World Cancer Society). (Reviewed in Journal of Clinical Oncology, 5(4):621-32). Despite reported LYPD3 expression, its exact function remains unclear, as animals lacking the LYPD3 gene are viable, fertile, and have no obvious defects in squamous epithelial development. Various molecules have been identified as binding partners of LYPD3. Laminin 1, laminin 5, and galectin-3 associate with LYPD3 to promote cell migration (Paret, Bourouba, Beer, Miyazaki, Schnolzer, Fiedler, and Zoller. International Journal of Cancer. 2005 Jul 10;115(5):724-33). Anterior gradient 2, AGR2, interacts with LYPD3 and promotes cancer growth, metastasis and resistance to therapy in pancreatic ductal adenocarcinoma (PDAC) (Arumugam, Deng, Bover, Wang, Logsdon and Ramachandran. Molecular Cancer Therapeutics. 2015 Apr;14(4):941-51). Under hypoxic conditions, LYPD 3 forms a complex with α6β4 integrin and matrix metalloproteinase 14 (MMP14), which promotes cancer cell motility through localized laminin 332 degradation. (Ngora, Galli, Miyazaki and Zoller. Neoplasia. 2012 Feb;14(2):95-107).

[0108] Desmoglein 3 (DSG3) encodes a calcium-binding transmembrane glycoprotein that is a member of the cadherin cell adhesion molecule superfamily of proteins. DSG3 is expressed in epithelia and mucosa in desmosomes, specialized structures for cell-cell adhesion. DSG3 has five extracellular cadherin domains (ECDs) that contain Ca2+ binding sites that are required for DSG3 cell-cell interactions (e.g., reviewed by Thomason, Scothern, McHarg, and Garrod, 2010, Biochemical Journal, 429(3):419-433). DSG3 cell-cell interactions are mediated by trans-homophilic interactions near their N-terminus. Loss of DSG3 in animals causes very severe erosion of the oral mucosa and hair loss at weaning, indicating how important this gene is for epithelial cell integrity in these tissues. Single-molecule atomic force microscopy experiments demonstrated homophilic trans-DSG3 binding via the extracellular cadherin domain (Heupel, Zillikens, Drenckhahn and Waschke.Journal of Immunology.August 1,2008,181(3) 1825-1834).

[0109] Illustrative, non-limiting examples of tissue-specific Wnt signal enhancing molecules include: 1) a fusion protein comprising a first domain comprising an R-spondin furin domain 1 or a variant thereof, and a second domain comprising an antibody or a fragment thereof that specifically binds to ASGR1 or ASGR2; 2) a fusion protein comprising a first domain comprising an R-spondin furin domain 1 or a variant thereof, and a second domain comprising an antibody or a fragment thereof that specifically binds to SLC10A1; 3) a fusion protein comprising a first domain comprising an R-spondin furin domain 1 or a variant thereof, and a second domain comprising an antibody or a fragment thereof that specifically binds to TFR2; 4) a fusion protein comprising a first domain comprising an R-spondin furin domain 1 or a variant thereof, and a second domain comprising an antibody or a fragment thereof that specifically binds to P The fusion protein includes a second domain comprising a ligand derivative, an antibody or a fragment thereof that specifically binds to TH1R; 5) a fusion protein includes a first domain comprising an R-spondin furin domain 1 or a variant thereof, and a second domain comprising a ligand derivative, an antibody or a fragment thereof that specifically binds to LYPD3; 6) a fusion protein includes a first domain comprising an R-spondin furin domain 1 or a variant thereof, and a second domain comprising a ligand derivative, an antibody or a fragment thereof that specifically binds to DSG3; and 7) a fusion protein includes a first domain comprising an R-spondin furin domain 1 or a variant thereof, and a second domain comprising a ligand derivative, an antibody or a fragment thereof that specifically binds to TFR1. In certain embodiments, the two domains are linked via a linker, for example, a polypeptide linker. In certain embodiments, the linker is albumin, for example, human serum albumin, and the targeting domain and the action domain are at the N-terminus and C-terminus of albumin. In certain embodiments, the tissue-specific Wnt signal enhancing molecule has a combined antibody (e.g., IgG) format that includes an antibody heavy chain and an antibody heavy chain (or a fragment or variant thereof of either or both chains). It further comprises one or more additional binding domains.In certain embodiments, the tissue-specific Wnt signal enhancing molecule has an additional antibody (e.g., IgG) format, the second domain comprises an antibody heavy chain and an antibody light chain (or a fragment or variant of either or both chains), and the first domain comprising R-spondin furin domain 1 or variant is added to one or both of the antibody heavy chain and / or light chain, for example, to either or both of the N-terminus and / or C-terminus of either or both chains. In certain embodiments, the first domain is added or fused to the heavy chain, for example, at either the N-terminus or C-terminus. In certain embodiments, the first domain is added or fused to the light chain, for example, at either the N-terminus or C-terminus.

[0110] Linker In certain embodiments, the targeting domain and the action domain are directly linked or fused to each other, while in other embodiments, they are separated by a linker, such as a polypeptide linker, or a non-peptidyl linker. For example, the linker is an Fc linker, such as a region of an antibody Fc domain that can dimerize with another Fc linker, for example, via one or more disulfide bonds. In another specific embodiment, the linker is albumin, such as human serum albumin, where the targeting domain and the action domain are at the N-terminus and C-terminus of albumin.

[0111] In certain embodiments, especially when linking two polypeptides, the linker is composed of amino acids linked together by peptide bonds. In certain embodiments, the linker comprises 1 to about 40 amino acid residues in length, 1 to about 20 amino acid residues, or 1 to about 10 amino acid residues. In certain embodiments, the amino acid residues in the linker are from among the 20 standard amino acids, and in certain embodiments, are selected from cysteine, glycine, alanine, proline, asparagine, glutamine, and / or serine. In certain embodiments, the linker comprises one or more unnatural amino acids. In some embodiments, peptidyl linkers are composed of a majority of sterically unhindered amino acids such as glycine, serine, and alanine linked by peptide bonds. Particular linkers include polyglycine, polyserine, and polyalanine, or any combination thereof. Some exemplary peptidyl linkers are poly(Gly)1-8 (SEQ ID NO:159-163, particularly (Gly)3, (Gly)4 (SEQ ID NO:159), (Gly)5 (SEQ ID NO:160)). and (Gly)7 (SEQ ID NO:162), as well as poly(Gly)4 Ser (SEQ ID NO:164), poly(Gly-Ala)2-4 (SEQ ID NO:165-167), and poly(Ala)1-8 (SEQ ID NO:168-172). Other specific examples of peptidyl linkers include (Gly)5 Lys (SEQ ID NO:173), and (Gly)5 LysArg (SEQ ID NO:174). To illustrate the above nomenclature, for example, (Gly)3 Lys(Gly)4 (SEQ ID NO:175) means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly (SEQ ID NO:175). Other combinations of Gly and Ala are also useful. Additionally, a peptidyl linker can also include a non-peptidyl segment, such as a six-carbon aliphatic molecule of the formula --CH2--CH2--CH2--CH2--CH2--CH2--. Peptidyl linkers can be modified to form derivatives as described herein.

[0112] Exemplary non-peptidyl linkers include alkyl linkers such as, for example, --NH--(CH2)s--C(O)-- (where s=2-20). These alkyl linkers may be further substituted with any non-sterically hindering group such as lower alkyl (e.g., C1-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, and the like. The compositions of the invention, such as non-peptide linkers or non-peptide half-life extending moieties, can be synthesized by conventional organic chemistry reactions. Chemical groups used to link the binding domains include carbamates; amides (amines + carboxylic acids); esters (alcohols + carboxylic acids), thioethers (haloalkanes + sulfhydryls; maleimides + sulfhydryls), Schiff bases (amines + aldehydes), ureas (amines + isocyanates), thioureas (amines + isothiocyanates), sulfonamides (amines + sulfonyl chlorides), disulfides; hydrazones, lipids, and the like, as known in the art.

[0113] The linkage between the domains may be linear or branched, typically linear, and may include a spacer, e.g., an alkyl spacer, which may include one or more unsaturated bonds, typically having from 1 to about 300 carbon atoms, more typically having from about 1 to 25 carbon atoms, and may be from about 3 to 12 carbon atoms. Spacers of this type may also include heteroatoms or functional groups, including amines, ethers, phosphodiesters, and the like. Specific structures of interest are as follows: (CH2CHO)n, where n is from 1 to about 12, (CH2CH2NH)n, where n is from 1 to about 12, and [(CH2)n(C=O)NH(CH2) m ] z where n and m are from 1 to about 6, z is from 1 to about 10, and [(CH2)nOPO3(CH2) m ] z wherein n and m are from 1 to about 6, and z is from 1 to about 10. Such linkers may include polyethylene glycol, which may be linear or branched.

[0114] In certain embodiments, the domains may be linked via homo- or heterobifunctional linkers. Exemplary identities include: azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartrate, N-γ-maleimidobutyryloxysuccinimide hydroxysulfosuccinimidyl-4-azidobenzoate, N-succinimidyl[4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, NHS-PEG-MAL; Cinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate; 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP); N,N'-(1,3-phenylene)bismaleimide; N,N'-ethylene-bis-(iodoacetamide); 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and the extended chain analog of MBS, succinimide 4-(p-maleimidophenyl)butyrate (SMPB). In certain embodiments, the succinimidyl group of these crosslinkers reacts with primary amines and the thiol-reactive maleimide forms a covalent bond with the thiol of a cysteine ​​residue.

[0115] Other reagents that are useful include: homobifunctional crosslinking reagents including bismaleimidohexane ("BMH"); p,p'-difluoro-m,m'-dinitrodiphenyl sulfone (which forms irreversible crosslinks with amino and phenolic groups); dimethyl adipate (specific for amino groups); phenol-1,4-disulfonyl chloride (which reacts primarily with amino groups); hexamethylene diisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts primarily with amino groups); didiazobenzidine (which reacts primarily with tyrosine and histidine); O-benzotriazolyloxytetramethylammonium hexafluorophosphate (HATU), dicyclohexylcarbodiimide, bromo-tris(pyrrolidino)phosphonium bromide (PyBroP); N,N-dimethylaminopyridine (DMAP); 4-pyrrolidinopyridine; N-hydroxybenzotriazole, and the like.

[0116] Wnt molecules, Norrin molecules, and Wnt signal enhancing molecules The present disclosure further relates to Wnt polypeptides, Norrin polypeptides, and Wnt signaling agonist molecules, and their use to enhance Wnt signaling and treat or prevent Wnt-related diseases or disorders, including those described herein. In certain embodiments, Wnt polypeptides, Norrin polypeptides, and Wnt signaling agonist molecules are provided to a subject, either alone or in combination with one or more tissue-specific Wnt signal enhancing molecules described herein.

[0117] Wnt polypeptides and polynucleotide sequences encoding Wnt are known in the art and include any and all Wnt polypeptides or polynucleotides, including mammalian Wnt polypeptides and polynucleotides, such as human Wnt polypeptides and polynucleotides.Exemplary Wnt polypeptides include Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11 and Wnt16, and any functional variants and fragments thereof.Wnt polypeptides include natural Wnt polypeptides, Wnt polypeptide variants, Wnt polypeptide fragments and chimeric Wnt polypeptides.In certain embodiments, Wnt polypeptides are natural human full-length mature Wnt proteins.

[0118] For example, human native sequence Wnt proteins of interest in the present application include, but are not limited to, Wnt1 (GenBank Accession No. NM_005430); Wnt-2 (GenBank Accession No. NM_003391); Wnt2B (Wnt-13) (GenBank Accession Nos. NM_004185 (isoform 1), NM_024494.2 (isoform 2)), Wnt3 (Reference Sequence: NM_030753), Wnt3A (GenBank Accession No. NM_033131), Wnt4 (GenBank Accession No. NM_030761), Wnt5A (GenBank Accession No. NM_003392), Wnt5B (GenBank Accession No. NM_032642), Wnt6 (GenBank Accession No. NM_032642). GenBank accession number NM_004625), Wnt7B (GenBank accession number NM_058238), Wnt8A (GenBank accession number NM_058244), Wnt8B (GenBank accession number NM_003393), Wnt9A (Wnt-14) (GenBank accession number NM_003395), Wnt15) (GenBank accession number NM_003396), Wnt10A (GenBank accession number NM_025216), Wnt10B (GenBank accession number NM_003394), Wnt11 (GenBank accession number NM_004626), and Wnt16 (GenBank accession number NM_016087). Although each member has a varying degree of sequence identity with the family, all encode small (i.e., 39-46 kD), acylated, palmitoylated, secreted glycoproteins that contain 23-24 conserved cysteine ​​residues whose spacing is highly conserved (McMahon, AP et al), Trends Genet. 1992;8:236-242; Miller, JR Genome Biol. 2002;3(1):3001.1-3001.15). Other native sequence Wnt polypeptides of interest include orthologs of the above from any mammal, including livestock and domestic animals, as well as animals such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, or laboratory or pet animals, such as mice, frogs, zebras, fruit flies, worms, etc.

[0119] Norrin polypeptides and Norrin-encoding polynucleotide sequences are also known in the art and include any type of Norrin polypeptide or polynucleotide, including mammalian Norrin polypeptides and polynucleotides, such as human Norrin polypeptides and polynucleotides, as well as functional variants and fragments thereof.

[0120] Wnt signaling agonist molecules include any kind of molecule that stimulates Wnt signaling. In certain embodiments, Wnt signaling agonist molecules are described in PCT Patent Application Publication No. WO 2016 / 040895. Wnt signaling agonists can be any molecule, such as a protein or a pharmaceutical (e.g., a small organic molecule), that directly activates canonical Wnt signaling through binding to one or more Fzd proteins and Lrp5 / 6, and that is water-soluble in certain embodiments. In certain embodiments, they are small molecules, which may be less than about 15 Kd. In other embodiments, they are polypeptides. Furthermore, certain wnt signaling agonists can include both polypeptide and non-polypeptide regions or domains.

[0121] In some embodiments of the present invention, the Wnt signaling agonist molecule is a polypeptide, which can contain separate or consecutive binding domains or elements for Fzd and Lrp5 / 6.The polypeptide Wnt signaling agonist can be a single chain, a dimer, or a higher order multimer.The Fzd binding domain / element and the Lrp5 / 6 binding domain / element can be directly linked or separated by a linker, such as a polypeptide linker, or a non-peptide linker.

[0122] In polypeptide embodiments, the Fzd-binding domain may be selected from any domain that binds to Fzd with high affinity, e.g., a KD of at least about 1×10-7M, at least about 1×10-8M, at least about 1×10-9M, or at least about 1×10-10M. Suitable Fzd-binding domains include, but are not limited to, de novo designed Fzd-binding proteins, antibody-derived binding proteins that specifically bind to one or more Fzd proteins, e.g., scFv, Fab, etc., and other portions of antibodies, nanobody-derived binding domains, knottin-based artificial scaffolds, Norrin and engineered binding fragments derived therefrom, naturally occurring Fzd-binding domains, and the like.

[0123] In some embodiments, the Fzd binding domain binds to one, two, three, four, five, or more different frizzled proteins, such as one or more of the human frizzled proteins Fzl, Fz2, Fz3, Fz4, Fz5, Fz6, Fz7, Fz8, Fz9, FzlO. In some embodiments, the antibody-based signaling agonist binds to Fzl, Fz2, Fz5, Fz7, and Fz8. In other embodiments, the frizzled binding moiety is selective for one or more frizzled proteins of interest, e.g., has at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold or more specificity for one or more desired frizzled proteins over other frizzled proteins.

[0124] In certain embodiments, the frizzled binding domain comprises the six CDR regions of the pan-specific frizzled antibody OMP-18R5 (vanticumab). In certain embodiments, the frizzled binding domain is an scFv comprising the six CDR regions of the pan-specific frizzled antibody OMP-18R5 (vanticumab). See, for example, U.S. Patent No. 8,507,442, which is specifically incorporated herein by reference. For example, the CDR sequences of OMP-18R5 comprise a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 176), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO: 177), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO: 178), (ii) a light chain CDR2 comprising SGDKLGKKYAS (SEQ ID NO: 179) or SGDNIGSFYVH (SEQ ID NO: 180), a light chain CDR2 comprising EKDNRPSG (SEQ ID NO: 181) or DKSNRPSG (SEQ ID NO: 182), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO: 183) or QSYANTLSL (SEQ ID NO: 184). In certain embodiments, the frizzled binding domain is an antibody or derivative thereof, including but not limited to, an ScFv, a minibody, a nanobody, and various antibody mimetics comprising the CDR sequences of SEQ ID NOs: 176-184. In certain embodiments, these CDR sequences contain one or more amino acid modifications compared to SEQ ID NOs: 176-184.

[0125] In other embodiments, the Fzd binding domain comprises the variable region sequence or CDRs from any of the numerous frizzled-specific antibodies known in the art and commercially available, or which can be generated de novo. Any of the frizzled polypeptides can be used as immunogens or in screening assays to develop antibodies. "Fz", "Fz protein" and "Fz receptor" are used herein to refer to proteins of the Frizzled receptor family. These proteins are seven transmembrane proteins that contain a CRD domain (Ingham, PW (1996) Trends Genet. 12:382-384; Yang-Snyder, J. et al. (1996) Curr. Biol. 6:1302-1306; Bhanot, P. et al. (1996) Nature 382:225-230). There are 10 known members of the Fz family (Fz1 to Fz10), any of which can function as a receptor for Wnts. Genbank accession numbers for human frizzled reference sequences are as follows: FZD1 (NM_003505), FZD2 (NM_001466), FZD3 (NM_145866), FZD4 (NM_012193), FZD5 (NM_003468), FZD6 (NM_003506), FZD7 (NM_003507), FZD8 (NM_031866), FZD9 (NM_003508), FZD10 (NM_007197).

[0076] Non-limiting examples of frizzled binding domains include antibodies available from Biolegend, such as clone CH3A4A7 (CD344) specific for human frizzled 4, clone W3C4E11 (CD349) specific for human Fz9, and antibodies available from Abeam, such as ab64636 specific for Fz7, ab83042 specific for human Fz4, ab77379 specific for human Fz7, ab75235 specific for human Fz8, and ab102956 specific for human Fz9. Other examples of suitable antibodies are described in, inter alia, U.S. Patent Application No. 20140105917, U.S. Patent Application No. 20130230521, U.S. Patent Application No. 20080267955, U.S. Patent Application No. 20080038272, U.S. Patent Application No. 20030044409.

[0126] The frizzled binding portion of the surrogate can be an artificial protein selected for structural homology to the frizzled binding region of the Wnt protein. Such proteins can be identified by screening structural databases for homology. In this way, the first protein, for example the microbial Bh1478 protein, was identified. The natural protein can then be engineered to provide amino acid substitutions that increase affinity and further selected by affinity maturation for increased affinity and selectivity in binding to the desired frizzled protein. Non-limiting examples of frizzled binding portions include the Fz27 and Fz27-B12 proteins shown in Figure 1 of PCT Patent Application Publication No. 2016 / 040895.

[0127] In certain polypeptide embodiments, the Lrp5 / 6 binding domain or element has high affinity, e.g., at least about 1×10 ”7 M, at least about 1 × 10 ”8 M, at least about 1 × 10 ”9 M, at least about 1 × 10 ”10 K of M D The Lrp5 / 6 binding domain may be selected from any domain that binds to Lrp5 / 6 at the C-terminal end. Suitable Lrp5 / 6 binding domains include, but are not limited to, de novo designed Lrp5 / 6 binding proteins, antibody-derived binding proteins that specifically bind to one or more Fzd proteins, such as scFv, Fab, etc., and other portions of antibodies, nanobody-derived binding domains, knottin-based artificial scaffolds, Norrin, DKK1, DKK2, DKK3, DKK4, sclerostin, and the like, naturally occurring Lrp5 / 6 binding proteins or polypeptides. In certain embodiments, the Lrp5 / 6 binding domain is the C-terminal portion of DKK1.

[0128]

[0079] The Lrp5 / 6 binding domain may, for example, be at least about 1 x 10 ”7 M, at least about 1 × 10 ”8 M, at least about 1 × 10 ”9 M, at least about 1 × 10 ”10 K of M1 DThe domain may be selected from any domain that binds to Lrp5 or Lrp6 with high affinity having a specific binding domain. "LRP", "LRP protein" and "LRP receptor" are used herein to refer to proteins of the low density lipoprotein receptor-related protein family. These receptors are single-pass transmembrane proteins that bind and internalize ligands in the process of receptor-mediated endocytosis. The LRP proteins LRP5 (GenBank Accession No. NM 002335.2) and LRP6 (GenBank Accession No. NM002336.2) are included in the Wnt receptor complex.

[0129] Suitable Lrp5 / 6 binding domains include, but are not limited to, de novo designed Lrp5 / 6 binding proteins, antibody-derived binding proteins that specifically bind to one or more Fzd proteins, such as scFv, Fab, etc., and other portions of antibodies; nanobody-derived binding domains; knottin-based artificial scaffolds; naturally occurring Lrp5 / 6, including, but not limited to, DKK1, DKK2, DKK3, DKK4, sclerostin; Wise; fusion proteins comprising any of the above; derivatives of any of the above; variants of any of the above; and biologically active fragments of any of the above. Lrp5 / 6 binding domains can be affinity selected to enhance binding.

[0130] Members of the Dickkopf (Dkk) gene family (see Krupnik et al. (1999) Gene 238(2):301-13) include Dkk-1, Dkk-2, Dkk-3, and Dkk-4, as well as the Dkk-3-related protein Soggy (Sgy). hDkk1-4 contain two distinct cysteine-rich domains in which the positions of the 10 cysteine ​​residues are highly conserved among family members. Exemplary sequences of human Dkk genes and proteins are publicly available, e.g., Genbank accession numbers NM_014419 (soggy-1), NM_014420 (DKK4), AF177394 (DKK-1), AF177395 (DKK-2), NM_015881 (DKK3), and NM_014421 (DKK2). In some embodiments of the invention, the Lrp6 binding moiety is a DKK1 peptide, including but not limited to the C-terminal domain of human DKK1. As shown in Figure 5, the C-terminal domain can comprise the sequence KMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKTKHRRKGSHGLEIFQRC YCGEGLSCRIQKDHHQASNSRLHTCQRH (SEQ ID NO: 185) (see Genbank Accession No. NP_036374), or a biologically active fragment thereof.

[0131] Binding of DKK proteins to LRP5 / 6 is described, for example, in Brott and Sokol Mol. Cell. Biol. 22(17), 6100-6110 (2002), and Li et al. J. Biol. Chem. 277(8), 5977-5981 (2002), each of which is specifically incorporated herein by reference. The corresponding region of human DKK2 (Genbank reference number NP_055236) may contain the sequence KMSHIKGHEGDPCLRSSDCIEGFCCARHFWTKICKPVQRKKGSHGLEIFQRCD CAKGLSCKVWKDATYSSKARLHVCQK (SEQ ID NO: 186), or a biologically active fragment thereof.

[0132]

[0083] Antibodies that specifically bind to Lrp5 or Lrp6 are known in the art and commercially available, or can be newly generated. Lrp5, Lrp6 or fragments thereof can be used as immunogens or in screening assays to develop antibodies. Examples of known antibodies include, but are not limited to, Gong et al. (2010) PLoS One.5(9):e12682; Ettenberg et al. (2010) Proc Natl Acad Sci US107(35):15473-8; commercially available from Santa Cruz Biotechnology antibody clone 1A12, which is made against synthetic LRP5 / 6 of human origin and binds to both full-length and proteolytic fragments of LRP6 and LRP5 of mouse and human origin; monoclonal antibody 2B11; Cell Signaling Technology antibody specific for LRP5(D80F2), catalog number 5731, and the like.

[0133] Polypeptides and binding domains may also include derivatives, variants, and biologically active fragments of the above polypeptides. A "variant" polypeptide refers to a biologically active polypeptide, as defined below, having less than 100% sequence identity with the provided sequence. Such variants include polypeptides that contain one or more amino acid modifications, such as insertions, deletions, or substitutions, compared to the provided sequence, such as those in which one or more amino acid residues are added to or within the N-terminus or C-terminus, the native sequence; those in which about 1-40 amino acid residues are deleted and optionally replaced by one or more amino acid residues; and derivatives of the above polypeptides in which amino acid residues are covalently modified such that the resulting product has a non-naturally occurring amino acid. In certain embodiments, biologically active variants will have an amino acid sequence that has at least about 90%, at least about 95%, or at least about 99% amino acid sequence identity with the native sequence polypeptide. A "functional variant" of a sequence is a compound that has qualitative biological properties in common with the initial sequence. "Functional variants" include, but are not limited to, fragments of sequences and variants of sequences, provided they have a common biological activity. The term "variant" encompasses both amino acid sequence variants of a polypeptide and covalent modifications thereof.

[0134] The Fzd binding domain and the Lrp5 / 6 binding domain may be adjacent in one globular domain, or may be separated by a linker, such as a polypeptide linker, including but not limited to any of those described herein, or a non-peptide linker. The length of the linker, and therefore the spacing between the binding domains, can be used to regulate the signal strength and can be selected depending on the desired application of the Wnt signaling agonist. The forced distance between the binding domains can vary, but in certain embodiments, it can be less than about 100 angstroms, less than about 90 angstroms, less than about 80 angstroms, less than about 70 angstroms, less than about 60 angstroms, or less than about 50 angstroms.

[0135] In some embodiments, the linker is a rigid linker, and in other embodiments, the linker is a flexible linker. When the linker is a peptide linker, in certain embodiments, it is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids long, and of sufficient length and amino acid composition to enforce the distance between the binding domains. In some embodiments, the linker comprises or consists of one or more glycine and / or serine residues.

[0136] The present disclosure also includes polynucleotides or nucleic acid sequences encoding one or more Wnt polypeptides, Norrin polypeptides, or Wnt signaling agonist molecules, as well as vectors including expression vectors that contain these polynucleotides, and cells that contain these vectors. In certain embodiments, the polynucleotides or nucleic acid sequences are DNA or RNA. In certain embodiments, the RNA is messenger RNA (mRNA). In certain embodiments, the RNA is a modified mRNA that contains one or more modified nucleosides. Modified mRNA that contains one or more modified nucleosides has been described to have advantages over unmodified mRNA, including improved stability, higher expression levels, and reduced immunogenicity. Non-limiting examples of modified mRNAs that may be used in accordance with the present invention include those described in, e.g., PCT Patent Application Publication Nos. WO2011 / 130624, WO2012 / 138453, WO2013151666, WO2013 / 071047, WO2013 / 078199, WO2012045075, WO2014081507, WO2014093924, WO2014164253, U.S. Pat. Nos. 8,278,036 (describes modified mRNAs containing pseudouridine), 8,691,966 (describes modified mRNAs containing pseudouridine and / or N1-methylpsudoridine), 8,835,108 (describes modified mRNAs containing 5-methylcytidine), and 8,748,089 (describes modified mRNAs containing pseudouridine or 1-methylpsudoridine). In certain embodiments, the modified mRNA sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule comprises at least one modification compared to an unmodified A, G, U, or C ribonucleoside. In certain embodiments, the at least one modified nucleoside comprises N1-methylpsudoridine and / or 5-methylcytidine. In certain embodiments, the modified mRNA comprises a 5' terminal cap sequence followed by a sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule followed by a 3' tailing sequence, such as a polyA or polyAG sequence.

[0137] In certain embodiments, the polynucleotide is a vector, e.g., an expression vector, and the expression vector comprises a promoter sequence, e.g., a polynucleotide sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule described herein, operably linked to a promoter. A sequence that drives the expression of the polynucleotide in a cell. In some embodiments, the vector is a viral vector, e.g., a virus that comprises a polynucleotide comprising an expression cassette that comprises a promoter operably linked to a DNA or RNA sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule. In certain embodiments, the expression cassette comprises 5' and / or 3' cellular or viral UTRs.

[0138] The present disclosure also includes functional fragments and variants of the polynucleotides described herein, including variants having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% polynucleotide sequence identity with the polynucleotides described herein. Such variants can include one or more nucleotide or nucleoside modifications, such as one or more nucleotide deletions, insertions or substitutions, compared to any sequence disclosed herein. In certain embodiments, the polynucleotides described herein are codon-optimized, for example, to enhance expression of the encoded polypeptide in a host cell. In certain embodiments, the polynucleotide variants include one or more modified nucleotides or nucleosides.

[0139] The present disclosure also includes cells comprising a polynucleotide or vector encoding a Wnt polypeptide, Norrin polypeptide, or Wnt signaling agonist molecule described herein. In certain embodiments, the cell is a host cell, such as, for example, a HEK293 cell, that can be used to produce a Wnt polypeptide, Norrin polypeptide, or Wnt signaling agonist molecule. Any host cell can be used in preparing the composition, including, but not limited to, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. In certain embodiments, the cell is xenogeneic or autologous to the subject treated with a Wnt polypeptide, Norrin polypeptide, or Wnt signaling agonist molecule described herein. In certain embodiments, the cell is obtained from the subject and transduced with a viral vector described herein. In certain embodiments, the transduced cell is delivered to the subject for treatment. The disclosure also includes pharmaceutical compositions comprising one or more Wnt polypeptides, Norrin polypeptides, or Wnt signaling agonist molecules, or one or more polynucleotides or vectors comprising sequences encoding a Wnt polypeptide, Norrin polypeptide, or Wnt signaling agonist molecule.

[0140] Pharmaceutical Compositions Also disclosed is a pharmaceutical composition comprising the tissue-specific Wnt signal enhancing molecule described herein and one or more pharma- ceutically acceptable diluents, carriers, or excipients.In certain embodiments, the pharmaceutical composition further comprises one or more Wnt polypeptides, Norrin polypeptides, or Wnt signaling agonist molecules described herein.

[0141] In further embodiments, pharmaceutical compositions are also disclosed that include a polynucleotide comprising a nucleic acid sequence encoding a tissue-specific Wnt signal enhancing molecule as described herein and one or more pharma- ceutically acceptable diluents, carriers, or excipients. In certain embodiments, the pharmaceutical composition further comprises one or more polynucleotides comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule as described herein. In certain embodiments, the polynucleotide is a DNA or an mRNA, such as a modified mRNA. In certain embodiments, the polynucleotide is a modified mRNA that further comprises a 5' cap sequence and / or a 3' tailing sequence, such as a polyA tail. In other embodiments, the polynucleotide is an expression cassette that includes a promoter operably linked to the coding sequence. In certain embodiments, the nucleic acid sequence encoding the tissue-specific Wnt signal enhancing molecule and the nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule are present in the same polynucleotide.

[0142] In further embodiments, pharmaceutical compositions are also disclosed that comprise a polynucleotide comprising a nucleic acid sequence encoding tissue-specific Wnt signal enhancer molecule as described herein and an expression vector, such as a viral vector, that comprises one or more pharma- ceutically acceptable diluents, carriers, or excipients.In certain embodiments, the pharmaceutical composition further comprises an expression vector, such as a viral vector, that comprises a polynucleotide comprising a nucleic acid sequence encoding Wnt polypeptide, Norrin polypeptide, or Wnt signaling agonist molecule as described herein.The sequence encoding tissue-specific Wnt signal enhancer molecule and the nucleic acid sequence encoding Wnt polypeptide, Norrin polypeptide, or Wnt signaling agonist molecule are present in the same polynucleotide, such as an expression cassette.

[0143] The present invention further contemplates a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid encoding a tissue-specific Wnt signal enhancing molecule as described herein, and one or more pharma- ceutically acceptable diluents, carriers, or excipients. In certain embodiments, the pharmaceutical composition further comprises a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule as described herein. In certain embodiments, the nucleic acid sequence encoding the tissue-specific Wnt signal enhancing molecule and the nucleic acid sequence encoding the Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule are present in the same polynucleotide, e.g., expression cassette, and / or in the same cell. In certain embodiments, the cell is a heterologous or autologous cell obtained from the subject to be treated. In certain embodiments, the cell is a stem cell, e.g., an adipose-derived stem cell or a hematopoietic stem cell.

[0144] The present disclosure contemplates a pharmaceutical composition comprising a first molecule for delivering tissue-specific Wnt signal enhancing molecules as a first active agent, and a second molecule for delivering Wnt polypeptide, Norrin polypeptide or Wnt signaling agonist as a second active agent. The first and second molecules can be the same or different types of molecules. For example, in certain embodiments, the first and second molecules can each be independently selected from the following types of molecules: polypeptide, small organic molecule, nucleic acid (optionally DNA or mRNA, optionally modified RNA) encoding the first or second active agent. A vector (optionally an expression vector or viral vector) comprising a nucleic acid sequence encoding the first or second active agent, and a cell (optionally an expression cassette) comprising a nucleic acid sequence encoding the first or second active agent.

[0145] The molecules, alone or in combination, can be combined with pharma- ceutically acceptable carriers, diluents and reagents useful for preparing formulations that are generally safe, non-toxic, and desirable, including excipients that are acceptable to mammals, e.g., humans. or primates, for use. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulation. Solutions or suspensions used in the formulation can include sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; detergents such as Tween 20 to prevent aggregation; compounds for isotonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In certain embodiments, the pharmaceutical compositions are sterile.

[0146] The pharmaceutical compositions may further include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some cases, the composition should be sterile and fluid to the extent that easy syringability exists. In certain embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, or, in the case of dispersions, by the maintenance of the required particle size, by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the internal composition can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0147] Sterile solution can be prepared by incorporating the tissue-specific Wnt signal enhancing molecule in the required amount in a suitable solvent with one or combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other components required from the above-listed ones.For the case of sterile powder for preparing sterile injectable solution, the preparation method is vacuum drying and lyophilization, which produces the powder of active ingredient plus any additional desired ingredient from the solution that is previously sterile filtered.

[0148] In one embodiment, the pharmaceutical composition is prepared with a carrier that protects the fusion protein from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be clear to those skilled in the art. Materials can also be commercially obtained. Liposomal suspensions can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0149] For ease of administration and uniformity of dosage, it may be advantageous to formulate pharmaceutical compositions into dosage unit form.Dosage unit form as used herein refers to a physically separate unit suitable as a unitary dosage for the subject to be treated.Each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect together with the necessary pharmaceutical carrier.The details of the dosage unit form of the present invention are determined and directly influenced by the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the technical field of compounding such active compound for individual treatment.

[0150] The pharmaceutical compositions can be included in a container, pack, or dispenser, such as a syringe, including a prefilled syringe, together with instructions for administration.

[0151] The pharmaceutical compositions of the present invention include any pharma- ceutically acceptable salts, esters, or salts of such esters, or any other compounds capable of providing biologically active tissue specificity upon administration to an animal, including a human, of the Wnt signal enhancing molecule.

[0152] The present invention includes pharma- ceutically acceptable salts of the tissue-specific Wnt signal enhancing molecules described herein. The term "pharma- ceutically acceptable salts" refers to physiologically and pharma- ceutical acceptable salts of the compounds of the present invention, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxic effects thereto. A variety of pharma- ceutical acceptable salts are known in the art, for example, "Remington's Pharmaceutical Sciences", 17th Edition, Alfonso R., for example, "Encyclopaedia of Pharmaceutical Technology", 3rd Edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J.Pharm.Sci.66:2 (1977). Also, for a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002).

[0153] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Metals used as cations include sodium, potassium, magnesium, calcium, and the like. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J.Pharma Sci., 1977, 66, 119). The base addition salts of the acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in a conventional manner. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acids for purposes of the present invention.

[0154] In some embodiments, the pharmaceutical composition provided herein comprises a therapeutically effective amount of the tissue-specific Wnt signal enhancing molecule described herein mixed with a pharma- ceutically acceptable carrier, diluent and / or excipient, such as saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins.Exemplary amino acids, polymers and sugars, etc. are octylphenoxy polyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene and glycol.Preferably, the formulation is stable at 4°C for at least 6 months.

[0155] In some embodiments, the pharmaceutical compositions provided herein comprise a buffer, such as phosphate buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those of skill in the art, such as those described in Good et al. (1966) Biochemistry 5: 467. The pH of the buffer can range from 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0156] Methods for increasing Wnt activity in cells The tissue-specific Wnt signal enhancing molecule exemplified herein for fusion protein can be used to increase Wnt signaling in target tissue or cell type. In certain embodiments, the Wnt signaling is standard Wnt signaling. Thus, in some aspects, the present invention provides a method for increasing Wnt or enhancing Wnt signaling in target tissue or cell, comprising contacting the target tissue or cell with an effective amount of the tissue-specific Wnt signal enhancing molecule of the present invention, the molecule comprising a targeting domain that binds to a cell surface receptor on the target tissue or cell in a tissue-specific or cell-specific manner. In some embodiments, the contacting occurs in vitro, ex vivo, or in vivo, for example, the tissue-specific Wnt signal enhancing molecule of the subject is administered or provided to the subject. In certain embodiments, the cell is a cultured cell, and the contacting occurs in vitro.

[0157] In certain embodiments, the method further comprises contacting the target tissue or cell with one or more Wnt polypeptides, Norrin polypeptides or Wnt signaling agonist molecules described herein. The present disclosure contemplates contacting the target tissue or cell with a first molecule for delivery of tissue-specific Wnt signal enhancing molecules as a first active agent and a second molecule for delivery of Wnt polypeptides, Norrin polypeptides or Wnt signaling agonists as a second active agent. The first and second molecules may be the same or different types of molecules. For example, in certain embodiments, the first and second molecules may each be independently selected from the following types of molecules: polypeptides, small organic molecules, nucleic acids (optionally DNA or mRNA, optionally modified RNA) encoding the first or second active agent, vectors (optionally expression vectors or viral vectors) comprising nucleic acid sequences encoding the first or second active agent, and cells (optionally expression cassettes) comprising nucleic acid sequences encoding the first or second active agent.

[0158] In a related aspect, the present invention provides a method for increasing Wnt signaling in a target tissue or cell, comprising contacting the target tissue or cell with an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a tissue-specific Wnt signaling enhancer molecule, the molecule comprising a targeting domain that binds to a cell surface receptor on the target tissue or cell in a tissue-specific or cell-specific manner. In certain embodiments, the target tissue or cell is also contacted with a polynucleotide comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist. In certain embodiments, the polynucleotide is DNA or mRNA, such as a modified mRNA. In certain embodiments, the polynucleotide is a modified mRNA, further comprising a 5' cap sequence and / or a 3' tailing sequence, such as a polyA tail. In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to the coding sequence. In certain embodiments, the nucleic acid sequence encoding the tissue-specific Wnt signaling enhancer molecule and the nucleic acid sequence encoding the Wnt polypeptide, the Norrin polypeptide, or the Wnt signaling agonist molecule are present in the same polynucleotide.

[0159] In a related aspect, the present invention provides a method for increasing Wnt signaling in a target tissue or cell, comprising contacting the target tissue or cell with an effective amount of a vector comprising a nucleic acid sequence encoding a tissue-specific Wnt signal enhancing molecule, the molecule comprising a targeting domain that binds to a cell surface receptor on the target tissue or cell in a tissue-specific or cell-specific manner. In certain embodiments, the target tissue or cell is also contacted with a vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide or a Wnt signaling agonist. In certain embodiments, the vector is an expression vector and may comprise a promoter operably linked to the nucleic acid sequence. In certain embodiments, the vector is a viral vector. In certain embodiments, the nucleic acid sequence encoding the tissue-specific Wnt signal enhancing molecule and the nucleic acid sequence encoding the Wnt polypeptide, the Norrin polypeptide or the Wnt signaling agonist molecule are present in the same vector, for example in the same expression cassette.

[0160] In a related aspect, the present invention provides a method for increasing Wnt signaling in a target tissue, comprising contacting the target tissue with an effective amount of cells comprising a nucleic acid sequence encoding a tissue-specific Wnt signaling enhancer molecule of the present invention, the molecule comprising a targeting domain that binds to a cell surface receptor on the target tissue or cell in a tissue-specific or cell-specific manner. In certain embodiments, the target tissue is also contacted with cells comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide or a Wnt signaling agonist. In certain embodiments, the nucleic acid sequence encoding the tissue-specific Wnt signaling enhancer molecule and the nucleic acid sequence encoding the Wnt polypeptide, a Norrin polypeptide or a Wnt signaling agonist molecule are present in the same cell. In certain embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated. In certain embodiments, the cell has been transduced with a vector comprising an expression cassette encoding a tissue-specific Wnt signaling enhancer molecule or a Wnt polypeptide, a Norrin polypeptide or a Wnt signaling agonist molecule. In certain embodiments, the cell is a stem cell, for example an adipose-derived stem cell or a hematopoietic stem cell.

[0161] Methods for treating illnesses and disorders The tissue-specific Wnt signal enhancer molecule exemplified herein with respect to a fusion protein can be used to treat a disease, disorder or condition, for example, by increasing Wnt signaling in a target cell, tissue or organ. Thus, in some aspects, the present invention provides a method for treating a disease or condition in a subject in need thereof, for example, a disease or disorder associated with decreased Wnt signaling or in which increased Wnt signaling would provide a therapeutic benefit, comprising contacting the subject with an effective amount of a composition of the present disclosure. In certain embodiments, the composition is a pharmaceutical composition comprising any of the following: a tissue-specific Wnt signal enhancer molecule, for example, a small molecule or a polypeptide; a polynucleotide comprising a nucleic acid sequence encoding a tissue-specific Wnt signal enhancer molecule, for example, DNA or mRNA, optionally modified mRNA; a vector comprising a nucleic acid sequence encoding a tissue-specific Wnt signal enhancer molecule; for example, an expression vector or a viral vector; or a cell comprising a nucleic acid sequence encoding a tissue-specific Wnt signal enhancer molecule, for example, a cell transduced with an expression vector or a viral vector encoding a tissue-specific Wnt signal enhancer molecule. In certain embodiments, the disease or condition is a pathological disease or disorder, or an injury, for example, an injury resulting from a wound. In certain embodiments, the wound may be the result of another therapeutic treatment. In certain embodiments, the disease or condition involves impaired tissue repair, healing or regeneration or would benefit from increased tissue repair, healing or regeneration. In some embodiments, the contacting occurs in vivo, i.e., the subject composition is administered to a subject.

[0162] In certain embodiments, the method further comprises contacting the subject with a pharmaceutical composition comprising one or more Wnt polypeptides, Norrin polypeptides or Wnt signaling agonist molecules described herein. The present disclosure contemplates contacting the subject with a first molecule for delivery of tissue-specific Wnt signal enhancing molecules as a first active agent and a second molecule for delivery of Wnt polypeptides, Norrin polypeptides or Wnt signaling agonists as a second active agent. The first and second molecules may be the same or different types of molecules. For example, in certain embodiments, the first and second molecules may each be independently selected from the following types of molecules: polypeptides, small organic molecules, nucleic acids (optionally DNA or mRNA, optionally modified RNA) encoding the first or second active agent, vectors (optionally expression vectors or viral vectors) comprising nucleic acid sequences encoding the first or second active agent, and cells (optionally expression cassettes) comprising nucleic acid sequences encoding the first or second active agent.

[0163] In a related embodiment, the present invention provides a method for treating a disease or condition, such as a disease or disorder associated with decreased Wnt signaling or in which increased Wnt signaling would provide therapeutic benefit, comprising contacting a subject with an effective amount of a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding a tissue-specific Wnt signaling enhancer molecule of the present invention, the molecule comprising a targeting domain that binds to a cell surface receptor on a target tissue or cell in a tissue-specific or cell-specific manner. In an embodiment, the subject is also contacted with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist. In certain embodiments, the polynucleotide is DNA or mRNA, such as a modified mRNA. In certain embodiments, the polynucleotide is a modified mRNA that further comprises a 5' cap sequence and / or a 3' tailing sequence, such as a polyA tail. In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to the coding sequence. In certain embodiments, the nucleic acid sequence encoding the tissue-specific Wnt signaling enhancer molecule and the nucleic acid sequence encoding the Wnt polypeptide, the Norrin polypeptide, or the Wnt signaling agonist molecule are present in the same polynucleotide.

[0164] In a related aspect, the present invention provides a method for treating a disease or condition, such as a disease or disorder associated with decreased Wnt signaling or in which increased Wnt signaling would provide therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding a tissue-specific Wnt signaling enhancer molecule of the present invention, the molecule comprising a targeting domain that binds to a cell surface receptor in a target tissue or cell in a tissue-specific or cell-specific manner. In certain embodiments, the subject is also contacted with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide or a Wnt signaling agonist. In certain embodiments, the vector is an expression vector and may comprise a promoter operably linked to the nucleic acid sequence. In certain embodiments, the vector is a viral vector. In certain embodiments, the nucleic acid sequence encoding the tissue-specific Wnt signaling enhancer molecule and the nucleic acid sequence encoding the Wnt polypeptide, the Norrin polypeptide or the Wnt signaling agonist molecule are present in the same vector, for example in the same expression cassette.

[0165] In a related aspect, the present invention provides a method for treating a disease or condition, such as a disease or disorder associated with decreased Wnt signaling or in which increased Wnt signaling would provide therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of cells comprising a nucleic acid sequence encoding a tissue-specific Wnt signaling enhancer molecule of the present invention, the molecule comprising a targeting domain that binds to a cell surface receptor in a target tissue or cell in a tissue-specific or cell-specific manner. In certain embodiments, the subject is also contacted with a cell comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist. In certain embodiments, the nucleic acid sequence encoding the tissue-specific Wnt signaling enhancer molecule and the nucleic acid sequence encoding the Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule are present in the same cell. In certain embodiments, the cell is a heterologous or autologous cell obtained from the subject to be treated. In certain embodiments, the cell has been transduced with a vector comprising an expression cassette encoding a tissue-specific Wnt signaling enhancer molecule or a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule. In certain embodiments, the cells are stem cells, such as adipose-derived stem cells or hematopoietic stem cells.

[0166] In another aspect, the present invention provides a method for treating a disease or condition associated with decreased Wnt signaling or in which increased Wnt signaling would provide therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist. In certain embodiments, the polynucleotide is DNA or mRNA, such as a modified mRNA. In certain embodiments, the polynucleotide is a modified mRNA, further comprising a 5' cap sequence and / or a 3' tailing sequence, such as a polyA tail. In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to a coding sequence.

[0167] In a related aspect, the present invention provides a method for treating a disease or condition, such as a disease or disorder associated with decreased Wnt signaling or in which increased Wnt signaling would provide therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide or a Wnt signaling agonist.In certain embodiments, the vector is an expression vector and may comprise a promoter operably linked to the nucleic acid sequence.In certain embodiments, the vector is a viral vector.

[0168] In a related aspect, the present invention provides a method for treating a disease or condition, such as a disease or disorder associated with decreased Wnt signaling or in which increased Wnt signaling would provide a therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of cells comprising a nucleic acid sequence encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist. In certain embodiments, the cells are heterologous or autologous cells obtained from the subject to be treated. In certain embodiments, the cells are transduced with a vector comprising an expression cassette encoding a Wnt polypeptide, a Norrin polypeptide, or a Wnt signaling agonist molecule. In certain embodiments, the cells are stem cells, such as adipose-derived stem cells or hematopoietic stem cells.

[0169] Wnt signaling plays an important role in stem cell development process and maintenance. Reactivation of Wnt signaling is associated with regeneration and repair of most tissues after injury and disease. Tissue-specific Wnt signaling enhancer molecules can provide healing and tissue repair benefits in response to injury and disease. Causes of tissue damage and loss include, but are not limited to, aging, degeneration, genetic conditions, infection and inflammation, trauma, toxin / metabolism-induced toxicity, or other pathological conditions. Wnt signals and enhancers of Wnt signals have been shown to activate adult tissue-resident stem cells. In some embodiments, the compounds of the present invention are administered for use in the treatment of diseased or damaged tissues, for use in tissue regeneration, and for use in cell growth and proliferation, and / or for use in tissue engineering.

[0170] Human diseases associated with mutations in the Wnt pathway provide strong evidence for the enhancement of Wnt signaling in the treatment and prevention of disease. Preclinical in vivo and in vitro studies provide further evidence of the involvement of Wnt signaling in many disease states, further supporting the use of tissue-specific Wnt signaling enhancer molecules in various human diseases. For example, the compositions of the present invention can be used to promote or increase bone growth or regeneration, bone grafting, fracture healing, osteoporosis and osteoporotic fracture treatment, spinal fusion, orthopedic device osseointegration, tendon-bone integration, tooth growth, regeneration, tooth implantation, periodontal disease, maxillofacial reconstruction, and osteonecrosis of the jaw. They can also be used to treat alopecia. Promoting the regeneration of sensory organs, such as the treatment of hearing loss, the treatment of vestibular function reduction, the treatment of macular degeneration, the treatment of vitreous retinopathy, diabetic retinopathy, or other retinal degenerative diseases, Fuchs' dystrophy, other corneal diseases, etc. Treatment of stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, and other conditions that affect the blood-brain barrier. Treatment of spinal cord injury and other spinal cord diseases. The compositions of the present invention can also be used to treat oral mucositis, intestinal mucositis, short bowel syndrome, inflammatory bowel disease (IBD), and other gastrointestinal disorders. Treatment of metabolic syndrome Treatment of diabetes, treatment of pancreatitis, conditions in which exocrine pancreatic tissue or endocrine pancreatic tissue is damaged. Conditions in which enhanced epidermal regeneration is desired, such as epidermal wound healing, treatment of diabetic foot ulcers, syndromes involving teeth, nails, or dermal hypoplasia, conditions in which angiogenesis is beneficial. Treatment of myocardial infarction, coronary artery disease, heart failure. Promotion of hematopoietic cell proliferation, such as promotion of hematopoietic stem cell transplantation from bone marrow, mobilized peripheral blood, treatment of immune deficiency, graft-versus-host disease, etc. Treatment of acute kidney injury, chronic kidney disease Treatment of pulmonary disease, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, promotion of regeneration of lung tissue. The compositions of the present invention can also be used to enhance regeneration of hepatocytes, e.g., liver regeneration, treatment of cirrhosis, enhancing liver transplantation, treatment of acute liver failure, treatment of chronic liver disease associated with Hepatitis C or Hepatitis B virus infection, or following antiviral drug therapy, alcoholic liver disease, non-alcoholic liver disease associated with steatosis or steatohepatitis, etc.

[0171] Human genetics, including loss-of-function or gain-of-function mutations in Wnt signaling components, show strong evidence in support of enhancing Wnt signals for bone growth. Conditions in which enhanced bone growth is desired include, but are not limited to, fractures, implants, ingrowth around prostheses, osteoporosis, osteoporotic fractures, spinal fusion, osteonecrosis of the jaw, dental implants, periodontal disease, maxillofacial reconstruction, etc. Tissue-specific Wnt signal enhancing molecules enhance and promote Wnt signals, which are important for promoting bone regeneration. Bone tissue regeneration methods benefit from the administration of the compounds of the present invention, which may be systemic or local. In some embodiments, bone marrow cells are exposed to the molecules of the present invention so that stem cells in the bone marrow are activated.

[0172] In some embodiments, bone regeneration is enhanced by contacting responsive cell populations, such as bone marrow, osteoprogenitor cells, osteoprogenitor cells, etc., with an effective amount of the molecules of the present invention. The method of bone tissue regeneration benefits from the administration of the compounds of the present invention, which may be systemic or local. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo. The molecules can be localized at the site of action, for example, by loading onto a matrix that is optionally biodegradable and optionally provides sustained release of the active agent. Matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, polymer microspheres, nanoparticles, bone cements, etc.

[0173] The composition comprising one or more molecules of the present invention can be used for the in vivo treatment of skeletal tissue defects. By "skeletal tissue defects" is meant defects of bone or other skeletal connective tissue at any site where it is desired to repair the bone or connective tissue, no matter what the cause of the defect (e.g., as a result of surgical intervention). Tumor removal, ulceration, implant, fracture, or other traumatic or degenerative conditions. The composition of the present invention can be used for defects or damage to cartilage tissue, such as degenerative wear and arthritis, trauma to the tissue, replacement of torn meniscus, as part of a regimen to restore cartilage function to connective tissue. Due to meniscectomy, joint loosening due to ligament tears, joint malalignment, fracture, or genetic disease.

[0174] The compositions of the present invention may also be used to treat periodontal disease. Periodontal disease is the leading cause of tooth loss and is associated with multiple systemic conditions. In some embodiments, the regeneration of teeth or the underlying bone is enhanced by contacting with a responsive cell population. In some such aspects, the contacting is performed in vivo. In other such aspects, the contacting is performed ex vivo, followed by transplantation of activated stem or progenitor cells. Molecules can be localized at the site of action, for example, by loading onto a matrix that is optionally biodegradable and optionally provides sustained release of the active agent. Matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, bone cements, polymeric microspheres, nanoparticles, and the like.

[0175] Studies have shown that Wnt signaling and R-spondin biology can promote the regeneration of sensory hair cells in the inner ear after injury, aging, or degeneration. Loss of sensory hair cells in the inner ear involved in hearing impairment or reduced vestibular function may also benefit from the compositions of the present invention. The inner ear houses the mechanosensitive hair cells necessary for the hearing organ to convert sound vibrations into electrical impulses. The vestibular organ, consisting of the semicircular canals (SSC), utricle, and saccule, also contains sensory hair cells to detect head position and movement. The compositions of the present invention can be used, for example, for injection. in matrices or other depot systems. or other topical applications to the ear for enhanced hearing regeneration.

[0176] The compositions of the present invention may also be used in the regeneration of retinal tissue. In adult mammalian retina, Müller glia cells can regenerate retinal cells, including photoreceptors, after, for example, neurotoxic injury in vivo. Wnt signaling and enhancers of Wnt signals can promote the proliferation of Müller glia-derived retinal progenitor cells after injury or during degeneration. The compositions of the present invention may also be used to regenerate tissues and other cell types in the eye. For example, age-related macular degeneration (AMD), other retinal degenerative diseases, corneal diseases, Fuchs' dystrophy, vitreoretinopathy, genetic diseases, etc. can benefit from the compositions of the present invention. AMD is characterized by gradually decreasing central vision and visual acuity. Fuchs' dystrophy is characterized by the progressive loss of corneal endothelial cells. Wnt signals and enhancement of Wnt signals can promote the regeneration of corneal endothelium, retinal epithelium, etc. in ocular tissues. In other embodiments, the compositions of the present invention can be used, for example, in injections; matrices or other depot systems; or other topical applications to the eye for the treatment of retinal regeneration and macular degeneration.

[0177] Specific populations of proliferative cells for homeostatic regeneration of hepatocytes, such as Axin2-positive cells in the pericentral region, have been identified through lineage tracing studies. Lineage tracing studies have also identified additional potential liver progenitor cells, including but not limited to Lgr-positive cells. Self-renewing hepatocytes and other potential progenitor cell populations, including Lgr5-positive cells and Axin2-positive cells, have been identified as capable of regenerating in response to Wnt signals and / or R-spondin following injury. Numerous preclinical models of acute liver injury and failure as well as chronic liver disease have shown that recovery and regeneration of hepatocytes benefit from enhancing Wnt signals. The compositions of the present invention can be used to treat acute liver failure, acute alcoholic liver injury, chronic liver disease with hepatitis C or B virus infection or antiviral drug treatment, chronic alcoholic liver disease, nonalcoholic fatty liver disease. Nonalcoholic steatohepatitis (NASH), treatment of liver cirrhosis, severe chronic liver disease from any cause, promoting regeneration of hepatocytes. Liver tissue regeneration methods benefit from administration of the compounds of the present invention, which can be systemic or local. These include, but are not limited to, systemic and local administration methods, such as injection into liver tissue, injection into a vein or into a blood vessel to the liver, implantation of a sustained release formulation, and the like.

[0178] Wnt signaling plays an important role in the regeneration of various epithelial tissues. A variety of epidermal conditions benefit from treatment with the compounds of the invention. Mucositis occurs when there is a breakdown of the rapidly dividing epithelial cells lining the gastrointestinal tract, leaving the mucosal tissue open to ulceration and infection. The portion of the epithelial lining that covers the mouth, called the oral mucosa, is one of the most sensitive parts of the body and is particularly vulnerable to chemotherapy and radiation. Oral mucositis is perhaps the most common and debilitating complication of cancer treatment, especially chemotherapy and radiation therapy. In addition, the compositions of the invention may also be beneficial in the treatment of intestinal mucositis, short bowel syndrome, inflammatory bowel disease (IBD), or other gastrointestinal disorders. Other epidermal conditions include epidermal wound healing, diabetic foot ulcers, dental syndrome, nails, or dermal hypoplasia, etc. The molecules of the invention can be used in all of these conditions in which regenerative cells are contacted with the compounds of the invention. The method of regenerating epithelial tissue benefits from the administration of the compounds of the invention, which may be systemic or local. The contact may be applied topically, for example, intradermally, subcutaneously, in a gel, as a lotion, cream, etc.

[0179] In addition to the skin and gastrointestinal tract, Wnt signaling and the enhancement and promotion of Wnt signaling also play an important role in the repair and regeneration of tissues, including the pancreas, kidney, and lung in preclinical models. Tissue-specific Wnt signaling enhancement molecules can benefit a variety of pathologies, including the exocrine and endocrine pancreas, kidney, or lung. The compositions of the present invention can be used to treat metabolic syndrome, treat diabetes, treat acute or chronic pancreatitis, treat exocrine pancreatic insufficiency, treat acute kidney injury, treat lung diseases, including but not limited to chronic kidney disease, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, and other conditions that cause loss of lung epithelial tissue. These tissue regeneration methods benefit from the administration of the compounds of the present invention, which can be systemic or local.

[0180] Epidermal Wnt signaling is important for adult hair follicle regeneration in cooperation with signaling through other developmental factors.Hair loss is a common problem, and androgenetic alopecia, often called androgenetic alopecia, is the most common form of hair loss in men.In some embodiments, hair follicle regeneration is enhanced by contacting responsive cell population with the molecule of the present invention.In some such embodiments, contacting is performed in vivo.In other such embodiments, contacting is performed ex vivo.The molecule can be localized at the site of action, for example, topical lotion, gel, cream, etc.

[0181] Stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, and other conditions that affect the blood-brain barrier (BBB) ​​can be treated with the tissue-specific Wnt signal enhancing molecules of the present invention. Angiogenesis is important to ensure the supply of oxygen and nutrients to many tissues in the body, and is particularly important to the CNS, as neural tissue is highly sensitive to hypoxia and ischemia. CNS endothelial cells that form the BBB differ from endothelial cells in non-neural tissues in that they are highly polarized cells that are held together by tight junctions and express specific transporters. Wnt signaling regulates CNS angiogenesis and / or function. Conditions in which the BBB is compromised can benefit from administration of the compounds of the present invention, which can be systemic or local, such as direct injection, intrathecal administration, implantation of sustained release formulations, etc. In addition, Wnt signaling is actively involved in neurogenesis and plays a role in neuroprotection after injury. The compositions of the present invention can also be used to treat spinal cord injury, other spinal cord diseases, stroke, traumatic brain injury, and the like.

[0182] Wnt signal also plays a role in angiogenesis.Tissue-specific Wnt signal enhancer molecules can be beneficial for conditions where angiogenesis is beneficial, such as the treatment of myocardial infarction, coronary artery disease, heart failure, and conditions from genetic diseases.These tissue regeneration methods benefit from the administration of the compound of the present invention, which can be systemic or local.

[0183] In certain embodiments, the method of the present invention promotes tissue regeneration, for example in damaged tissue or tissue that is subject to tissue or cell loss or loss. Loss or damage can be anything that reduces cell number, including disease or injury. For example, accidents, autoimmune disorders, therapeutic side effects or pathologies can constitute trauma. Tissue regeneration increases cell number in tissue, preferably allowing the connection between cells of tissue to be re-established, and more preferably allowing the functionality of tissue to be restored.

[0184] In certain embodiments, the composition is administered parenterally, for example, intravenously, orally, rectally, or by injection. In some embodiments, it is administered locally, for example, topically or intramuscularly. In some embodiments, the composition is administered to a target tissue, for example, bone, joint, ear tissue, eye tissue, gastrointestinal tract, skin, wound site, or spinal cord. The method of the present invention can be performed in vivo or ex vivo. In some embodiments, contacting the target cell or tissue with the tissue-specific Wnt signal enhancing molecule is performed ex vivo, with subsequent transplantation of the cell or tissue (e.g., activated stem or progenitor cell) into a subject. Those skilled in the art can determine the appropriate administration site and route based on the disease or disorder being treated.

[0185] The dosage and administration schedule may depend on various factors that can be easily determined by a physician, such as the nature of the disease or disorder, the characteristics of the subject, and the medical history of the subject. In certain embodiments, the amount of tissue-specific Wnt signal enhancing molecule, e.g., fusion protein, administered or provided to a subject ranges from about 0.01 mg / kg to about 50 mg / kg, 0.1 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 50 mg / kg of the subject's body weight.

[0186] In certain embodiments, the subject can be any mammal, for example, a human, rodent (eg, mouse, rat, gerbil), rabbit, cat, dog, goat, sheep, pig, horse, cow, or primate.

[0187] In some embodiments, the methods of the invention provide a therapeutic benefit, e.g., prevention of the onset of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc. In some embodiments, the methods include detecting that a therapeutic benefit has been achieved. One of skill in the art will recognize that such measures of therapeutic efficacy are applicable to the particular disease being modified, and appropriate detection methods to use to measure therapeutic efficacy.

[0188] In certain embodiments, the present disclosure provides a method for treating or preventing a disease or disorder that is related to a decrease in Wnt signaling or would benefit from an increase in Wnt signaling activity in bone tissue, such as any of the conditions disclosed herein in which bone growth is desired, comprising providing a pharmaceutical composition comprising a Wnt signaling enhancer molecule comprising a targeting domain that binds to bone tissue, such as a targeting domain that specifically binds to PTH1R, to a subject in need thereof, wherein the Wnt signaling enhancer molecule increases or enhances signaling in the bone tissue of the subject.In certain embodiments, the pharmaceutical composition is administered orally or systemically, such as parenterally.In certain embodiments, the Wnt signaling enhancer molecule comprises an action domain that comprises R-spondin furin domain 1 or a fragment or variant thereof, and optionally a mutant furin domain 2 or a fragment or variant thereof.

[0189] In certain embodiments, the present disclosure provides a method for treating or preventing a disease or disorder that is associated with a decrease in Wnt signaling or would benefit from an increase in Wnt signaling activity in liver tissue, such as any of the diseases or disorders disclosed herein that would benefit from liver regeneration, comprising providing a pharmaceutical composition comprising a Wnt signaling enhancer molecule comprising a targeting domain that binds to liver tissue, such as a targeting domain that specifically binds to ASGR1, ASGR2, TFR2 or SLC10A1, to a subject in need thereof, wherein the Wnt signaling enhancer molecule increases or enhances the Wnt signaling in liver tissue of the subject.In certain embodiments, the pharmaceutical composition is administered orally or systemically, such as parenterally.In certain embodiments, the Wnt signaling enhancer molecule comprises an action domain that comprises R-spondin furin domain 1 or a fragment or variant thereof, and optionally a mutant furin domain 2 or a fragment or variant thereof.

[0190] In certain embodiments, the present disclosure provides a method for treating or preventing a disease or disorder that is related to reduced Wnt signaling or would benefit from increased Wnt signaling activity in oral mucosal tissue (such as oral mucositis), comprising providing a pharmaceutical composition comprising a Wnt signaling enhancer molecule comprising a targeting domain that binds to oral mucosal tissue, such as a targeting domain that specifically binds to LYPD3 or DSG3, to a subject in need thereof, wherein the Wnt signaling enhancer molecule increases or enhances the Wnt signaling in the oral mucosal tissue of the subject. In certain embodiments, the pharmaceutical composition is administered orally or systemically, for example parenterally. In certain embodiments, the Wnt signaling enhancer molecule comprises an action domain that comprises R-spondin furin domain 1 or a fragment or variant thereof, and optionally a mutant furin domain 2 or a fragment or variant thereof.

[0191] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to herein and / or listed in the Application Data Sheets are hereby incorporated by reference in their entireties.

[0192] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications can be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. EXAMPLES

[0193] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as the invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0194] Common methods in molecular biology, cell biology and biochemistry are called "Molecular "Cloning: A Laboratory Manual, 3rd Ed." (Sambrook et al., Harbor Laboratory Press 2001), "Short Protocols in Molecular Biology, 4th Ed." (Ausubel et al. eds., John Wiley & Sons 1999), "Protein Methods" (Bollag et al. et al., John Wiley & Sons 1996), "Nonviral Vectors for Gene Therapy" (Wagner et al. eds., Academic Press 1999), "Viral Vectors" (Kaplift & Loewy eds., Academic Press 1995), "Immunology Methods Manual" (I. Lefkovits ed., Academic Press 1997), and "Cell and Tissue Culture: Laboratory Procedures in Biotechnology" (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0195] In the following examples, a recombinant human Rspo2 preparation consisting of a fragment (S36-E143) containing the Fu1 and Fu2 domains was used as a positive control. For the Wnt signal enhancers in scFv format (Figures 8, 11, 12, 15, and 16), a monomeric form of Rspo2 was used (identified by SEQ ID NOs: 33 and 34, with a short tag to aid protein purification). For the Wnt signal enhancers in the accompanying IgG format (Figures 13, 14, 17, 18, and 19), a fusion of the same Rspo2 Fu1 and Fu2 domains in frame with a human IgG Fc fragment was used. Since no significant differences were observed in vitro between these two forms when tested in parallel under the same experimental conditions, both are generally referred to as Rspo2 positive controls without further specification.

[0196] A brief summary of the various constructs and sequences described in the Examples and accompanying figures is provided in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0197] Example 1 ASGR1-specific enhancement of WNT signaling in the SCFV format Tissue-specific enhancement of Wnt signaling was first demonstrated using a tissue-specific Wnt signaling enhancer molecule that contains an scFv antibody against human ASGR1 (designed based on patent WO2014 / 023709A1, clone 4F3) fused to mutant human Rspo2 (amino acid residues) with two point mutations (F105A and F109A) (α-ASGR-mtRspo2). These mutations in human Rspo2 reduce / abolish binding to LGR4-6 without compromising the interaction with ZNRF3 / RNF43, as illustrated in Figure 1, and allow Rspo2 to function as an action domain, as illustrated in Figure 2. As a negative control for the target domain (see Figure 2), an scFv antibody against green fluorescent protein (GFP) was fused to the same mutant human Rspo2 (α-GFP-mtRspo2). As a positive control for Wnt signal enhancement activity, GFP antibody was fused to wild-type human Rspo2 (amino acid residues 37-143) (α-GFP-Rspo2). These constructs are illustrated in FIG. 5A, and their amino acid sequences and encoding polynucleotide sequences are shown as follows: α-GFP-mtRspo2 (SEQ ID NOs: 8 and 7, respectively), α-ASGR-mt-Rspo2 (SEQ ID NOs: 10 and 9, respectively). These tissue-specific Wnt signal enhancement molecules also contained a signaling peptide at the N-terminus for secretion and a FLAG tag at the C-terminus for detection, followed by a (His)8 tag for affinity purification. Expression constructs encoding the tissue-specific Wnt signal enhancement fusion proteins and controls were generated by standard molecular cloning techniques.

[0198] Two cell lines, including human hepatoma Huh-7 and human epidermoid carcinoma A431 cells, expressing the luciferase gene controlled by a Wnt-responsive promoter (Super Top Flash reporter assay, STF), were used to measure Wnt signaling activity. As demonstrated by quantitative PCR analysis shown in Figure 5B, both cell lines expressed E3 ligase targeted by R-spondin, but liver-specific gene ASGR1 / 2 expression was only detected in Huh-7.

[0199] Transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the supplier's recommended procedures to test the activity of constructs targeting ASGR in Huh-7 reporter cells. Wnt3a-conditioned medium (prepared using the supplier's recommended procedures in the ATCC L-Wnt-3A cell line) was added to constitute 10% of the total medium volume 3 hours after transfection. Forty hours after transfection, cells were assayed for luciferase activity using a standard luciferase assay-readout protocol (e.g., Stop and Glo Dual Luciferase Assay Kit (Promega)), which includes cell lysis followed by the addition of the luciferase substrate, luciferin. Luciferase-mediated conversion of the luciferin substrate to oxyluciferin resulted in luminescence, which was read by a plate reader, and luminescence was quantified. The results are shown in Figure 5C. Compared to mock-transfected controls ("no DNA"), constructs expressing anti-ASGR1 antibodies fused to mutant Rspo2 showed an approximately 20-fold increase in luciferase activity, much higher than anti-GFP fused to the same mutant, suggesting that the ASGR1 antibody portion of the construct (the targeting domain) was responsible for the increased activity.

[0200] Western blot analysis was performed to confirm the expression and stability of the various fusion proteins under the experimental conditions. 10 μl of culture supernatant 40 hours after transfection (the same time point at which luciferase activity was measured) was analyzed with anti-FLAG monoclonal antibody, M2 (Sigma_Aldrich). As shown in Figure 5C, all proteins were detectable at similar levels. Therefore, the differences in Wnt signal enhancement activity between the tested constructs are likely to reflect protein activity, and not expression levels or protein stability.

[0201] A431 cells were used to test the ASGR1 dependency of anti-ASGR1-based constructs. Anti-ASGR1 mutant Rspo2 constructs were co-transfected with human TFR2 or human ASGR1 in A431 cells in parallel with anti-GFP fusions with wild-type or mutant Rspo2. Transfections were supplemented with 10% Wnt3a-conditioned medium, and luciferase activity was assayed 40 h after transfection. Supernatants from the same cells were harvested for Western blot. As shown in Figure 5D, with ASGR1 co-transfection, the anti-ASGR1 constructs showed several-fold higher activity than the negative control. This effect was not observed with TFR2 co-transfection. This suggested that the specific Wnt-enhancing activity observed here was dependent on the presence of the specific receptor that is targeted by the design of the fusion molecules. These results demonstrate that ASGR and TFR2-targeting constructs can be used for the specific targeting of Wnt signal-enhancing molecules to tissues such as the liver where they are expressed.

[0202] Example 2 Mechanisms of behavioral domains and modulation of the dynamic range of behavior To verify the mechanism of action of the fusion construct design of tissue-specific Wnt signal enhancer molecules, additional mutations were introduced into Rspo2 (which functions as an action domain), and the activity of these constructs was analyzed in Huh-7 cells in the same transient transfection-based reporter assay described in Example 1. The role of ZNRF3 / RNF43 interaction was first tested by introducing point mutations (in addition to the F105A and F109A mutations) into residues in Rspo2 known to bind ZNRF3 / RNF43, such as S47, N50, R65, R69, and Q70. The amino acid sequence of α-GFP-mtRspo2 containing the F105A / F109A / S47A / N50A mutations is provided in SEQ ID NO:12, and the coding polynucleotide sequence is provided in SEQ ID NO:11. The amino acid sequence of α-ASGR1-mtRspo2 containing F105A / F109A / S47A / N50A mutations is provided in SEQ ID NO: 14, and the encoding polynucleotide sequence is provided in SEQ ID NO: 13. The amino acid sequence of α-GFP-mtRspo2 containing F105A / F109A / R65A / R69A / Q70A mutations is provided in SEQ ID NO: 16, and the encoding polynucleotide sequence is provided in SEQ ID NO: 15. The amino acid sequence of α-ASGR1-mtRspo2 containing F105A / F109A / R65A / R69A / Q70A mutations is provided in SEQ ID NO: 18, and the encoding polynucleotide sequence is provided in SEQ ID NO: 17. As shown in FIG. 6A, introduction of S47A / N50A or R65A / R69A / Q70A mutations abolished the Wnt signal enhancing activity of both anti-GFP and anti-ASGR1 constructs. These results are consistent with a mechanism in which these two E3 ligases are targeted by their action domains.

[0203] To test the possibility of increasing the activity of mutant Rspo2, an attempt was made to alleviate the defect in LGR interaction by limiting the mutations to single amino acid residues F105A or F109A instead of the initial F105A / F109A double mutation. The amino acid sequence of α-GFP-mtRspo2 containing only the F105A mutation is provided in SEQ ID NO:20, and the coding polynucleotide sequence is provided in SEQ ID NO:19. The amino acid sequence of α-ASGR1-mtRspo2 containing only the F105A mutation is provided in SEQ ID NO:22, and the coding polynucleotide sequence is provided in SEQ ID NO:21. The amino acid sequence of α-GFP-mtRspo2 containing only the F109A mutation is provided in SEQ ID NO:24, and the coding polynucleotide sequence is provided in SEQ ID NO:23. The amino acid sequence of α-ASGR1-mtRspo2 containing only the F109A mutation is provided in SEQ ID NO:26, and the coding polynucleotide sequence is provided in SEQ ID NO:25. These constructs were tested in Huh-7 reporter cells by transient transfection assays. As shown in Figure 6B, the F109A single mutant had higher activity than the double mutant, although this was at the expense of a higher basal activity in the anti-GFP control, this approach suggested that the dynamic range of activity of the fusion constructs could be fine-tuned by mutations affecting the R-spondin-LGR interaction.

[0204] In addition to alanine ("A"), other amino acid residues can also be used as mutations to replace residues important for LGR protein interaction. As an example, FIG. 6C demonstrated the use of Rspo2 F106R / F109A mutant as an action domain. Fusion of this Rspo2 mutant to an anti-TFR1 targeting domain resulted in clear Wnt signal enhancing activity in Huh-7 cells in a transient transfection-based STF assay (in the presence of 10% Wnt3a conditioned medium) compared to the corresponding anti-GFP control. The amino acid sequence of α-TFR1-mtRspo2 containing the F105A / F109A mutation is provided in SEQ ID NO:28, and the encoding polynucleotide sequence is provided in SEQ ID NO:27. The amino acid sequence of α-GFP-mtRspo2 containing the F105R / F109A mutation is provided in SEQ ID NO:30, and the encoding polynucleotide sequence is provided in SEQ ID NO:29. The amino acid sequence of α-TFR1-mtRspo2 containing the F105R / F109A mutations is provided in SEQ ID NO:32, and the encoding polynucleotide sequence is provided in SEQ ID NO:31.

[0205] Example 3 Validation of targeting strategies using other cell surface receptors To verify that the anti-ASGR1-based design of tissue-specific Wnt signal enhancer molecules is not just a specific case, but rather an example representing a general principle, a mutant (F105A / F109A) Rspo2 fragment (amino acid residues 37-143) was fused to an scFv antibody against another cell surface receptor, human transferrin receptor 1 (TFR1) (designed based on patent WO2016 / 081640, clone 7A4). The amino acid sequence of the construct is provided in SEQ ID NO: 28. Using the transient transfection and reporter assays described in Example 1, this new construct was demonstrated to have activity equal to or exceeding that of a positive control anti-GFP (SEQ ID NO: 6, Figures 7A and 7B) fused to wild-type Rspo2. This not only supported the generalization of the targeting strategy, but also proved the ample potential of this approach to significantly enhance specific Wnt signal enhancer activity, as long as a targeting domain recognizing an appropriate cell surface receptor is used. The wide distribution of the TFR1 receptor also provided an opportunity to use this fusion construct to screen for tissues / cells sensitive to this Wnt signal enhancement strategy.

[0206] Example 4 Purified fusion proteins (SCFV format) as tissue / cell-specific WNT enhancers To directly test the activity of the designed fusion proteins, constructs were subcloned into the baculovirus transfer vector pAcGP67-A and expressed and purified from SF9 / hi5 cells as previously described (Janda et al., 2017 Nature). The expressed proteins were purified via the His-tag using complete his-tag purification resin (Sigma-Aldrich) according to the vendor's recommended procedures and then further purified by size-exclusion column (S200, GE Healthcare) fractionation. Figure 8A shows a representative example of a Coomassie-stained gel image of purified fusion proteins (anti-TFR1 constructs were not further purified by size-exclusion column) with the indicated target and action domains, with an estimated purity of >90%.

[0207] FIG. 8B (left graph) shows a comparison of anti-ASGR1 constructs (F105A / F109A double mutant with Rspo2 (SEQ ID NO: 10) and F109A single mutant with Rspo2 (SEQ ID NO: 26)) with anti-GFP negative control (F105A / F109A double mutant (SEQ ID NO: 8)) on targeted Huh-7 cells. Anti-ASGR1 fused to Rspo2 double mutant at a final concentration of 10 μM was about 3-fold more active than the anti-GFP control at a concentration of 100 μM, clearly demonstrating a specific enhancement of activity by the specific antibody. In agreement with the results of the transient transfection assay, the fusion protein containing Rspo2 single F109A mutant (SEQ ID NO: 26) was even more active. FIG. 8B (right graph) shows the maximum efficacy (E) of ASGR1-targeting fusions with a his-tagged Rspo2 construct (comprising amino acid residues S36 to E143) purified by the same procedure (SEQ ID NO: 34, and the encoding polynucleotide sequence provided in SEQ ID NO: 33). max The results show that the targeted Rspo2 double mutant produces a functional Rspo2 protein comparable to E max This suggests that while the activity of the targeted Rspo2 single mutant Emax can reach 100 μM, the activity of the targeted Rspo2 single mutant Emax can be even higher. In contrast to 1-3 μM for the Rspo2 protein, higher concentrations (10-100 μM) can reach 100 μM. max at these concentrations, the negative control (anti-GFP fusion in FIG. 8B, left panel) had little activity.

[0208] To verify the specificity of tissue / cell targeting, the activity of purified fusion proteins was compared in three cell lines, namely human liver Huh-7 cells, human colorectal adenocarcinoma HT29 cells, and mouse liver FL83B cells, which are luciferase reporters for Wnt signaling (shown from left to right in Figure 8C). Liver and intestinal / rectal epithelial cells are known to be sensitive to R-spondin-mediated upregulation of Wnt signaling, and Rspo proteins are highly conserved between mice and humans. Not surprisingly, all three cell lines responded well to Rspo2. Significant activity from anti-ASGR1-based fusion proteins was observed on Huh-7 cells, but little activity on HT29 cells. Intestinal epithelial cells were the most sensitive to Rspo proteins. The lack of stimulation of HT29 cells by fusion proteins targeting ASGR1 is a good indication that they may be much less likely to have off-target effects when applied in vivo. In contrast, the fusion protein targeting TFR1 was much more active even at a very low concentration of 0.1 μM, further demonstrating the potency and efficacy of this protein. This activity is likely specifically dependent on the presence of the receptor, as this human TFR1-targeting construct showed no activity against mouse FL83B cells, indicating that the targeted binder is specific for the human receptor. Some response to the fusion protein targeting human ASGR1 was observed in mouse hepatocytes, suggesting cross-species reactivity of the antibody.

[0209] Example 5 Active WNT signal enhancer in full-length IGG format demonstrated by transient transfection Antibodies in full IgG format are associated with superior pharmacokinetic properties and stability, among other advantages. To demonstrate that the tissue-specific Wnt signal enhancing molecules described in the previous examples are active in full IgG format, these tissue-specific Wnt signal enhancing fusion proteins were converted to full IgG format. The TFR1 binder was selected as a proof of concept for Wnt signal enhancing molecules in IgG format due to its highest activity from the previous examples of this application. As shown in the diagram in Figure 9A, mutant (F105R / F109A) Rspo2 was fused to the N-terminus of the light or heavy chain of the TFR1 antibody (or GFP antibody as a negative control, both as IgG2). The activity of these molecules was determined by transient transfection into 293 cells with a reporter that responds to Wnt signaling. For the construct having the Rspo2(F105R / F109A) domain added to the N-terminus of the heavy chain of α-GFP IgG, the amino acid sequence of the α-GFP light chain is provided in SEQ ID NO: 36, the coding polynucleotide sequence of which is provided in SEQ ID NO: 35, the amino acid sequence of Rspo2(F105R / F109A), α-GFP heavy chain IgG2 is provided in SEQ ID NO: 38, the coding polynucleotide sequence of which is provided in SEQ ID NO: 37. For the construct having the Rspo2(F105R / F109A) domain added to the N-terminus of the heavy chain of α-TFR1 IgG, the amino acid sequence of the α-TFR1 light chain is provided in SEQ ID NO: 40, the coding polynucleotide sequence of which is provided in SEQ ID NO: 39, the amino acid sequence of Rspo2(F105R / F109A), α-TFR1 heavy chain IgG2 is provided in SEQ ID NO: 42, the coding polynucleotide sequence of which is provided in SEQ ID NO: 41. For the construct with the Rspo2(F105R / F109A) domain added to the N-terminus of the light chain of α-GFP IgG, the amino acid sequence of the α-GFP double-chain IgG2 is provided in SEQ ID NO: 46 and its encoding polynucleotide sequence is provided in SEQ ID NO: 45, and the amino acid sequence of the Rspo2(F105R / F109A), α-GFP light chain is provided in SEQ ID NO: 44 and its encoding polynucleotide sequence is provided in SEQ ID NO: 43.For the construct having the Rspo2(F105R / F109A) domain added to the N-terminus of the light chain of α-TFR1 IgG, the amino acid sequence of the α-TFR1 two-chain IgG2 is provided in SEQ ID NO:50, its encoding polynucleotide sequence is provided in SEQ ID NO:49, and the amino acid sequence of the Rspo2(F105R / F109A), α-TFR1 light chain is provided in SEQ ID NO:48, its encoding polynucleotide sequence is provided in SEQ ID NO:47.

[0210] As shown in Figure 9B, the full IgG format retained the strong Wnt signal enhancing activity observed in the scFv format, especially when the Rspo2 mutant was fused to the heavy chain. These results provided one viable example of how to apply the design of receptor-targeted Wnt enhancers to other scaffolds.

[0211] Example 6 Additional RSPO mutants to support targeted WNT signal-enhancing activity In addition to the two hydrophobic residues known to be most important for the interaction between Rspo and LGR proteins (F105 and F109 for human Rspo2), additional residues may also contribute to the interaction. These residues include, for example, K58, H76, S77, R86, N91, and R121 of Rspo2. Mutations at these residues can be used in combination with Fs105 / F109 mutations to abolish / compromise the interaction of Rspo with LGR proteins. The amino acid sequences of the various constructs and the polynucleotide sequences encoding them are provided as follows: anti-ASGR1, Rspo2(F105R / F109A), SEQ ID NOs:52 and 51; anti-GFP, Rspo2(R86E / F105R / F109A), SEQ ID NOs:54 and 53; anti-ASGR1, Rspo2(R86E / F105R / F109A), SEQ ID NOs:56 and 55; anti-TFR1, Rspo2(R86E / F105R / F109A), SEQ ID NOs:58 and 57; anti-GFP, Rspo2(R86E / F105R / F109A / R121E), SEQ ID NOs:60 and 59. ; anti-ASGR1, Rspo2(R86E / F105R / F109A / R121E), sequence numbers 62 and 61; anti-TFR1, Rspo2(R86E / F105R / F109A / R121E), sequence numbers 64 and 63; anti-GFP, Rspo2(K58E / R86E / F105R / F109A / R121E), sequence numbers 66 and 65; anti-ASGR1, Rspo2(K58E / R86E / F105R / F109A / R121E), sequence numbers 68 and 67; anti-TFR1, Rspo2(K58E / R86E / F105R / F109A / R121E), sequence numbers 70 and 69.

[0212] As shown in Figure 10, mutations in these LGR-interacting residues can be used in various combinations to generate action domains that support Wnt signal-enhancing activity in a transient transfection-based STF assay in Huh-7 cells in the presence of 10% Wnt3a-conditioned medium.

[0213] Example 7 WNT signal-enhancing activity of additional recombinant proteins in SCFV format The Wnt signal enhancing activity of scFv anti-TFR1 based constructs observed by transient transfection experiments (Figure 6C) was further verified using purified proteins. Plasmid vectors encoding fusion proteins of anti-hTFR1-Rspo2 (F105R / F109A) and anti-GFP control (SEQ ID NOs: 30 and 32, respectively) were transfected into Expi293F cells (Thermo Fisher Scientific) and purified using cOmplete his-tag purification resin (Sigma-Aldrich) according to the vendor's recommended procedure. Figure 11A shows a Coomassie stained gel image of the purified proteins. As shown in Figure 11B, the TFR1 target recombinant protein showed much stronger activity than the anti-GFP fusion control. Furthermore, the E of this target molecule max is equivalent to that of the Rspo2 positive control.

[0214] Example 8 Cell surface receptor dependence demonstrated with purified proteins To further demonstrate the dependency of the designed Wnt signal enhancer molecules on the presence of specific cell surface receptors, human ASGR1 targeting molecules (anti-ASGR1-Rspo2(F105A / F109A); ​​SEQ ID NO: 10) were tested in STF assays on A431 cells that do not naturally express ASGR1 receptors. Cells were first transiently transfected in 96-well plates with vectors expressing either human ASGR1 or human TFR2 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommended procedures. After 24 hours, the transfected cells were treated with hASGR1 targeting fusion proteins, together with Rspo2 as a positive control. Transient transfection with ASGR1 expression vectors was found to render A431 cells responsive, whereas control transfection with TFR2 expression vectors did not respond to the Wnt signal enhancer molecules ( FIG. 12 ).

[0215] Example 9 WNT signal enhancing activity of purified protein in attached IGG format To further validate the Wnt signal enhancing activity of the TFR1-targeted IgG molecules demonstrated by the transient transfection experiments (Figure 9), these molecules were expressed as recombinant proteins using Expi293F cells (Thermo Fisher Scientific). The recombinant proteins were first purified by Protein A affinity resin (standard procedure) and then polished by size exclusion column (S200, GE Healthcare) fractionation. The purified proteins were directly tested on Huh-7 and 293T cells using the STF assay, both of which express the human TFR1 receptor. As shown in Figure 13, the TFR1-targeted molecules showed 3-4 orders of magnitude better potency and higher E than the anti-GFP fusion control. max showed.

[0216] Example 10 Structure-activity relationship analysis of additional IGG scaffolds We compared multiple methods of attaching the action domain using targeting domains in full-length IgG format. A summary of the activity of the various IgG2 scaffolds tested is shown in Figures 14A-14B, where mutant Rspo2(F105R / F109A) is attached to the N-terminus of the heavy chain, the N-terminus of the light chain, and the C-terminus of the light chain of either anti-ASGR1 or anti-TFR1 as the targeting domain, or anti-GFP as a control antibody. Some constructs used are described in Example 5, and additional constructs tested included the following: a Rspo2(F105R / F109A) domain added to the N-terminus of the heavy chain of α-ASGR1 IgG; the light chain is provided in SEQ ID NO:72 and its encoding polynucleotide sequence is provided in SEQ ID NO:71, the amino acid sequence of Rspo2(F105R / F109A)α-ASGR1 heavy chain IgG is provided in SEQ ID NO:74 and its encoding polynucleotide sequence is provided in SEQ ID NO:73; a Rspo2(F105R / F109A) domain added to the C-terminus of the light chain of α-GFP IgG2 (wherein the amino acid sequence of α-GFP heavy chain IgG2 is provided in SEQ ID NO:46) and its encoding polynucleotide sequence is provided in SEQ ID NO:45, Rspo2(F105R / F109A), the amino acid sequence of the α-GFP light chain is provided in SEQ ID NO:76 and its encoding polynucleotide sequence is provided in SEQ ID NO:76; The Rspo2(F105R / F109A) domain added to the C-terminus of the light chain of IgG2 (wherein the amino acid sequence of the α-ASGR1 dual-chain IgG2 is provided in SEQ ID NO:80) and its encoding polynucleotide sequence is provided in SEQ ID NO:79, the amino acid sequence of Rspo2(F105R / F109A), α-ASGR1 light chain is provided in SEQ ID NO:78 and its encoding polynucleotide sequence is provided in SEQ ID NO:78; the Rspo2(F105R / F109A) domain added to the C-terminus of the light chain of α-TFR1 IgG2 (wherein the amino acid sequence of the α-TFR1 dual-chain IgG2 is provided in SEQ ID NO:50) and its encoding polynucleotide sequence is provided in SEQ ID NO:49; the amino acid sequence of Rspo2(F105R / F109A), α-TFR1 light chain is provided in SEQ ID NO:82 and its encoding polynucleotide sequence is provided in SEQ ID NO:81.

[0217] These proteins were overexpressed in Expi193T cells by transient transfection and purified by Protein A affinity resin followed by size exclusion chromatography. In Huh-7 cells (Figure 14A), all target molecules showed clearly better activity than the corresponding anti-GFP fusion controls. The anti-TFR1 fusion constructs were generally more active than the anti-ASGR1 fusion proteins, which is consistent with the observation that these target domains are in scFv format (Figure 8C). The dynamic range (potency and E between target and control molecules in the same format) was significantly greater than that of the control molecules. max The difference in the STF activity (difference in STF activity) is overall greater for N-terminal heavy and N-terminal light chain attachment molecules than for C-terminal light chain attachment molecules. This suggests that while multiple formats are amenable to further development, some formats may be more preferable. The STF activity of these molecules was also compared in human 293T cells expressing only TFR1 and not the ASGR1 receptor (Figure 14B). As expected, only the anti-TFR1 fusion protein remained significantly more potent than the anti-GAS fusion control, suggesting that the specific Wnt signal enhancement activity of the designed molecules remains dependent on the presence of specific cell surface receptors in the IgG format. This concept was further validated using mouse FL83B cells that express neither human ASGR1 nor human TFR1 (Figure 14C). No differences were observed between the target molecule and the anti-GFP control fusion protein in FL83B cells. The attachment location of the Rspo mutants may also be at the C-terminus of the IgG heavy chain (data not shown).

[0218] IgG1 is another immunoglobulin isotype frequently used for therapeutic antibody development. Multiple methods of attaching mutant Rspo2 action domains to different termini of the N297G effector-less mutant form of IgG1 (Jacobsen FW et al., The Journal of Biological Chemistry 2017, 292:1865) were compared. Some constructs used are described in Example 5, and additional constructs tested included: Rspo2(F105R / F109A) domain added to the N-terminus of the heavy chain of anti-GFP IgG1 (the amino acid sequence of the anti-GFP light chain is provided in SEQ ID NO:36 and its encoding polynucleotide sequence is provided in SEQ ID NO:35; Rspo2(F105R / F109A), the amino acid sequence of the anti-GFP heavy chain is provided in SEQ ID NO:84 and its encoding polynucleotide sequence is provided in SEQ ID NO:83); anti-ASGR1 Rspo2(F105R / F109A) domain added to the N-terminus of the heavy chain of IgG1(N297G) (the amino acid sequence of the anti-ASGR1 light chain is provided in SEQ ID NO:72 and its encoding polynucleotide sequence is set forth in SEQ ID NO:71, the amino acid sequence of the Rspo2(F105R / F109A), anti-ASGR1 heavy chain is provided in SEQ ID NO:86 and its encoding polynucleotide sequence is provided in SEQ ID NO:85); Rspo2(F105R / F109A) domain added to the N-terminus of the heavy chain of anti-TFR1 IgG1(N297G) (the amino acid sequence of the anti-TFR1 light chain is provided in SEQ ID NO:40 and its encoding polynucleotide sequence is provided in SEQ ID NO:39, the amino acid sequence of the Rspo2(F105R / F109A), anti-TFR1 heavy chain is provided in SEQ ID NO:88 and its encoding polynucleotide sequence is provided in SEQ ID NO:87); anti-GFP Rspo2(F105R / F109A) domain added to the N-terminus of IgG1 light chain (N297G) (the amino acid sequence of the anti-GFP heavy chain is provided in SEQ ID NO:90 and its encoding polynucleotide sequence is provided in SEQ ID NO:89, and Rspo2(F105R / F109A), the amino acid sequence of the anti-GFP light chain is provided in SEQ ID NO:44 and its encoding polynucleotide sequence is provided in SEQ ID NO:43);Rspo2(F105R / F109A) domain added to the N-terminus of the light chain of anti-TFR1 IgG1(N297G) (the amino acid sequence of the anti-TFR1 heavy chain is provided in SEQ ID NO:92, the coding polynucleotide sequence of which is provided in SEQ ID NO:91, the amino acid sequence of the Rspo2(F105R / F109A), anti-TFR1 light chain is provided in SEQ ID NO:48, the coding polynucleotide sequence of which is provided in SEQ ID NO:47); Rspo2(F105R / F109A) domain added to the C-terminus of the light chain of anti-ASGR1 IgG1 (the amino acid sequence of the anti-ASGR1 heavy chain is provided in SEQ ID NO:94, the coding polynucleotide sequence of which is provided in SEQ ID NO:93, the amino acid sequence of the Rspo2(F105R / F109A), anti-ASGR1 light chain is provided in SEQ ID NO:78, the coding polynucleotide sequence of which is provided in SEQ ID NO:77);

[0219] Following the same protein purification and activity testing procedures as described for the attached IgG2 protein, it was found to have the action domain (mutant Rspo2) attached to the N-terminus of the anti-ASGR1 or anti-TFR1 heavy or light chain. The α1β in IgG1(N297G) isotype also supported specific Wnt signal enhancement activity in Huh-7 cells in contrast to the corresponding anti-GFP control (Figure 14D). Such specific activity was lost in 293T cells with anti-ASGR1 fusions but was preserved with anti-TFR1 fusions, consistent with the expected dependency on the presence of the target receptor (Figure 14E). Thus, multiple immunoglobulin isotypes with multiple sites for attachment of action domains were demonstrated to be a suitable format for tissue-specific Wnt signal enhancement molecules.

[0220] Example 11 Structure-activity relationship analysis of four R-spondins as action domains Humans have four R-spondin proteins with high sequence homology, especially within the furin domain (Figure 4). To directly compare their abilities in enhancing Wnt signaling in vitro, the Fu1-Fu2 domains of each of the four human R-spondins were expressed as fusions to the C-terminus of scFv binders to ASGR1, TFR1 and GFP in Expi293F cells by transient transfection. Standard His-tag and size-exclusion chromatography purification procedures. The amino acid sequence and coding polynucleotide sequence of the anti-GFP, Rspo1 wild-type fusion protein are provided in SEQ ID NOs: 96 and 95, respectively. The amino acid sequence and coding polynucleotide sequence of the anti-GFP, Rspo2 wild-type fusion protein are provided in SEQ ID NOs: 6 and 5, respectively. The amino acid sequence and coding polynucleotide sequence of the anti-GFP, Rspo3 wild-type fusion protein are provided in SEQ ID NOs: 98 and 97, respectively. The amino acid and encoding polynucleotide sequences of anti-GFP, Rspo4 wild-type fusion proteins are provided in SEQ ID NOs: 100 and 99, respectively. The purified proteins were then tested by STF assay in Huh-7 and 293T cells in the presence of Wnt3a-conditioned medium. As shown in FIG. 15A, Rspo2 and 3 were more potent than Rspo4 and 1, consistent with previous reports.

[0221] Given the structural and functional conservation among R-spondin molecules, not only Rspo2 but also other Rspo molecules may be suitable to support the construction of action domains of tissue-specific Wnt signal enhancing proteins. Shown in Figure 15B-C is a side-by-side comparison of a series of human Rspo2- and Rspo3-based constructs tested in STF assays in Huh-7 and HEK293T cells. These constructs include the polypeptides disclosed in SEQ ID NOs: 8, 10, 28, 30, 32, and 52. In addition, the constructs tested also included the following (shown in SEQ ID NOs. corresponding to their amino acid sequences and encoding the polynucleotide sequences): anti-GFP, Rspo3(F106A / F110A), SEQ ID NOs. 102 and 101; anti-GFP, Rspo3(F106R / F110A), SEQ ID NOs. 104 and 103; anti-ASGR1, Rspo3(F106A / F110A), SEQ ID NOs. 106 and 105; anti-ASGR1, Rspo3(F106R / F110A), SEQ ID NOs. 108 and 107; anti-TFR1, Rspo3(F106A / F110A), SEQ ID NOs. 110 and 109; anti-TFR1, Rspo3(F106R / F110A), SEQ ID NOs. 112 and 111.

[0222] In the context of specific fusion to the particular tissue-targeting binders used here, the Rspo3 mutant showed the highest efficacy, while other Rspo variants also showed significant tissue targeting.

[0223] Example 12 Diverse RSPO variants support specific WNT signal-enhancing activities in the supplemented IGG format To further demonstrate the diversity of mutations and their combinations that can support the construction of Wnt signal-enhancing molecules, we generated a series of additional Rspo3 mutant-based molecules fused to anti-GFP or anti-ASGR1 and tested their activity in Huh-7 cells using the STF assay. These mutations included F106R / F110A(RA), F106R / F110R(RR), F106E / F110E(EE), F106R / F110A(RE), F106E / F110A(EA), R60E / R88E / N93A / F106R / F110A(EEARA). R60, R88, and N93 are residues that are involved in LGR interaction based on homology with other Rspo proteins (Figure 16A). The amino acid and coding polynucleotide sequences of these constructs are as follows: anti-GFP Rspo3 wild type, SEQ ID NOs: 98 and 97; anti-GFP Rspo3 RA, SEQ ID NOs: 104 and 103; anti-GFP Rspo3 RR, SEQ ID NOs: 114 and 113; anti-GFP Rspo3 EE, SEQ ID NOs: 116 and 115; anti-GFP Rspo3 RE, SEQ ID NOs: 118 and 117; anti-GFP Rspo3 EA, SEQ ID NOs: 120 and 119; anti-GFP Rspo3 EEARA, SEQ ID NOs: 122 and 121; anti-ASGR1 Rspo3 RA, SEQ ID NOs: 108 and 107; anti-ASGR1 Rspo3 RR, SEQ ID NOs: 124 and 123; anti-ASGR1 Rspo3 EE, SEQ ID NOs: 126 and 125; anti-ASGR1 Rspo3 RE, SEQ ID NOs: 128 and 127; anti-ASGR1 Rspo3 EA, SEQ ID NOs: 130 and 129; anti-ASGR1 Rspo3 EEARA, sequence numbers 132 and 131.

[0224] As shown in Figure 16B, all of these combinations significantly reduced the activity of the protein compared to wild-type Rspo3 fused to the anti-GFP control, consistent with their disruption of the Rspo-LGR interaction. When fused to the anti-ASGR1 targeting domain, all mutants showed enhanced activity, although the dynamic range (difference between the target activity and that of the anti-GFP control fusion protein) varied depending on the specific mutation selected. They all support the construction for tissue-specific Wnt signal enhancement molecular design (Figure 16C).

[0225] Example 13 Non-RSPO-based domains of action To demonstrate the feasibility of using non-Rspo-based structures as action domains, we designed "Fab-IgG" fusion proteins in which the Fab fragment of anti-human ZNRF3 mAb (Ab2 from patent WO2013054307A2) was fused to anti-ASGR1, anti-TFR1, or anti-GFP IgG (Figure 17A). The anti-ZNRF3-anti-GFP constructs contained the following polypeptides with the indicated polypeptide and coding polynucleotide sequences: anti-GFP light chain (S176K) (SEQ ID NOs: 134 and 133), anti-ZNRF3 light chain (S176E) (SEQ ID NOs: 136 and 135), and anti-ZNRF3-anti-GFP fusion heavy chain (SEQ ID NOs: 138 and 137). The anti-ZNRF3-anti-ASGR1 constructs contained the following polypeptides with the indicated polypeptide sequences and coding polynucleotide sequences: anti-ASGR1 light chain (S176K) (SEQ ID NOs: 140 and 139), anti-ZNRF3 light chain (S176E) (SEQ ID NOs: 136 and 135), and anti-ZNRF3-anti-ASGR1 fusion heavy chain (SEQ ID NOs: 142 and 141). The anti-ZNRF3-anti-TFR1 constructs contained the following polypeptides with the indicated polypeptide sequences and coding polynucleotide sequences: anti-TFR1 light chain (S176K) (SEQ ID NOs: 144 and 143), anti-ZNRF3 light chain (S176E) (SEQ ID NOs: 1136 and 135), and anti-ZNRF3-anti-TFR1 fusion heavy chain (SEQ ID NOs: 146 and 145).

[0226] These proteins were transiently transfected into Expi293F cells, purified by Protein A affinity resin followed by size exclusion chromatography, and then tested in Huh-7 cells by STF assay in the presence of 30% Wnt3a conditioned medium. As shown in Figure 17B, both anti-ASGR1 and anti-TFR1 "targeted" anti-ZNRF3 modules showed activity exceeding that of the anti-GFP fusion protein, validating the possibility of constructing tissue-specific Wnt signal enhancer molecules using pure binders for ZNRF3 / RNF43 E3 ligase independent of Rspo structure.

[0227] Example 14 In addition to liver-targeted WNT enhancers, tissue-targeted WNT enhancers The examples provided above utilized ASGR1 binders to generate Wnt signaling enhancing molecules with the ability to target the liver for various applications. TFR1 binders may also target the liver, as well as the broader range of tissues in which it is expressed. To provide tissue targets beyond the liver, additional tissue-specific cell surface molecules were identified by searching the public gene expression database (https: / / www.proteinatlas.org / ). LYPD3, Ly6 / PLAUR domain-containing protein 3, and DSG3, desmoglein 3, were selected as target molecules because they are highly abundantly and specifically expressed in mucosal epithelial cells from oral mucosa, skin and tonsils. Furthermore, their specific antibody sequences were previously published (US20170158775A1, mAb clone M31-B01 was selected as a LYPD3 binder; US20100092457A1, mAb clone DF364c was selected as a DSG3 binder).

[0228] To obtain a mucosal epithelial cell-specific WNT enhancer, the Rspo2(F105R / F109A) mutant was fused to the N-terminus of either anti-LYPD3 or anti-DSG3 heavy chain in the form of an "effectorless" IgG1 (Lo M et al., 2017 The Journal of Biological Chemistry, 292). The anti-GFP Rspo2(F105R / F109A) construct contained the following polypeptide encoding polynucleotide sequences with the indicated polypeptide sequences: anti-GFP light chain (SEQ ID NOs: 36 and 35) and Rspo2(F105R / F109A), anti-GFP heavy chain IgG2 (SEQ ID NOs: 38 and 37). The anti-LYPD3 Rspo2(F105R / F109A) construct contained the following polypeptides having the indicated polypeptide sequences and encoding polynucleotide sequences: anti-LYPD3 light chain (SEQ ID NOs: 148 and 147) and Rspo2(F105R / F109A), anti-LYPD3 heavy chain LALA-PG (SEQ ID NOs: 150 and 149). The anti-DSG3 Rspo2(F105R / F109A) construct contained the following polypeptides having the indicated polypeptide sequences and encoding polynucleotide sequences: anti-DSG3 light chain (SEQ ID NOs: 152 and 151), and Rspo2(F105R / F109A), anti-DSG3 heavy chain LALA-PG (SEQ ID NOs: 154 and 153).

[0229] Fusion proteins were expressed from Expi293F cells (Thermo Fisher Scientific) and purified using Protein A resin followed by a size-exclusion column (S200, GE The proteins were purified using a ELISA kit (Healthcare) fractionation system with a typical estimated purity of >90%. The Wnt signal-enhancing activity of these proteins was tested in two oral mucosa cell lines, CAL27 and SCC25, as well as the control A431 cell line.

[0230] As shown in Figure 18A, LYPD3 and DSG3 genes were highly expressed in CAL27 and SCC25 cells, whereas very low expression was observed in A431 cells. Both Rspo2 mutant fusion proteins targeting LYPD3 and DSG3 showed much stronger activity than the anti-GFP control in CAL27 cells. In SCC25 cells, the DSG3-targeted molecule was much more active than the anti-GFP control, whereas that of the LYPD3-targeted protein was less pronounced. This may be a reflection of the lower LYPD3 receptor levels on SCC25 cells. These results confirm that tissue-specific antibodies can enhance Rspo mutant activity against target cells when used as a targeting domain. In contrast, the activity of LYPD3 and DSG3-targeted molecules was indistinguishable from the anti-GFP control fusion protein in A431 cells lacking expression of LYPD3 and DSG3, clearly indicating that specific binding to cell surface receptors is required for enhanced activity. In addition to enhanced tissue-specific activity, anti-LYPD3 and anti-DSG3 fusion proteins were more potent than the Rspo2 positive control in target CAL17 cells and showed better EC 50 and E better or equal to the Rspo2 positive control max showed.

[0231] Example 15 In vivo induction of WNT-responsive genes by tissue-specific WNT signal enhancers To investigate the in vivo activity of the designed Wnt signal enhancer, the induction of Axin2, a Wnt-responsive gene, was examined. As shown in Figure 19A, AAV-hASGR1 was first injected intravenously into 8-week-old male mice at a titer of 1E11 per animal. This resulted in human ASGR1 gene expression in mouse liver. Seven days later, purified proteins were injected intravenously into eight groups at specific doses. Either alone or in combination with Wnt signal, Rspo2 positive control at 0.46 mg / kg, anti-GFP-Rspo2(F105R / F109A) at 1 mg / kg, and anti-ASGR1-Rspo2(F105R / F109A) at 3 mg / kg agonist 18R5-Dkk1c (Janda et al., 2017 Nature). After 8 hours, the mice were euthanized and liver samples were taken for quantitative PCR analysis of gene expression. The anti-GFP constructs contained the following polypeptides having the indicated polypeptide and encoding polynucleotide sequences: anti-GFP light chain (SEQ ID NOs: 36 and 35) and anti-GFP heavy chain IgG1 (LALA-PG) (SEQ ID NOs: 156 and 155). The anti-GFP Rspo2 (F105R / F109A), N-HC construct contained the polypeptides provided in SEQ ID NOs: 35-38. The anti-ASGR1 IgG2 N-HC construct contained the polypeptides provided in SEQ ID NOs: 71-74. The 18R5-Dkk1c construct had the polypeptide sequence disclosed in SEQ ID NO: 158 and was encoded by the polynucleotide sequence disclosed in SEQ ID NO: 157.

[0232] Figure 19B shows the expression levels of ectopic hASGR1 in mouse liver, which were comparable across all experimental groups. Treatment of mice with Rspo2 positive control protein alone induced a modest but statistically significant induction of Axin2 gene expression, consistent with the in vivo function of Rspo protein (Figure 19C, left). Interestingly, treatment with anti-ASGR1-Rspo2 (F105 / F109) alone also induced a trend increase in Axin2 levels that was not observed with the control anti-GFP fusion protein. 18R5-Dkk1c is a surrogate for Wnt. At 3 mg / kg, it alone did not cause any significant changes in Axin2 gene expression. However, a clear synergy was observed between Rspo2 and 18R5-Dkk1c (Figure 19C, right), consistent with the Wnt signal-enhancing activity of Rspo protein. Such synergy was also observed with anti-ASGR1 fusion proteins but not with anti-GFP controls, suggesting that induction of Axin2, which represents enhanced Wnt signaling in mouse liver, is specifically dependent on the anti-ASGR1 targeting domain. References: Clevers H, Loh KM and Nusse R.2014, An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science 346(6205):1248012. Knight MN and Hankenson KD.R-spondins:novel matricellular regulators of the skeleton.Matrix Biology 37:157-161. Yan JJ, Liao JZ, Lin JS and He XX.2015,Active radar guides missile to its target:receptor-based targeted treatment of hepatocellular carcinoma by nanoparticulate systems.Tumor Biology 36:55-67. Stockert RJ,Morell AG and Ashwell G.1991,Structural characteristics and regulation of the asialoglycoprotein receptor.Targeted Diagnostic and Therapy 4:41-64. D’Souza AA and Devarajan PV.2015,Asialoglycoprotein receptor mediated hepatocyte targeting-strategies and applications.Journal of Controlled Release,203:126-139. Yan H,Zhong G,Xu G,He W,Jing Z,Gao Z,Huang Y,Qi Y,Peng B,Wang H,Fu L,Song M,Chen P,Gao W,Ren B,Sun Y,Cai T,Feng X,Sui J,Li W.2012,Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.eLife,1:e00049. Worthen CA and Enns CA.2014,The role of hepatic transferrin receptor 2 in the regulation of iron homeostasis in the body.Frontiers in Pharmacology 5:34 Mannstadt M,Juppner H,and Gardella TJ.1999,Receptors for PTH and PTHrP:their biological importance and functional properties.American Journal of Physiology 277:F665-F675. Janda CY,Dang LT,You C,Chang J,de Lau W,Zhong ZA,Yan KS,Marecic O,Siepe D,Li X,Moody JD,Williams BO,Clevers H,Piehler J,Baker D,Kuo CJ,and Garcia KC.2017,Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signaling.Nature 545:234-237. Lo M,Kim HS,Tong RK,Bainbridge TW,Vernes JM,Zhang Y,Lin YL,Chung S,Dennis MS,Zuchero YJ,Watts RJ,Couch JA,Meng YG,Atwal JK,Brezski RJ,Spiess C,Ernst JA.2017,Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice.J BiolChem.292:3900-3908. Jacobsen FW,Stevenson R,Li C,Salimi-Moosavi H,Liu L,Wen J,Luo Q,Daris K,Buck L,Miller S,Ho SY,Wang W,Chen Q,Walker K,Wypych J,Narhi L,Gunasekaran K.2017 Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability.J BiolChem.292:1865-1875. Paret C,Bourouba M,Beer A,Miyazaki K,Schnolzer M,Fiedler S,Zoller M.International Journal of Cancer.2005 Jul 10;115(5):724-33.Ly6 family member C4.4A binds laminins 1 and 5,associates with galectin-3 and supports cell migration. Arumugam T,Deng D,Bover L,Wang H,Logsdon CD,Ramachandran V.Molecular Cancer Therapeutics.2015 Apr;14(4):941-51.New Blocking Antibodies against Novel AGR2-C4.4A Pathway Reduce Growth and Metastasis of Pancreatic Tumors and Increase Survival in Mice. Ngora H1,Galli UM,Miyazaki K,Zoller M.Neoplasia.2012 Feb;14(2):95-107.Membrane-bound and exosomal metastasis-associated C4.4A promotes migration by associating with the α(6)β(4) integrin and MT1-MMP. Wolfgang-Moritz Heupel,Detlef Zillikens,Detlev Drenckhahn and Jens Waschke Pemphigus.Vulgaris IgG Directly Inhibit Desmoglein 3-Mediated Transinteraction.Journal of Immunology.August 1,2008,181(3)1825-1834.

Claims

1. A tissue-specific Wnt signal enhancing molecule, or a pharma- ceutically acceptable salt thereof, comprising: (a) a first domain comprising two R-spondin-2 polypeptides or variants thereof, wherein the R-spondin-2 polypeptide or variants thereof comprises a furin1 domain of the R-spondin-2 polypeptide, and the R-spondin-2 polypeptide comprises amino acid substitutions at F105 and F109 of human R-spondin-2; and (b) a second domain comprising an antibody that binds to asialoglycoprotein receptor 1 (ASGR1), the antibody comprising two heavy chain polypeptides and two light chain polypeptides; wherein the molecule comprises i) two polypeptides each having 100% identity to SEQ ID NO:72 and two polypeptides each having 100% identity to SEQ ID NO:74; ii) two polypeptides each having 100% identity to SEQ ID NO:78 and two polypeptides each having 100% identity to SEQ ID NO:80; iii) two polypeptides each having 100% identity to SEQ ID NO:72 and two polypeptides each having 100% identity to SEQ ID NO:86; or iv) two polypeptides each having 100% identity to SEQ ID NO:78 and two polypeptides each having 100% identity to SEQ ID NO:94 or a pharma- ceutically acceptable salt thereof.

2. The molecule of claim 1, wherein the molecule comprises two polypeptides of SEQ ID NO:72 and two polypeptides of SEQ ID NO:

86.

3. The molecule of claim 1 or 2, wherein the molecule is a fusion protein.

4. The molecule of claim 3, wherein the fusion protein comprises a first domain comprising a first polypeptide sequence and a second domain comprising a second polypeptide sequence, the first domain and the second domain being linked by a linker moiety.

5. The molecule of claim 4 , wherein the linker moiety is a peptide linker sequence.

6. The molecule of claim 5 , wherein the linker sequence comprises one or more amino acids selected from the group consisting of glycine, asparagine, serine, threonine and alanine.

7. (i) an effective amount of a molecule according to any one of claims 1 to 6; (ii) a pharma- ceutically acceptable diluent, adjuvant or carrier; and 23. A pharmaceutical composition comprising:

8. 8. The pharmaceutical composition of claim 7 for use in the treatment of a disease or disorder associated with decreased Wnt signaling.

9. The pharmaceutical composition for use according to claim 8, wherein the disease or disorder is a disease or disorder of the liver tissue.

10. 10. The pharmaceutical composition for use according to claim 9, wherein the disease or disorder is selected from the group consisting of acute liver failure, acute alcoholic liver disease, chronic liver disease due to Hepatitis C virus (HCV), chronic liver disease due to Hepatitis B virus (HBV), chronic alcoholic liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), cirrhosis, and chronic liver failure due to any cause.

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