Use of a frizzled receptor polypeptide for the treatment of metabolic bone diseases

Abstract

The present disclosure generally relates to the use of a frizzled receptor polypeptide for treatment of one or more bone-related disorders. In an embodiment, a method of treating a bone disorder by inhibition of a WNT signaling pathway in a human subject in need thereof is provided, the method comprising administering to the human subject a therapeutically effective amount of an inhibitory frizzled (FZD) receptor polypeptide.

Description

ATTY DKT NO: ASHI-007WOUSE OF A FRIZZLED RECEPTOR POLYPEPTIDE FOR THE TREATMENT OF METABOLIC BONE DISEASES REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (ASHI-007WO_SEQLIST; Size: 72,307 bytes; and Date of Creation: February 26, 2026) is herein incorporated by reference in its entirety.

[0002] CROSS REFERENCE TO RELATED APPLICATIONSThis application, pursuant to 35 U.S.C. § 119 (e), claims priority to the filing dates of United States Provisional Patent Application Serial No. 63 / 765,289 filed February 28, 2025, and United States Provisional Patent Application Serial No. 63 / 863,272 filed August 13, 2025, the disclosures of which applications are herein incorporated by reference.FIELD OF THE DISCLOSURE

[0003] The disclosure relates generally to administration of a frizzled (FZD) receptor polypeptide for the treatment of metabolic bone diseases.BACKGROUND

[0004] The Wingless-related integration site or Wingless and Int-1 or Wingless-Int (“WNT”) signaling pathway plays key roles in the control of development, homeostasis and regeneration of many essential organs and tissues including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste bud, ovary, cochlea and many other tissues (see Clevers, Loh, and Nusse, Science 2014 Oct 3;346(6205):1248012). In particular, the WNT signaling pathway plays a significant role in controlling skeletal formation (see Regard JB, Zhong Z, Williams BO, Yang Y. Cold Spring Harb Perspect Biol. 2012 Dec l;4(12):a007997). The canonical WNT / p-catenin signaling pathway promotes bone formation by stimulating osteoblast development and therefore targeting of WNT signaling pathways is an important area of therapeutic development for metabolic bone disorders that are associated with osteoblast function. However, the impact of WNT / p-catenin signaling pathway on osteoclast- related disorders remains largely unexplored. Osteoclasts are responsible for bone resorption, and understanding how WNT signaling affects these cells could open new avenues for treating disorders such as osteopetrosis and other bone resorption related disorders.ATTY DKT NO: ASHI-007WOSUMMARY OF THE INVENTION

[0005] The disclosure is based, in part, on the use of FZD receptor polypeptides. More specifically, the present disclosure relates to administering a composition comprising a therapeutically effective amount of a FZD receptor polypeptide to a subject suffering from or at risk of a metabolic disease, such as a metabolic bone disease. The present disclosure also relates to administering a composition comprising a therapeutically effective amount of an inhibitory FZD receptor polypeptide to a subject suffering from or at risk of a metabolic disease, such as a metabolic bone disease.

[0006] I’he inventors consider that FZD receptor polypeptides may affect the activities of osteoclasts and / or osteoblasts, for example by increasing osteoclast activity and / or decreasing osteoblast activity. This property is useful, for example, in addressing disorders featuring an imbalance of osteoclast activity and osteoblast activity, such as an imbalance of bone resorption and bone formation.

[0007] In one aspect, the present disclosure provides a method of treating a bone disorder by modulation of WNT signaling pathway in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a frizzled (FZD) receptor polypeptide.

[0008] In another aspect, the present disclosure provides a method of treating a bone disorder by inhibition of a WNT signaling pathway in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of an inhibitory frizzled (FZD) receptor polypeptide.

[0009] In some embodiments, the bone disorder is a bone resorption-related disorder. In some embodiments, the bone resorption-related disorder is caused by an imbalance of osteoblast and osteoclast activity. In some embodiments, the bone resorption-related disorder is characterized by an imbalance of osteoblast and osteoclast activity. In some embodiments, the bone resorption-related disorder is associated with dysfunctional osteoclast activity. In some embodiments, the bone disorder is osteopetrosis. In some embodiments, osteopetrosis is autosomal dominant osteopetrosis type II (ADO2). In some embodiments, osteopetrosis is autosomal recessive osteopetrosis (ARO). In some embodiments, ARO comprises attenuated ARO. In some embodiments, osteopetrosis comprises intermediate autosomal osteopetrosis.

[0010] In another aspect, the present disclosure provides a method of improving homeostasis of bone turnover in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a FZD receptor polypeptide.ATTY DKT NO: ASHI-007WQ

[0011] In some embodiments, the improvement of homeostasis of bone turnover comprises a restoration of balance between bone resorption and bone formation as determined by improvement in bone metrics as assessed by dual energy X-ray absorptiometry (DXA) and / or computed tomography scan (CT).

[0012] In another aspect, the present disclosure provides a method of modulating homeostasis of bone turnover in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitory frizzled (FZD) receptor polypeptide.

[0013] In some embodiments, the modulation of homeostasis of bone turnover comprises a restoration of balance between bone resorption and bone formation as determined by improvement in bone metrics as assessed by dual energy X-ray absorptiometry (DXA) and / or computed tomography scan (CT).

[0014] In yet another aspect, the present disclosure provides a method of modulating osteoclast activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a FZD receptor polypeptide.

[0015] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an inhibitory frizzled (FZD) receptor polypeptide. In some embodiments, the modulation of osteoclast activity comprises an increase in osteoclast function and / or activity, osteoclast activity, osteoclast activation, and / or stimulation of osteoclast differentiation. In some embodiments, the modulation of osteoclast activity comprises an increase in one or more of osteoclast function, osteoclast activity, osteoclast activation, and stimulation of osteoclast differentiation. In some embodiments, the modulation of osteoclast activity is at neutral pH.

[0016] In some embodiments, the subject, at baseline, is characterized by bone quality as measured by bone mineral density (BMD) and / or morphometry. In some embodiments, the subject, at baseline, is characterized by bone quality as measured by bone mineral density (BMD) and / or morphometry (e.g., such as analysis of internal bone architecture and / or bone geometry). In some embodiments, the subject, at baseline, is characterized by an increase in BMD, for example an increase in BMD relative to a normal control. In some embodiments, the subject, at baseline, is characterized by an elevated BMD relative to a healthy control range. In some embodiments, the BMD is assessed by dual energy X-ray absorptiometry (DXA), computed tomography scan (CT), and / or radiography. In some embodiments, the BMD is assessed by dual energy X-ray absorptiometry (DXA), computed tomography scan (CT), QCT (Quantitative Computed Tomography), HR-pQCT (High-Resolution Peripheral QuantitativeATTY DKT NO: ASHI-007WOComputed Tomography) and / or radiography. Other biomarkers of interest may include one or more of: serum PTH (parathyroid hormone), serum calcium, urine calcium, and urine calcium / creatinine ratio.

[0017] In some embodiments, the diagnosis of osteopetrosis is made by BMD, radiography, and / or morphometry of the bones of the subject. In some embodiments, the subject, at baseline, is characterized by the level of one or more biochemical biomarkers. In some embodiments, the subject, at baseline, has altered levels of one or more biochemical biomarkers as compared to a healthy reference subject (e.g., a subject without a bone disorder). In some embodiments, the subject, at baseline, has altered levels of one or more biochemical biomarkers as compared to a healthy reference range (e.g., the range of values found in healthy subjects without a bone disorder). In some embodiments, the subject, at baseline, has altered levels of one or more biochemical biomarkers measured in serum and / or urine. In some embodiments, the one or more biochemical biomarkers comprises one or more bone resorption biomarkers and / or one or more bone formation biomarkers. In some embodiments, the one or more bone resorption biomarkers is selected from the group consisting of C-terminal telopeptide of type 1 collagen (CTX), Tartrate- resistant acid phosphatase (TRAP), N-terminal telopeptide (NTX), the ratio of CTX / TRAP, the ratio of NTX / TRAP, and osteocalcin. In some embodiments, the one or more bone formation biomarkers is selected from the group consisting of: procollagen type 1 N- terminal propeptide (P1NP), bone-specific alkaline phosphatase, and osteocalcin. Other biomarkers of interest may include one or more of: serum PTH (parathyroid hormone), serum calcium, urine calcium, and urine calcium / creatinine ratio.

[0018] In some embodiments, the subject has been diagnosed with osteopetrosis. In some embodiments, the diagnosis of osteopetrosis is made using characteristic radiographic pathognomonic features, in addition to generalized osteosclerosis, that are particularly apparent in the vertebrae of the spine and the long bones of the arms and legs. In some embodiments, these features can include a bone-in-bone appearance described as “endobones”, and / or club- shaped flaring of the metaphysis with abnormal cortical thinning resulting in an Erlenmyer flask-like appearance that is also described as “Erlenmyer flask bone deformity”, and / or variable sclerosis of the superior and inferior end plates of vertebrae from mild, to an anvil appearance, to uniformly dense, sometimes described as “sandwich vertebrae” or “rugger jersey spine”. In some embodiments, the subject has been diagnosed with ADO2 and this diagnosis is characterized by BMD, radiography, and / or morphometry of the subject. In some embodiments, the subject has been diagnosed with ADO2 and has an autosomal dominantATTY DKT NO: ASHI-007WOmutation in one or more genes important for osteoclast function and / or activity. Tn some embodiments, the subject has been diagnosed with AD02 and has an autosomal dominant mutation in chloride voltage-gated channel 7 (CLCN7), TCIRG1, or PLEKHM1. In some embodiments, the diagnosis of AD02 is characterized by BMD and / or morphometry of the subject.

[0019] In some embodiments, the subject has been diagnosed with autosomal recessive osteopetrosis (ARO). In some embodiments, the subject has been diagnosed with ARO and has an autosomal recessive mutation in one or more genes that are important for formation, function, and activity of osteoclasts. In some embodiments, the subject has been diagnosed with ARO and has an autosomal recessive mutation in one or more genes selected from the group consisting of TCIRG1, CLCN7, OSTM1, PLEKHM1, SNX10, and CAII. In some embodiments, the subject has been diagnosed with ARO and has an autosomal recessive mutation in genes that are associated with conditions where osteoclast function, differentiation or formation is impaired (e.g., TNFSF11 or TNFRSF11 A). In some embodiments, the subject has been diagnosed with ARO and has an autosomal recessive mutation in TNFSF11 or TNFRSF11A. In some embodiments, the diagnosis of ARO is characterized by BMD, morphometry, and / or radiography of the subject. In some embodiments, the diagnosis of osteopetrosis is accompanied by and associated with a reduced level of one or more boneresorption biomarkers in the subject as compared to the level of one or more bone-resorption biomarkers in a healthy reference subject. In some embodiments, the diagnosis of osteopetrosis is accompanied by and associated with a reduced level of one or more bone-resorption biomarkers in the subject as compared to the level of one or more bone-resorption biomarkers in a healthy reference range. In some embodiments, the diagnosis of osteopetrosis is accompanied by and associated with a reduced ratio of CTX / TRAP relative to a healthy reference range.

[0020] In some embodiments, the subject, at baseline, experiences one or more of the following: increased likelihood of bone fracture, increased likelihood of brittle bones, osteonecrosis, osteomyelitis, stunted growth, skeletal deformity, cytopenia, pancytopenia, macrocephaly, hepatosplenomegaly, abnormal cortical bone morphology, excessive sclerosis of the vertebral endplates, bone pain, cranial neuropathies, and narrowed medullary cavities, as compared to an untreated subject having a bone disorder.

[0021] In some embodiments, the subject, at baseline, experiences one or more of the following: increased likelihood of bone fracture, increased likelihood of brittle bones,ATTY DKT NO: ASHI-007WOosteonecrosis, osteomyelitis, stunted growth, skeletal deformity, pancytopenia, macrocephaly, hepatosplenomegaly, abnormal cortical bone morphology, excessive sclerosis of the vertebral endplates, bone pain, cranial neuropathies and narrowed medullary cavities, as compared to a subject not having a bone disorder.

[0022] In some embodiments, the administration of the FZD receptor polypeptide results in improvement in bone quality as measured by bone mineral density (BMD) and / or morphometry. In some embodiments, the administration of the FZD receptor polypeptide results in an increased level of one or more bone-resorption biomarkers in the subject as compared to the baseline level of the one or more bone-resorption biomarkers. In some embodiments, the bone resorption markers may be selected from the group consisting of C- terminal telopeptide of type 1 collagen (CTX), Tartrate- resistant acid phosphatase (TRAP), N- terminal telopeptide (NTX), the ratio of CTX / TRAP, the ratio of NTX / TRAP, and osteocalcin.

[0023] In some embodiments, the FZD receptor polypeptide comprises a fragment of an extracellular domain of a human FZD receptor and a human Fc domain. In some embodiments, the fragment of the extracellular domain of the human FZD receptor comprises Fri domain of the human FZD receptor.

[0024] In some embodiments, the Fri domain of the human FZD receptor comprises the Fri domain of human FZD8. In some embodiments, the Fri domain of human FZD8 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the Fri domain of the human FZD receptor comprises the Fri domain of human FZD4. In some embodiments, the Fri domain of human FZD4 comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the Fri domain of the human FZD receptor comprises the Fri domain of human FZD5. In some embodiments, the Fri domain of human FZD5 comprises the amino acid sequence of SEQ ID NO: 15.

[0025] In some embodiments, the human Fc is human IgGl Fc comprising the amino acid sequence of SEQ ID NO: 34.

[0026] In some embodiments, the FZD receptor polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 60-67. In some embodiments, the FZD receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 60.

[0027] In some embodiments, the subject is a human subject. In some embodiments, the subject is an infant (i.e., aged less than 2 years), child (i.e., aged 2 to 18 years), adolescent (i.e. aged 10-19 years), or an adult (aged over 18 years).ATTY DKT NO: ASHI-007WOBRIEF DESCRIPTION OF THE DRAWINGS

[0028] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. Included in the drawings are the following figures.

[0029] FIGS. 1A-1C: Analysis of BMD using DXA at 4 Weeks and 12 Weeks. (A) Whole body aBMD; (B) Spine aBMD; (C) Femur aBMD.

[0030] FIGS. 2A-2D: Analysis of BMD using microCT. (A) Trabecular BV / TV; (B) Cortical BV / TV; (C) Tb.N; (D) Tb.Sp.

[0031] FIG. 3: Analysis of Ip of the Femur.DETAILED DESCRIPTION

[0032] The disclosure is based, in part, on the discovery that a frizzled (FZD) receptor polypeptide can be used for the treatment of one or more bone related diseases associated with the dysfunction of osteoclast.

[0033] In some embodiments, the present application provides therapeutic methods for a FZD receptor polypeptide (e.g., a FZD receptor polypeptide bound to an Fc domain).I. Definitions

[0034] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0035] The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0036] All publications, patents and other references mentioned herein are hereby incorporated by reference in their entirety.

[0037] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.ATTY DKT NO: ASHI-007WO

[0038] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0039] As used herein, all the amino acid positions in an Fc domain or hinge region are numbered according to EU numbering.

[0040] As used herein, the terms “WNT” or “Wnt,” or the plural “WNTs” or “Wnts” refer to a group of secreted, cysteine-rich glycoproteins which bind to a co-receptor complex of FZD receptors and low-density lipoprotein receptor-related proteins 5 or 6 (LRP5 / 6) and regulate expression of a number of target genes through the Wnt signaling pathway.

[0041] As used herein, the term “frizzled receptor” or “FZD receptor” are used interchangeably and depending on context, refers to gene or protein member of the Frizzled family. Frizzled proteins are involved in the activation of Disheveled protein in the cytosol. Frizzled or FZD refers to any of Frizzled 1 “FZD1”, Frizzled 2 “FZD2”, Frizzled 3 “FZD3”, Frizzled 4 “FZD4”, Frizzled 5 “FZD5” Frizzled 6 “FZD6”, Frizzled 7 “FZD7”, Frizzled 8 “FZD8”, Frizzled 9 “FZD9” and Frizzled 10 “FZD10”.

[0042] As used herein, the term “soluble receptor” or “FZD soluble receptor” refers to an N- terminal extracellular fragment of a FZD receptor protein preceding the first transmembrane domain of the receptor that can be secreted from a cell in soluble form. FZD soluble receptors comprising the entire N-terminal extracellular domain (ECD) as well as smaller fragments are included in the term. FZD soluble receptors comprising the Fri domain are also included in the term.

[0043] As used herein, the term “bone disorder” refers to a disorder, disease, or condition in a mammal in which there is an imbalance in the ratio of bone formation to bone resorption, resulting in an abnormal mass of bone. By way of non-limiting examples, bone disorder comprises osteopetrosis, osteoporosis, osteopenia, Paget’s disease, osteogenesis imperfecta, osteonecrosis, osteoarthritis, osteomyelitis, fibrous dysplasia, bone cancer and tumors, osteomalacia, and rickets. In some embodiments of the disclosure, a bone disorder relates to inefficient breakdown and remodeling of bone tissue by cells called osteoclasts, leading to more dense but structurally abnormal bones that are prone to fracture. In certain embodiments, a bone disorder is a bone resorption-related disorder.

[0044] As used herein, the term “bone-resorption” refers to dissolution and degradation of the bone matrix, both mineral and organic components.

[0045] As used herein, the term “homeostasis” in the context of bones refers to a balance between bone formation and bone resorption. It is maintained by three key bone cells:ATTY DKT NO: ASHI-007WOosteoblasts, osteocytes, and osteoclasts, which work together to ensure that the bone’ s structure and vitality are maintained.

[0046] As used herein, the terms “bone turnover’’ and “bone remodeling” refer to the continuous process of bone resorption and bone formation which is essential for maintaining bone health.

[0047] As used herein, the term “osteoclast” refers to a specialized cell that resorbs bone and ensures development and continuous remodeling of the skeleton.

[0048] The term “osteoclast activity" refers to the function of osteoclasts in bone-resorption and bone turnover and remodeling.

[0049] As used herein, the tern “baseline” refers to characterization of a subject, via clinical observation or via level of one or more biomarkers, established prior to treatment or at a point during treatment with an agent (e.g., an anti-FZD receptor antibody) disclosed herein or composition thereof. For example, a subject’s baseline bone mineral density is measured or established prior to treatment or at a point during treatment with an agent (e.g., anti-FZD receptor antibody) disclosed herein or a composition thereof. In another example, the level of one or more biomarkers (e.g., level of degradation fragments or cleaved products relevant to bone remodeling) is measured or established prior to treatment or at a point during treatment with an agent (e.g., anti-FZD receptor antibody) disclosed herein or a composition thereof. In another example, diagnostic and / or clinical findings for a subject (e.g., increased likelihood of bone fracture, increased likelihood of brittle bones, stunted growth, skeletal deformity, cytopenia, pancytopenia, macrocephaly, hepalosplenomegaly, abnormal cortical bone morphology, excessive sclerosis of the vertebral endplates, and cranial neuropathies), along with symptoms such as bone pain, are observed, measured, or established prior to treatment or at a point during treatment with an agent (e.g., anti-FZD receptor antibody) disclosed herein or a composition thereof.

[0050] As used herein, the terms “dosage” or “dosing” refer to the prescribed administration of an agent (e.g., a FZD receptor polypeptide) in a specific amount, number, and / or frequency of doses over a specified period of time.

[0051] As used herein, the terms “modulation” or “modulate” refer to regulation of a biological activity. In the context of a gene and / or protein expression, these terms refer to the alteration of the regulation, expression, or activity of a gene and / or protein. Modulation may be increasing or reducing (decreasing) the expression and / or activity of one or more genes and / or proteins.ATTY DKT NO: ASHI-007WO

[0052] As used herein, the phrases “percent identity,” “percent sequence identity,” “% identity,” or “identical” in the context of amino acid sequences or nucleic acid sequences refer to the extent to which two sequences e.g., two polypeptides or two nucleic acids have the same respective amino acid or nucleotide at the same positions in an alignment. As used herein, “percent identity” between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Similarly, percent “identity” between a nucleic acid sequence and a reference sequence is defined as the percentage of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. For a discussion of basic issues in searching sequence databases see Altschul et al., (1994) NATURE GENETICS 6:119-129, which is fully incorporated by reference herein. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0053] As used herein, the term “polynucleotide" or “nucleic acid molecule" or “nucleotide sequence” refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native intemucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single- stranded, doublestranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation.

[0054] The term “polypeptide fragment” as used herein refers to a polypeptide that has a deletion, e.g., an amino-terminal and / or carboxy-terminal deletion compared to a full-length polypeptide. In certain embodiments, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, at least 6, at least 7, at leastATTY DKT NO: ASHI-007WO8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25 , at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, or at least 70 amino acids long.

[0055] As used herein, the term “gene” refers to a transcription unit and regulatory regions that are adjacent (e.g., located upstream and downstream), and operably linked, to the transcription unit. A transcription unit is a series of nucleotides that are transcribed into an RNA molecule. A transcription unit may include a coding region. A “coding region” is a nucleotide sequence that encodes an unprocessed preRNA (i.e., an RNA molecule that includes both exons and introns) that is subsequently processed to an mRNA. A transcription unit may encode a non-coding RNA. A non-coding RNA is an RNA molecule that is not translated into a protein. Examples of non-coding RNAs include microRNA. The boundaries of a transcription unit are generally determined by an initiation site at its 5' end and a transcription terminator at its 3' end. A “regulatory region” is a nucleotide sequence that regulates expression of a transcription unit to which it is operably linked. Nonlimiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, transcription terminators, and poly(A) signals. A regulatory region located upstream of a transcription unit may be referred to as a 5' UTR, and a regulatory region located downstream of a transcription unit may be referred to as a 3' UTR. A regulatory region may be transcribed and be part of an unprocessed preRNA.

[0056] With regard to gene designations, single genes have often been denoted by multiple symbols. In the context of this document, gene symbols, whether they be human or non-human, may be designated by either upper-case or lower case letters. Neither the use of one particular symbol nor the adoption of lower or upper case symbols is intended to limit the scope of the gene in the context of these disclosures. All gene identification numbers identified herein (GenelD) are derived from the National Center for Biotechnology Information “Entrez Gene” or KEGG web site unless identified otherwise.

[0057] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.

[0058] As used herein, the term “effective amount” refers to the amount of an active agent (e.g., a FZD receptor polypeptide) sufficient to effect beneficial or desired results (e.g., aATTY DKT NO: ASHI-007WOdesired prophylactic or therapeutic effect). An effective amount can be administered in one or more administration(s), application(s) or dosage(s) and is not intended to be limited to a particular formulation or administration route.

[0059] As used herein, the term “pharmaceutical composition’’ refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0060] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] ,

[0061] As used herein, the term “treating” includes any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, augmenting, enhancing, increasing, supplementing, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.

[0062] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. The term administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces clinical and diagnostic findings and symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The deliveryATTY DKT NO: ASHI-007WOcan be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0063] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0064] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0065] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular polypeptide, that polypeptide can be used in various embodiments of compositions of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0066] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0067] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should beATTY DKT NO: ASHI-007WOunderstood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0068] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0069] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0070] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0071] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.IL WNT Signaling Pathway

[0072] WNT proteins regulate expression of a number of target genes through the WNT signaling pathway. In humans, the WNTs include WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, and WNT9B. In the absence of WNT ligands, P-catenin, a downstream effector of the canonical WNT pathway, is phosphorylated by a protein complex containing glycogen synthase kinase- 33 (GSK-3P). The phosphorylated P-catenin is constantly degraded, to prevent its accumulation. Upon binding of certain WNTs to the FZD-LRP5 / 6 co-receptors, phosphorylation of P-catenin is inhibited, which prevents the degradation of P-catenin and results in its accumulation. P-catenin is then translocated into the nucleus, where it associates with T cell factor for DNA binding and thus regulates expression of target genes including but not limited to VEGF.

[0073] The seven-pass transmembrane receptor Frizzled (“FZD”) is critical for nearly all WNT signaling, and the N-terminal FZD cysteine rich domain (CRD) serves as the WNT binding domain. WNT signaling pathway plays a significant role in controlling skeletal development and homeostasis. In particular, WNT signaling controls multiple aspects ofATTY DKT NO: ASHI-007WOskeletal development, controlling the differentiation and function of mesenchymal stem cells (MSCs), chondrocytes, osteoblasts, and osteoclasts (see Regard JB, Zhong Z, Williams BO, Yang Y. Wnt signaling in bone development and disease: making stronger bone with Wnts. Cold Spring Harb Perspect Biol. 2012 Dec l;4(12):a007997). Targeting of WNT signaling pathway has drawn attention for treatment of some bone disorders in which WNT signaling is directly dysregulated and activated, particularly with respect to osteoblast function. However, the impact of WNT / 0-catenin signaling pathway on osteoclast-related disorders remains largely unexplored. Further research is needed to elucidate how modulating WNT signaling would affect osteoclast function, activity, and / or differentiation.A. Frizzled (FZD) Receptors

[0074] The frizzled (FZD) receptors are an important class of seven transmembrane receptors of the G-protein coupled receptor (GPCR) superfamily and contain a large extracellular N- terminal ligand binding domain with 10 conserved cysteines, known as a cysteine-rich domain (CRD) or Fri domain. These FZD receptors are involved in many important biological processes such as development, cell proliferation, survival, migration and stem cell maintenance. The signaling pathways are activated when WNT ligands interact with the FZD receptors and control stem and progenitor cell renewal and cell differentiation during embryonic development and tissue homeostasis in adult animals. There are ten human FZD receptors: FZD1-10. Different FZD CRDs have different binding affinities for specific WNTs (see Wu CH, Nusse R. J Biol Chem. 2002 Nov l;277(44):41762-9), and FZD receptors have been grouped into those that activate the canonical 0-catenin pathway and those that activate non-canonical pathways (Miller et al., 1999, Oncogene 18:7860-72).

[0075] Provided herein are methods of treating a bone disorder (e.g., by modulation of WNT signaling pathway) in a subject in need thereof by administering to the subject a therapeutically effective amount of a FZD receptor polypeptide. The full-length amino acid and nucleotide sequences for FZD1-10 are known in the art. TABLE 1 provides amino acid sequences of human FZD receptors 1-10 (includes variants). Sequences provided in TABLE 1 may comprise a signaling peptide sequence. It is well known in the art that the signaling peptide sequence can be cleaved off leaving a mature protein.

[0076] TABLE 1 discloses amino acid sequences of human FZD receptors.TABLE 1 : Sequences of Human FZD ReceptorsATTY DKT NO: ASHI-007WOATTY DKT NO: ASHI-007WOATTY DKT NO: ASHI-007WOATTY DKT NO: ASHI-007WOATTY DKT NO: ASHI-007WOATTY DKT NO: ASHI-007WOATTY DKT NO: ASHI-007WOB. Osteoclasts and Osteoblasts

[0077] Bone remodeling is a continuous renewal process for skeletal system and is carried out by osteoclasts and osteoblasts. Bones are constantly being remodeled, and the process is carefully controlled to ensure that bones stay strong and healthy. Osteoclasts are involved in bone remodeling, a normal process in which old bone is removed and new bone is created to replace it. Osteoclasts can differentiate from cells of the monocyte / macrophage lineage upon stimulation of factors such as monocyte / macrophage colony stimulating factor (M-CSF) and receptor activation of NF-KB ligand (RANKL) (see Feng X, Teitelbaum SL. Osteoclasts: New Insights. Bone Res. 2013 Mar 29;1(1):11-26). Osteoclast activity is intricately regulated by their differentiation process, which is influenced by various factors and signaling pathways. The acidification of the resorption lacuna is a critical process in bone resorption earned out by osteoclasts. This process involves the active transport of protons (H+ions) into the lacuna, creating an acidic environment that dissolves the mineral components of bone.

[0078] Osteoblasts are specialized cells responsible for bone formation. They originate from mesenchymal stem cells, skeletal stem cells, chondrocytes and other cells lining bone surfaces (see Mizoguchi T, Ono N. The diverse origin of bone-forming osteoblasts. J Bone Miner Res.2021 Aug;36(8):1432-1447) and play a crucial role in synthesizing and secreting the bone matrix, which consists primarily of collagen and other proteins. Osteoblasts also regulate the mineralization process that give bones their strength and rigidity. These cells are essential for maintaining bone health and remodeling, responding to mechanical stress, and repairing microdamage in the bone tissue. Once their bone-forming activity is complete, osteoblasts can become embedded in the matrix as osteocytes, which help maintain bone tissue, or they can remain on the bone surface as lining cells.

[0079] While enhanced osteoclast activity plays a major role in diseases such as osteoporosis, Paget's disease, lytic bone metastases, multiple myeloma, hyperparathyroidism, rheumatoid arthritis, periodontitis, and hypercalcemia of malignancy, decreased osteoclast activity plays a significant role in the pathogenesis of osteopetrosis. Insufficient osteoclast activity leads to inadequate bone resorption and can cause osteopetrosis, a disease in which the bones of the sufferer become more dense with an abnormal structure that renders them susceptible to fracture. A continuous, orderly and functional supply of both osteoblasts and osteoclasts is essential for skeletal homeostasis, as increased or decreased production of osteoclasts or osteoblasts and / or changes in the rate of their apoptosis and / or changes in their function and / or activity are largely responsible for the imbalance between bone resoiption and formation thatATTY DKT NO: ASHI-007WOunderlies several bone disorders. Thus, there exists a need for the identification and characterization of agents that can balance the activity of osteoblast and / or osteoclasts and provide therapeutic benefits for patients suffering from bone related disorders. Osteoclast activation and / or osteoblast inhibition may be indicated, for example, by an increase in medullary volume and a decrease in periosteal volume.

[0080] In some embodiments, the present disclosure provides a method of modulating osteoclast activity by administration of a FZD receptor polypeptide. In certain embodiments, modulating osteoclast activity comprises an increase in osteoclast activity. In certain embodiments, the modulation of osteoclast activity can comprise an increase in activation of an osteoclast. In certain embodiments, the modulation of osteoclast activity can comprise stimulation of osteoclast differentiation. In certain embodiments, the modulation of osteoclast activity can comprise enhancing the acidification of lacunae. In certain embodiments, the modulation of an osteoclast can comprise an increase in the activity or function of a differentiated osteoclast. In certain embodiments, modulation of osteoclast activity can comprise increased functionality at neutral pH.

[0081] In some embodiments, the present disclosure provides a method of modulating osteoblast and osteoclast activity by administration of a FZD receptor polypeptide. In certain embodiments, modulating osteoblast activity comprises a decrease in osteoblast activity.

[0082] A number of human bone disorders are associated with mutations in genes that have important roles in osteoclast function. Bone disorders disclosed herein may be caused by mutations in these genes. By way of non-limiting example, these genes can comprise chloride voltage-gated channel 7 (CLCN7), T cell immune regulator 1 (TCIRG1), osteoclastogenesis associated transmembrane protein 1 (OSTM1), Pleckstrin homology and RUN domain containing Ml (PLEKHM1), sorting nexin 10 (SNX10), tumor necrosis factor ligand superfamily member 11 (TNFSF11), tumor necrosis factor receptor superfamily member Ila (TNFRSF11A), and Carbonic Anhydrase 2 (CAII). General description on these genes is provided below.

[0083] Chloride Voltage-Gated Channel 7 (CLCN7). The CLCN7 gene belongs to the CLC family of genes, which provide instructions for making chloride channels. These channels transport negatively charged chlorine atoms (chloride ions) and play a key role in a cell’s ability to generate and transmit electrical signals. Some CLC channels regulate the flow of chloride ions across cell membranes, while others transport chloride ions within cells. The CLCN7 gene provides instructions for making a chloride channel called CLC-7. These channels are abundantATTY DKT NO; ASHI-007WOin cells throughout the body. They are particularly important for the resorptive function of osteoclasts. CLCN7 is required for acidification of the osteoclast lacunae at the surface of bone resorption, via C1- / H+ exchange. Mutations in CLCN7 display dominant-negative effects. Two forms of osteopetrosis associated with significant osteoclast dysfunction include autosomal dominant osteopetrosis type II, or AD02, and the autosomal recessive osteopetrosis (ARO, also known as infantile malignant osteopetrosis). Both AD02 and ARO can result from mutations in CLCN7, with at least 75% of AD02 associated with autosomal dominant mutations in CLCN7, and approximately 15% of ARO associated with autosomal recessive mutations. External Ids for CLCN7 gene: HGNC: 2025: NCB1 Gene: 1186; Ensembl: ENSG00000103249; OMIM®: 602727; UniProtKB / Swiss-Prot: P51798.

[0084] T Cell Immune Regulator 1 (TCIRG1). The TCIRG1 gene encodes several protein isoforms, with 2 main isoforms. The full-length isoform a (OC116) encodes the A3 subunit of vacuolar H(+)- ATPase, which is involved in regulation of the pH of intracellular compartments and organelles of eukaryotic cells, including the pH of osteoclasts. The shorter isoform b (TIRC7) encodes a T-cell-specific membrane protein that plays an essential role in T- lymphocyte activation and immune response. Mutations in this gene are associated with autosomal recessive osteopetrosis (>50% of ARO, MedlinePlus Genetics and Penna, Villa and Capo Dis. Model Meeh. 2021) and mutations have also been identified in one family experiencing autosomal dominant osteopetrosis (ADO2) (Jodeh et. al. J. Clin. Endo. Metab.2024). External Ids for TCIRG1 gene; HGNC: 11647; NCBI Gene: 10312; Ensembl: ENSG00000110719; OMIM®; 604592; UniProtKB / Swiss-Prot; Q13488.

[0085] Osteoclastogenesis Associated Transmembrane Protein 1 (OSTM1). OSTM1 gene encodes a protein that may be involved in the degradation of G proteins via the ubiquitindependent proteasome pathway. The encoded protein binds to members of subfamily A of the regulator of the G-protein signaling (RGS) family through an N-terminal leucine-rich region. This protein also has a central RING finger-like domain and E3 ubiquitin ligase activity. This protein is highly conserved from flies to humans. Defects in this gene may cause the autosomal recessive, infantile malignant form of osteopetrosis, in part at least via its role as an accessory protein to CLCN7. External Ids for OSTM1 gene: HGNC: 21652; NCBI Gene: 28962; Ensembl; ENSG00000081087; OMIM®: 607649; UniProtKB / Swiss-Prot: Q86WC4.

[0086] Pleckstrin Homology And RUN Domain Containing Ml (PLEKHM1). PLEKHM1 gene encodes a protein that is essential for bone resorption, and may play a critical role in vesicular transport in the osteoclast. Mutations in this gene are associated with autosomalATTY DKT NO: ASHI-007WOrecessive osteopetrosis type 6 (0PTB6) and autosomal dominant osteopetrosis (AD02). External Ids for PLEKHM1 gene: HGNC: 29017; NCBI Gene: 9842; Ensembl: ENSG00000225190; OMIM®: 611466; UniProtKB / Swiss-Prot: Q9Y4G2.

[0087] Sorting Nexin 10 (SNX10). SNX10 gene encodes a member of the sorting nexin family. Members of this family contain a phox (PX) domain, which is a phosphoinositide binding domain, and are involved in intracellular trafficking. This protein does not contain a coiled coil region, like some family members. This gene may play a role in regulating endosome homeostasis. Studies have shown that SNX10 mediates bone resorption and gastric acidification by regulating vesicular trafficking (see Ye L et al., PLoS Genet. 2015 Mar 26;ll(3):el005057). External Ids for SNX10 gene: HGNC: 14974; NCBI Gene: 29887; Ensembl: ENSG00000086300; OMIM®: 614780; UniProtKB / Swiss-Prot: Q9Y5X0.

[0088] Tumor Necrosis Factor Superfamily Member 11 (TNFSF11). TNFSF11 also known as RANKL is a gene that encodes a member of the tumor necrosis factor (TNF) cytokine family which is a ligand for osteoprotegerin and functions as a key factor for osteoclast differentiation and activation. This protein was shown to be a dendritic cell survival factor and is involved in the regulation of T cell-dependent immune response. T cell activation was reported to induce expression of this gene and lead to an increase of osteoclastogenesis and bone loss. This protein was shown to activate antiapoptotic kinase AKT / PKB through a signaling complex involving SRC kinase and tumor necrosis factor receptor-associated factor (TRAF) 6, which indicated this protein may have a role in the regulation of cell apoptosis. Targeted disruption of the related gene in mice led to severe osteopetrosis and a lack of osteoclasts. Most autosomal- recessive osteopetrosis (ARO) are classified as osteoclast-rich, but recently a subset of osteoclast-poor ARO has been recognized as due to a defect in TNFSF11 (see Guerrini MM, et al., Am J Hum Genet. 2008 Jul;83(l):64-76). External Ids for TNFSF11 gene: HGNC: 11926; NCBI Gene: 8600; Ensembl: ENSG00000120659; OMIM®: 602642; UniProtKB / Swiss-Prot: 014788.

[0089] Tumor Necrosis Factor Receptor Superfamily Member Ila (TNFRSF11A). TNFRSF11 A also known as RANK encodes a member of the TNF-receptor superfamily. This receptor can interact with various TRAF family proteins, through which this receptor induces the activation of NF-kappa B and MAPK8 / JNK. This receptor and its ligand are important regulators of the interaction between T cells and dendritic cells. This receptor is also an essential mediator for osteoclast and lymph node development. Mutations at this locus have been associated with familial expansile osteolysis, autosomal recessive osteopetrosis (ARO,ATTY DKT NO: ASHI-007WOosteoclast-poor), and Paget disease of bone. Externa] Ids for TNFRSFHA gene: HGNC: 11908; NCBI Gene: 8792; Ensembl: ENSG00000141655; OMIM®: 603499; UniProtKB / S wi ss-Prot : Q Y 6Q6.

[0090] Carbonic Anhydrase 2 (CAII). The protein encoded by CAII gene (also known as CA2 gene) is one of several isozymes of carbonic anhydrase, which catalyzes reversible hydration of carbon dioxide. Defects in this enzyme are associated with osteopetrosis and renal tubular acidosis. In osteoclasts, CAII facilitates proton production, leading to the acidification of resorption lacunae (see Jahn K, Kelkar S et al., Osteocytes Acidify Their Microenvironment in Response to PTHrP In Vitro and in Lactating Mice In Vivo. J Bone Miner Res. 2017 Aug;32(8):1761-1772). External Ids for CA2 gene: HGNC: 1373; NCBI Gene: 760; Ensembl: ENS G00000104267; OMIM®: 611492; UniProtKB / Swiss-Prot: P00918.

[0091] Other Genes. Mutations in other genes implicated in autosomal recessive osteopetrosis, functionally important in osteoclasts for processes such as trafficking, ruffled border formation, proteolytic cleavage and osteoclast function include SLC29A3, KINDLIN3, Integrin-beta, LRRK1, CTSK, MITF, TRAF6, RELA and NEMO.

[0092] In one aspect, the present disclosure provides a FZD receptor polypeptide for use in treating a bone disorder (e.g., by modulation of WNT signaling pathway) in a subject in need thereof. In another aspect, the present disclosure provides a FZD receptor polypeptide for use in restoring homeostasis of bone turnover in a subject in need thereof. In yet another aspect, the present disclosure provides a FZD receptor polypeptide for use in modulating osteoclast activity in a subject in need thereof. In yet another aspect, the present disclosure provides a FZD receptor polypeptide for use in modulating both osteoblast and osteoclast activity in the context of a disease or mutational background where osteoclast function is impaired. In some embodiments, the subject is characterized by a functional defect in one or more genes associated with osteoclast function and / or osteoclast activity. In some embodiments, the one or more genes associated with osteoclast function and / or osteoclast activity comprises CLCN7 or TCIRG1. In some embodiments, a subject may be diagnosed with autosomal dominant osteopetrosis, based on clinical findings such as generalized osteosclerosis predominating in sites such as the spine, pelvis and long bones, and / or pathognomonic radiographic features, or other analysis of bone mineral density and / or other similar findings, without the above mutations being identified, or without a specific causal mutation being identified or characterized.ATTY DKT NO: ASHI-007WO

[0093] In some embodiments, a subject to be selected for a FZD receptor polypeptide treatment is characterized by a functional defect in one or more proteins expressed by genes associated with osteoclast function and / or osteoclast activity as described herein. In some embodiments, the subject is characterized by altered function of one or more mutant genes. In some embodiments, the subject is characterized by loss of normal function of one or more mutant genes. In some embodiments, the subject is characterized by modulated (e.g., an increase or decrease) expression of one or more mutant genes. In some embodiments, the subject is characterized by mislocalization of one or more mutant genes. In some embodiments, a subject may be selected based on other evidence of presence of osteopetrosis (e.g. bone imaging and clinical parameters) without a target mutant gene being identified.III. Frizzled (FZD) Receptor Polypeptides

[0094] As used herein, the term “FZD receptor polypeptide” or “Frizzled receptor polypeptide” refers to an N-terminal extracellular fragment (or a portion thereof) of a FZD receptor preceding the first transmembrane domain of the receptor that can be secreted from a cell in soluble form. FZD soluble receptors include FZD soluble receptors comprising the entire N-terminal ECD or Fri domain fused in-frame to other functional and structural proteins including, but not limited to, a human Fc. In some embodiments, the FZD receptor polypeptide comprises a Fri domain of a FZD receptor protein.

[0095] In some embodiments, FZD soluble receptor polypeptides comprising the Fri domain exhibit altered biological activity (e.g., increased half-life) compared to soluble receptors comprising the entire extracellular domain.

[0096] The predicted Fri domains for each of the human FZD receptors 1-10 proteins are provided in SEQ ID NOs: 11-20. The predicted minimal Fri domains for each of the human FZD receptors 1-10 proteins are provided as SEQ ID NOs: 22-31. Those of skill in the art may differ in their understanding of the exact amino acids corresponding to the Fri domains. Thus, the N-terminus and / or C-terminus of the domains outlined above and herein may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids.

[0097] In certain embodiments, the FZD receptor polypeptide comprises a Fri domain of a human FZD receptor, or a fragment or variant of the Fri domain that binds or more Wnt proteins. In certain embodiments, the human FZD protein is FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10. In certain embodiments, the human FZD protein is FZD4. In certain embodiments, the human FZD protein is FZD5. In certain embodiments, the human FZD protein is FZD8.ATTY DKT NO: ASHI-007WO

[0098] In some embodiments, the FZD receptor polypeptide comprises a Fri domain comprising the minimal Fri domain of FZD receptor 1 (SEQ ID NO: 22). In some embodiments, the FZD receptor polypeptide comprises a Fri domain comprising the minimal Fri domain of FZD receptor 2 (SEQ ID NO: 23). In some embodiments, the FZD receptor polypeptide comprises a Fri domain comprising the minimal Fri domain of FZD receptor 3 (SEQ ID NO: 24). In some embodiments, the FZD receptor polypeptide comprises a Fri domain comprising the minimal Fri domain of FZD receptor 4 (SEQ ID NO: 25). In some embodiments, the FZD receptor polypeptide comprises a Fri domain comprising the minimal Fri domain of FZD receptor 5 (SEQ ID NO: 26). In some embodiments, the FZD receptor polypeptide comprises a Fri domain comprising the minimal Fri domain of FZD receptor 6 (SEQ ID NO: 27). In some embodiments, the FZD receptor polypeptide comprises a Fri domain comprising the minimal Fri domain of FZD receptor 7 (SEQ ID NO: 28). In some embodiments, the FZD receptor polypeptide comprises a Fri domain comprising the minimal Fri domain of FZD receptor 8 (SEQ ID NO: 29). In some embodiments, the FZD receptor polypeptide comprises a Fri domain comprising the minimal Fri domain of FZD receptor 9 (SEQ ID NO: 30). In some embodiments, the FZD receptor polypeptide comprises a Fri domain comprising the minimal Fri domain of FZD receptor 10 (SEQ ID NO: 31).

[0099] In some embodiments, the FZD receptor polypeptide comprises a Fri domain consisting essentially of the Fri domain of FZD receptor 1. In some embodiments, the FZD receptor polypeptide comprises a Fri domain consisting essentially of the Fri domain of FZD receptor 2. In some embodiments, the FZD receptor polypeptide comprises a Fri domain consisting essentially of the Fri domain of FZD receptor 3. In some embodiments, the FZD receptor polypeptide comprises a Fri domain consisting essentially of the Fri domain of FZD receptor 4. In some embodiments, the FZD receptor polypeptide comprises a Fri domain consisting essentially of the Fri domain of FZD receptor 5. In some embodiments, the FZD receptor polypeptide comprises a Fri domain consisting essentially of the Fri domain of FZD receptor 6. In some embodiments, the FZD receptor polypeptide comprises a Fri domain consisting essentially of the Fri domain of FZD receptor 7. In some embodiments, the FZD receptor polypeptide comprises a Fri domain consisting essentially of the Fri domain of FZD receptor 8. In some embodiments, the FZD receptor polypeptide comprises a Fri domain consisting essentially of the Fri domain of FZD receptor 9. In some embodiments, the FZD receptor polypeptide comprises a Fri domain consisting essentially of the Fri domain of FZD receptor 10.ATTY DKT NO: ASHI-007WO

[0100] In some embodiments, the FZD receptor polypeptide comprises a variant of any one of the aforementioned FZD Fri domain sequences that comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) conservative substitutions and is capable of binding Wnt protein(s).Fc Domain

[0101] In some embodiments, the FZD receptor polypeptide further comprises a non-FZD receptor polypeptide. In some embodiments, a FZD receptor polypeptide may include FZD extracellular domain or Fri domains linked to other non-FZD functional and structural polypeptides, including, but not limited to, a human Fc domain. In some embodiments, the non-FZD receptor polypeptide comprises a human Fc domain.

[0102] The Fc domain can be from any of the classes of immunoglobulin, IgG, IgA, IgM, IgD and IgE. Depending upon the circumstances, the IgG Fc domain is derived from an IgGl, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the IgG Fc domain is derived from a human IgGl Fc domain. In certain embodiments, the Fc domain is a human IgGl Fc domain. In certain embodiments, the Fc domain is a human IgG2 Fc domain. In certain embodiments, the Fc domain is a wild-type Fc domain. In certain embodiments, the Fc domain is a mutated Fc domain. In certain embodiments, the Fc domain is truncated at the N-terminal end. In certain embodiments, the Fc domain is truncated at the C-terminal end. In certain embodiments, the Fc domain is truncated at the N-terminal end by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, (e.g., in the hinge domain). In certain embodiments, the Fc domain is truncated at the C-terminal end by 1, 2, 3, or more amino acids. In some embodiments, the Fc domain is truncated at the C- terminal end by 1 amino acid. Particularly preferred is an IgGl Fc domain.

[0103] TABLE 2 provides amino acid sequences of exemplary Fc domains.TABLE 2: Amino Acid Sequences of Exemplary Fc domains.ATTY DKT NO: ASHI-007WO

[0104] In certain embodiments, the non-FZD polypeptide comprises SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36. In certain embodiments, the non-FZD polypeptide consists essentially of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36. In certain embodiments, the non-FZD polypeptide consists essentially of SEQ ID NO: 33 or SEQ ID NO: 34.

[0105] Linker. In certain embodiments, a FZD receptor is fused to the IgG Fc domain via a linker polypeptide. In certain embodiments, the linker polypeptide has an optimized lengthATTY DKT NO: ASHI-007WOand / or amino acid composition and / or can be flexible or rigid. In certain embodiments, the linker polypeptide includes 4-50 amino acid residues. In certain embodiments, the linker polypeptide consists of 4-50 amino acid residues. In certain embodiments, the linker polypeptide consists of 4-20 amino acid residues.

[0106] In certain embodiments, the linker polypeptide is absent, i.e., the protein sequence of the FZD receptor is linked to the hinge of the Fc domain or directly to the Fc domain via a peptide bond. In certain embodiments, the linker polypeptide is “short,” e.g., consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, the linker polypeptide consists of about 12 or fewer amino acid residues. In certain embodiments, the linker polypeptide is “long,” e.g., consists of 15, 20 or 25 amino acid residues. In some embodiments, the linker polypeptide consists of about 3 to about 15, for example, 8, 9 or 10 contiguous amino acid residues. The linker polypeptide suitable for linking the FZD receptor to the Fc domain may include, as at least a part of a full length linker polypeptide, a (GS)n(SEQ ID NO: 37), (GGS)n(SEQ ID NO: 38), (GGGS)n(SEQ ID NO: 39), (GGSG)„ (SEQ ID NO: 40), (GGSGG)n (SEQ ID NO: 41), and (GGGGS)n(SEQ ID NO: 42) sequence, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Exemplary linker polypeptide sequences in the FZD receptor polypeptide are provided in TABLE 3.TABLE 3: Linker PolypeptidesATTY DKT NO: ASHI-007WO

[0107] In certain embodiments, the FZD receptor polypeptide described herein blocks binding of at least one ligand (e.g. WNT) to a Frizzled receptor. In certain embodiments, the ligand is a WNT protein. In certain embodiments, the WNT pathway inhibitor (e.g., a FZD receptor polypeptide) blocks binding of at least one human WNT protein to one or more of its receptors. In some embodiments, the WNT pathway inhibitor (e.g., a FZD receptor polypeptide) blocks binding of at least one WN T to at least one FZD protein. In some embodiments, the WN T pathway inhibitor (e.g., a FZD receptor polypeptide) blocks binding of at least one WNT protein to FZD receptor 1, FZD receptor 2, FZD receptor 3, FZD receptor 4, FDZ receptor 5, FDZ receptor 6, FDZ receptor 7, FDZ receptor 8, FDZ receptor 9, and / or FZD receptor 10. In certain embodiments, the blocking of binding of at least one ligand (e.g., WNT) to at least one FZD receptor protein is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In certain embodiments, a WNT pathway inhibitor (e.g., a FZD receptor polypeptide) that blocks binding of at least one WNT protein to at least one FZD receptor protein further inhibits WNT pathway signaling and / or P-catenin signaling. In certain embodiments, a WNT pathway inhibitor (e.g., a FZD receptor polypeptide) that blocks binding of at least one human WNT to at least one FZD receptor protein is a soluble receptor. In certain embodiments, a WNT pathway inhibitor (e.g., a FZD receptor polypeptide) that blocks binding of at least one human WNT to at least one FZD protein is a FZD-Fc soluble receptor. In certain embodiments, a WNT pathway inhibitor (e.g., a FZD receptor polypeptide)ATTY DKT NO: ASHI-007WOthat blocks binding of at least one human WNT to at least one FZD receptor protein is a FZD8- Fc soluble receptor.

[0108] Non-limiting examples of FZD receptor polypeptides have been described in, for example, U.S. Patent No. 7,723,477, U.S. Patent No. 7,947,277, and U.S. Patent Publication No. 2011 / 0305695.

[0109] In certain embodiments, the FZD receptor polypeptide described herein inhibits WNT pathway signaling. It is understood that a WNT pathway inhibitor that inhibits WNT pathway signaling may, in certain embodiments, inhibit signaling by one or more receptors in the WNT signaling pathway but not necessarily inhibit signaling by all receptors. In certain embodiments, WNT pathway signaling by all human receptors may be inhibited. In certain embodiments, WNT pathway signaling by one or more receptors selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FDZ5, FDZ6, FDZ7, FDZ8, FDZ9, and FZD 10 is inhibited.

[0110] In certain embodiments, the inhibition of WNT pathway signaling by a WNT pathway inhibitor (e.g. , FZD receptor polypeptide) is a reduction in the level of WNT pathway signaling of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In some embodiments, a WNT pathway inhibitor that inhibits WNT pathway signaling is a soluble receptor. In some embodiments, a WNT pathway inhibitor that inhibits Wnt pathway signaling is a FZD-Fc soluble receptor. In some embodiments, a WNT pathway inhibitor that inhibits WNT pathway signaling is a FZD8- Fc soluble receptor.A. Ipafricept (Fzd8-Fc, OMP-54F28)

[0111] Ipafricept is a fusion protein (or a decoy receptor) comprising a cysteine-rich domain of frizzled family receptor 8 (Fzd8) fused to a human immunoglobulin Fc domain that competes with the native Fzd8 receptor for its ligands and antagonizes Wnt signaling (see Le PN el al., “Targeting the Wnt pathway in human cancers: therapeutic targeting with a focus on OMP-54F28,” Pharmacol Ther. 2015 Feb;146:l-ll). TABLE 4 provides amino acid sequences for ipafricept.TABLE 4: Ipafricept amino acid sequence (with or without signal sequence). Bolded amino acid residues denote FZD receptor component within the FZD receptor fused to Fc domain.ATTY DKT NO: ASHI-007WQ

[0112] In certain embodiments, a WNT pathway inhibitor (e.g., a FZD receptor polypeptide) that blocks binding of at least one human WNT to at least one FZD protein is soluble receptor OMP-54F28. In certain embodiments, the methods disclosed herein comprise administering a FZD receptor polypeptide comprising the amino acid sequence of S EQ ID NO: 60.

[0113] In some embodiments, the methods disclosed herein comprise administering a receptor polypeptide comprising an amino acid sequence having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to sequence provided in SEQ ID NO: 60. In some embodiments, the methods disclosed herein comprise administering the receptor polypeptide provided in SEQ ID NO: 60, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions.

[0114] In some embodiments, the FZD receptor polypeptide disclosed herein comprises the same extracellular fragment of a FZD receptor as ipafricept. In some embodiments, the polypeptide comprises the same Fc region as ipafricept. In some embodiments, the polypeptide comprises ipafricept. In some embodiments, the polypeptide consists of ipafricept.ATTY DKT NO: ASHI-007WO

[0115] B. Other FZD Receptor Polypeptides

[0116] In some embodiments, the FZD receptor polypeptides comprise any number of FZD receptor polypeptides, which are known in the art and / or are commercially available, or can be generated de novo. TABLE 5 provides amino acid sequences of exemplary known FZD receptor polypeptides.TABLE 5: Amino Acid Sequences of FZD Receptor PolypeptidesATTY DKT NO: ASHI-007WOATTY DKT NO: ASHI-007WO

[0117] In certain embodiments, the methods disclosed herein comprise administering a FZD receptor polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 60-67. In certain embodiments, the methods disclosed herein comprise administering a FZD receptor polypeptide comprising the amino acid sequence of SEQ ID NO: 61. In certain embodiments, the methods disclosed herein comprise administering a FZD receptor polypeptide comprising the amino acid sequence of SEQ ID NO: 62. In certain embodiments, the methods disclosed herein comprise administering a FZD receptor polypeptide comprising the amino acid sequence of SEQ ID NO: 63. In certain embodiments, the methods disclosed herein comprise administering a FZD receptor polypeptide comprising the amino acid sequence of SEQ ID NO: 64. In certain embodiments, the methods disclosed herein comprise administering a FZD receptor polypeptide comprising the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the methods disclosed herein comprise administering a FZD receptor polypeptide comprising the amino acid sequence of SEQ ID NO: 66. In certain embodiments, the methods disclosed herein comprise administering a FZD receptor polypeptide comprising the amino acid sequence of SEQ ID NO: 67.

[0118] In some embodiments, the methods disclosed herein comprise administering a receptor polypeptide comprising an amino acid sequence having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to sequence provided in SEQ ID NO: 61. In some embodiments, the methods disclosed herein comprise administering the receptor polypeptide provided in SEQ ID NO: 61, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions.

[0119] In some embodiments, the methods disclosed herein comprise administering a receptor polypeptide comprising an amino acid sequence having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, aboutATTY DKT NO: ASHI-007WO98%, or about 99% identity to sequence provided in SEQ ID NO: 62. In some embodiments, the methods disclosed herein comprise administering the receptor polypeptide provided in SEQ ID NO: 62, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions.

[0120] In some embodiments, the methods disclosed herein comprise administering a receptor polypeptide comprising an amino acid sequence having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to sequence provided in SEQ ID NO: 63. In some embodiments, the methods disclosed herein comprise administering the receptor polypeptide provided in SEQ ID NO: 63, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions.

[0121] In some embodiments, the methods disclosed herein comprise administering a receptor polypeptide comprising an amino acid sequence having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to sequence provided in SEQ ID NO: 64. In some embodiments, the methods disclosed herein comprise administering the receptor polypeptide provided in SEQ ID NO: 64, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions.

[0122] In some embodiments, the methods disclosed herein comprise administering a receptor polypeptide comprising an amino acid sequence having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to sequence provided in SEQ ID NO: 65. In some embodiments, the methods disclosed herein comprise administering the receptor polypeptide provided in SEQ ID NO: 65, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions.

[0123] In some embodiments, the methods disclosed herein comprise administering a receptor polypeptide comprising an amino acid sequence having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to sequence provided in SEQ ID NO: 66. In some embodiments, the methods disclosed herein comprise administering the receptor polypeptide provided in SEQ ID NO: 66, with up to 1, 2, 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 21, 22, 23, 24, or 25 amino acid substitutions.ATTY DKT NO: ASHI-007WO

[0124] In some embodiments, the methods disclosed herein comprise administering a receptor polypeptide comprising an amino acid sequence having at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to sequence provided in SEQ ID NO: 67. In some embodiments, the methods disclosed herein comprise administering the receptor polypeptide provided in SEQ ID NO: 67, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions.IV. Osteopetrosis

[0125] Osteopetrosis encompasses a heterogeneous group of bone disorders that are characterized by increased bone density with abnormal bone structure. They are predominantly caused by defective osteoclast-mediated bone resorption, with mutations in genes that encode proteins involved in the acidification of the lacunae between the bone matrix and osteoclasts accounting for the vast majority of osteopetrosis cases. As rates of resorption and formation in bone are coupled and involve cross-talk between osteoclasts and osteoblasts, it is likely that both bone resorption and formation rates are altered in many or most cases, with an imbalance of too much bone tissue formation and too little resoption. Two forms of osteopetrosis associated with significant osteoclast dysfunction include autosomal dominant osteopetrosis type II, or AD02, and the autosomal recessive osteopetrosis (ARO, also known as infantile malignant osteopetrosis). AD02, the most common form of osteopetrosis (incidence approximately 1 / 20,000 (Polgreen, Imel and Econs, 2023; Osteopetrosis: MedlinePlus Genetics)), is associated with abundant but hypofunctional osteoclasts, most commonly arising from or associated with missense mutations in CLCN7. The penetrance of these mutations is variable and there are large differences in disease severity, even between members of the same family. The most common complications are fractures, pain and osteonecrosis / osteomyelitis, particularly of the jaw. Severe disease can result in vision loss and bone marrow failure (see Alam I, Gerard-O'Riley RL, Acton D, Hardman SL, Murphy M, Alvarez MB, Blosser RJ, Sinn A, Srour EF, Kacena MA, Econs MJ. FASEB J. 2022 Sep;36(9):e22471). The predominant gene associated with ADO2 is CLCN7, which encodes the chloride channel 7 (C1C-7) protein, that is necessary for the acidification of the lacunae of osteoclasts that enables their resorptive function (see Polgreen LE et al., Autosomal dominant osteopetrosis. Bone. 2023 May;170:l 16723). In ADO2, the dominant negative effect occurs when a mutant form of C1C- 7 protein interferes with the function of the normal CLC-7 protein. CLC-7 functions as a dimer, i.e., two CLC-7 proteins pair up to form a functional unit. When one of these proteins isATTY DKT NO: ASHI-007WOmutated, it can pair with a normal CLC-7 protein. The mutant CLC-7 protein disrupts the function of the normal protein, for example by causing incorrect localization within the osteoclast, resulting in a dysfunctional dimer. This impairs the chloride ion and proton (H+) exchange which is necessary for acidification of osteoclast lacunae on the bone surface and resorptive activity. The dysfunctional dimer results in osteoclasts that cannot resorb bone effectively, resulting in defective bone remodeling and structurally-abnormal increased bone density. AD02 is presently incurable, whereas a preclinical study suggested the potential use of interferon gamma-lb (IFN-G) for its modest beneficial effects (see Alam et al., Interferon Gamma, but not Calcitriol Improves the Osteopetrotic Phenotypes in AD02 Mice. J Bone Miner Res. 2015 Nov;30(l I):2005-13), a clinical trial in human subjects failed to show significant benefit (Imel et al JBMR 2019). SIS-101-ADO, an siRNA therapeutic, was granted orphan drug designation by the United States Food and Drug Administration for the treatment of autosomal dominant osteopetrosis in May 2023 with particular mutations in CLCN7.

[0126] The incidence of ARO is considerably lower (approximately 1 / 250,000) and it is more severe, appearing in early infancy and can involve life-threatening defects in bone marrow function along with vision loss, hearing loss, cranial neuropathies and paralysis of facial muscles. Different mutations in genes that impact osteoclast function have been identified, including TCIRG1 (at least 50% of ARO) and CLCN7 (approximately 15% of ARO), which are the primary genes affected in the osteoclast-rich forms of ARO, along with mutations of much lower prevalence in genes such as OSTM1, PLEKHM1, SNX10, and CAIL Other rare mutations can impact osteoclast generation and maturation to result in osteoclast-poor ARO, including mutations in the RANKL signaling pathway (e.g. TNFSF11, TNFRSF11A). Individuals with an additional form of osteopetrosis, intermediate autosomal osteopetrosis (IAO), can show either an autosomal dominant or recessive inheritance pattern with hypofunctional or diminished numbers of osteoclasts. The incidence of this condition is unknown but it is less frequent than ADO2, and individuals with this form of the disorder typically do not have life-threatening bone marrow abnormalities but can experience anemia and an increased risk of fracture.

[0127] Attenuated autosomal recessive osteopetrosis: Some patients with ARO may present with a less severe, attenuated form of the disease with diagnosis in childhood but survival into early adulthood with pathological fractures and abnormal bone morphology. Biallelic missense mutations in the genes associated with ARO may result in some retention of proteinATTY DKT NO: ASHI-007WOfunctionality in these patients. These patients may still require hematopoietic stem cell transplantation in early adulthood.

[0128] A study has shown that canonical WNT signaling through FZD8 receptor and |3- catenin negatively regulates osteoclast differentiation and / or activity and reduces bone resorption independent of osteoprotegerin (see Albers J et al., “Canonical Wnt signaling inhibits osteoclast ogenesis independent of osteoprotegerin,” J Cell Biol. 2013 Feb 18;200(4):537-49). The study highlights phenotypic analysis of Fzd8-deficient mice which show a profound osteoporotic phenotype driven by excess osteoclast differentiation and / or activity. The study found that FZD8 inhibition paradoxically induces osteoclast differentiation and / or activity.V. Diagnosis of Bone Related Disorders

[0129] Clinically, a subject with a bone disorder often presents with features such as increased likelihood of bone fracture, increased likelihood of brittle bones, stunted growth, skeletal deformity, cytopenia, pancytopenia, macrocephaly, hepatosplenomegaly, abnormal cortical bone morphology, excessive sclerosis of the vertebral endplates, bone pain, narrowed medullary cavities and cranial neuropathies as compared to a healthy reference subject (e.g., a matched reference subject), or as compared to a healthy reference range (e.g., the range of values in matched reference subjects that do not have a bone disorder).

[0130] Detection and / or diagnosis of bone related disorders (e.g., osteopetrosis) primarily involves a combination of clinical evaluation, radiographic imaging, and genetic testing. Bone disorders can be detected and diagnosed using standard methods known in the art. These include clinical assessment of the subject and / or measurement of biochemical and molecular markers correlative of bone metabolism.

[0131] Radiographic imaging is crucial, revealing characteristic features like increased bone density, “bone-within-bone” appearance, and “sandwich vertebrae” (see Calvin C Wu et al. , Diagnosis and Management of Osteopetrosis: Consensus Guidelines From the Osteopetrosis Working Group, The Journal of Clinical Endocrinology & Metabolism, Volume 102, Issue 9, 1 September 2017, Pages 3111-3123; Stark, Z. et al. , Osteopetrosis. Orphanet J Rare Dis 4, 5 (2009)). Genetic testing can be used to further support the diagnosis by identifying mutations in the CLCN7 gene, which is commonly associated with AD02. Genetic testing can also be used to further support the diagnosis by identifying mutations in other less prevalent mutations in genes such as TCIRG1 and PLEKHM1.ATTY DKT NO: ASHI-007WO

[0132] In some embodiments, the basis for diagnosis of a bone disorder can be bone mineral density and / or bone density. A number of techniques can be used to assess bone mineral density and / or bone density. One widely used technique is dual energy X-ray absorptiometry (DXA or DEXA). The x-ray beam in this technique has at least two distinct radiation energies that allow for measurement of two tissue types of differing density (e.g., bone and soft tissue). Measurements of total body bone mineral content and density are also possible with DXA and can be useful for assessment of bone mineral accumulation during growth and development and for body composition analysis. Another method for assessing bone mineral density and quality is computed tomography (CT), an imaging procedure that uses X-rays to create detailed cross-sectional images of the area being scanned. Both the DXA and CT can be used to assess apparent bone mineral density calculated as bone mineral content per unit area (g / cm2).

[0133] In some embodiments, DXA can be used to assess bone mineral density in whole body. In some embodiments, DXA can be used to assess bone mineral density in femur and / or spine.

[0134] Another method for assessing bone mineral content and / or bone mineral density is computed tomography scan. Measurement of bone mineral density by computed tomography (CT) can be done using well known procedures. In some embodiments, CT is used to assess bone volume (BV) relative to total tissue volume (TV). In some embodiments, the ratio of BV / TV is used to measure density of bones (e.g., trabecular bone) wherein a higher BV / TV ratio indicates presence of a greater amount of bone tissue present within the total volume.

[0135] Another method for assessing bone mineral content and / or bone density and bone quality is quantitative computed tomography (CT or QCT). A related analysis, peripheral quantitative computed tomography (pQCT) assesses bone properties in the limbs. Density of a selected area on the CT image is compared to the densities of a set of known standards, known as calibration phantoms, for example, a series of tubes filled with different concentrations of calcium solution imaged within the field of view of the CT.

[0136] In some embodiments, the bone structure of a subject can be directly assessed by magnetic resonance imaging (MRI).

[0137] Other types of techniques that can be used to measure bone density include, as examples, single energy X-ray absorptiometry (SXA), which is generally used to measure the bone density in the wrist or heel; radiographic absorptiometry (RA), which uses an X-ray of the hand and a small metal wedge to calculate bone density; dual photon absorptiometry (DPA), which measures the spine, hip or total body; and single photon absorptiometry (SPA), which generally measures bone density in the wrist.ATTY DKT NO: ASHI-007WO

[0138] In addition to measurements of bone density, assays for biochemical markers can provide an indication of the state of bone remodeling. These markers can be used independently or as an adjunct to bone density measurements. Markers of bone formation and / or resorption are detectable in the urine and / or serum. A significant class of bone resorption markers are known to a person skilled in the art. When bone is being degraded or being formed, these peptides and proteins are released into circulation, metabolized in the liver, and / or excreted in the urine.

[0139] Exemplary markers include, among others, C-terminal telopeptide of type 1 collagen (CTX), osteocalcin, Tartrate-resistant acid phosphatase (TRAP), Procollagen type 1 N-terminal propeptide (P1NP), N-terminal telopeptide (NTX), ratio of CTX / TRAP, and / or ratio of NTX / TRAP to assess activity per unit osteoclast, bone-specific alkaline phosphatase, and osteocalcin.

[0140] In some embodiments, the bone resorption biomarkers and / or bone formation biomarkers are used for diagnostic purposes. In some embodiments, the bone resorption biomarkers and / or bone formation biomarkers are used to evaluate a subject’s response to the FZD receptor polypeptide disclosed herein. In some embodiments, the bone resorption biomarkers are selected from the group consisting of C-terminal telopeptide of type 1 collagen (CTX), Tartrate- resistant acid phosphatase (TRAP), N-terminal telopeptide (NTX), the ratio of CTX / TRAP, the ratio of NTX / TRAP, and osteocalcin. In some embodiments, the bone formation biomarkers are selected from the group consisting of procollagen type 1 N-terminal propeptide (P1NP), bone-specific alkaline phosphatase, and osteocalcin. In some embodiments a biomarker of interest is one or more of: serum PTH, serum calcium, urine calcium, and urine calcium / creatinine ratio.

[0141] In some embodiments, an animal model of osteopetrosis (e.g., ADO2) can be used to determine efficacy and dosages for therapeutic purposes. One animal model is a murine model of AD02 osteopetrosis and has been well characterized in the art (see Alam et al Bone 2014; Alam et al JBMR 2015; Alam et al FASEB J. 2022).

[0142] In addition to the in vivo analyses for assessing impact on bone remodeling and resorption, various in vitro assays can be used to examine the effect of the FZD receptor polypeptide treatment on osteoclast activity.ATTY DKT NO: ASHI-007WOVI. Therapeutic Applications

[0143] It is contemplated that a FZD receptor polypeptide disclosed herein can be administered to a mammal, generally a human, in a pharmaceutically acceptable dosage form via one or more of the administrations approaches described herein.

[0144] It is contemplated that the FZD receptor polypeptides described herein can be used in the treatment of a bone disorder (e.g., a bone resorption-related disorder) which may be achieved by administering to the subject an effective amount of a FZD receptor polypeptide disclosed herein. In some embodiments, the bone disorder (e.g., bone resorption-related disorder) is associated with dysfunctional osteoclast activity.

[0145] The present disclosure provides a method of treating a bone disorder by modulation of WNT signaling pathway in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a polypeptide that specifically binds to at least one FZD receptor ligand (e.g., FZD receptor polypeptide) disclosed herein or an effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the bone disorder is a bone resorption-related disorder. In some embodiments, the bone disorder is caused by an imbalance of osteoclast and osteoblast activity. In some embodiments, the bone disorder is osteopetrosis. Osteopetrosis can be autosomal dominant osteopetrosis or autosomal recessive osteopetrosis. In some embodiments, the osteopetrosis is an autosomal dominant osteopetrosis (e.g., type II).

[0146] The present disclosure also provides a method of restoring homeostasis of bone turnover in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a FZD receptor polypeptide disclosed herein or an effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the restoration of homeostasis of bone turnover comprises a restoration of balance between bone resorption and bone formation.

[0147] The present disclosure also provides a method of modulating osteoclast activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a FZD receptor polypeptide disclosed herein or an effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the modulation of osteoclast activity comprises an increase in osteoclast function and / or activity. In some embodiments, the modulation of osteoclast activity comprises an increase in activation of an osteoclast. In some embodiments, the modulation of osteoclast activity comprises stimulation of osteoclast differentiation. In some embodiments, the modulation of osteoclast activityATTY DKT NO: ASHI-007WOcomprises an increase in activation of an osteoclast and stimulation of osteoclast differentiation. In some embodiments, the modulation of osteoclast activity comprises an increase in one or more of osteoclast function, osteoclast activity, osteoclast activation, and stimulation of osteoclast differentiation.

[0148] In some embodiments, the present disclosure provides a method of modulating osteoblast and osteoclast activity by administration of a FZD receptor polypeptide. In certain embodiments, modulating osteoblast activity comprises a decrease in osteoblast activity.

[0149] In some embodiments, the administration of a FZD receptor polypeptide for use in any of the methods disclosed or described herein results in improvement in bone quality as measured by bone mineral density and / or bone mineral content. In some embodiments, the administration of a FZD receptor polypeptide results in reduction of bone mineral density and / or changes in morphometry to a level or structure that reduces fracture risk and / or improves bone biomarkers profiles in a subject with osteopetrosis. In some embodiments, the reduction in bone mineral density is assessed by well known methods such as Z scores. In some embodiments, the administration of a FZD receptor polypeptide to a subject results in a reduction in Z score in the subject as compared to the subject's Z score prior to administration of the FZD receptor polypeptide. In some embodiments, the administration of a FZD receptor polypeptide results in reduction of bone mineral density and / or changes in morphometry while preserving or improving bone strength in the subject as compared to prior to administration of the FZD receptor polypeptide. In some embodiments, the preservation of bone strength is assessed by well- known methods e.g., polar moment of inertia from CT data. In some embodiments, the administration of a FZD receptor polypeptide activates dysfunctional osteoclasts and regulates the imbalance in bone homeostasis to restore an appropriate balance between bone resorption and synthesis.

[0150] In some embodiments, the administration of a FZD receptor polypeptide to a subject results in improvement in bone quality as measured by bone density, morphometry, radiography, and / or bone mineral content in comparison to prior to administration of the FZD receptor polypeptide.

[0151] In some embodiments, therapeutic effects of administration of the polypeptide comprise reduced bone mineral density (BMD), such as reduced whole body areal BMD (aBMD), reduced cortical aBMD, reduced femur aBMD, reduced spine aBMD, and / or reduced trabecular aBMD; reduced bone volume, optionally reduced trabecular bone volume and / or reduced cortical bone volume; reduced bone volume as a fraction of total volume (B V / TV), forATTY DKT NO: ASHI-007WOexample reduced BV / TV for the trabecular bone; reduced number of trabeculae (Tb.N); decreased cortical and / or trabecular and / or medullary bone; increased trabecular spacing (Tb.S); improved bone quality, such as improved cortical bone quality; preservation of bone while rescuing osteopetrotic phenotype; increased osteoclast activity; decreased osteoblast activity; improved bone homeostasis; increased medullary volume; and / or decreased periosteal volume.

[0152] In some embodiments, effects of administration of the polypeptide comprise no significant difference in polar moment of inertia (Ip) of the femur and / or tibia.

[0153] In some embodiments, the subject receives the polypeptide for at least 4 weeks, optionally for at least 12 weeks. In some embodiments, therapeutic effects of administration of the polypeptide occur by 4 weeks after the start of administration. In some embodiments, therapeutic effects of administration of the polypeptide occur by 12 weeks after the start of administration.

[0154] In some embodiments the Fzd receptor polypeptide is administered for treatment of a bone disorder at a dose that activates osteoclast activity, but which does not substantially inhibit osteoblast activity. In some embodiments, a Fzd receptor polypeptide is administered for treatment of a bone disorder at a dose that activates osteoclast activity and also inhibits osteoblast activity. In some such embodiments the bone disorder is a condition in a mammal in which there is inadequate bone resorption, resulting in an abnormally dense but brittle bones. In some embodiments the bone disorder is osteopetrosis. The present disclosure further provides a therapeutically effective amount of a frizzled (FZD) receptor polypeptide as disclosed herein, for use in a method of therapy.

[0155] The present disclosure further provides a therapeutically effective amount of a frizzled (FZD) receptor polypeptide as disclosed herein, for use in a method of therapy, wherein the method of therapy comprises a method disclosed herein.VII. Pharmaceutical Compositions

[0156] The present disclosure also features pharmaceutical compositions comprising one or more of the FZD receptor polypeptides described herein. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipient(s) or carrier(s) can also be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).ATTY DKT NO: ASHI-007WO

[0157] A FZD receptor polypeptide suitable for use with the disclosed methods is formulated for any suitable route of administration to a subject including, but not limited to injection (e.g., intravenous injection), in some embodiments. Injection includes, e.g., subcutaneous injection, peritoneal injection, intravenous injection, or intramuscular injection. In some embodiments, the FZD receptor polypeptide of the disclosure are formulated for subcutaneous administration. In some embodiments, the FZD receptor polypeptides of the disclosure are formulated for peritoneal administration. In some embodiments, the FZD receptor polypeptides of the disclosure are formulated for intravenous administration (e.g., intravenous injection or infusion). In some embodiments, the FZD receptor polypeptides of the disclosure are formulated for intramuscular administration. In some embodiments, administration is in one, two, three, four, five, six, seven, or more injection sites. In some embodiments, administration is in one injection site. In some embodiments, administration is in two injection sites. In some embodiments, administration is in three injection sites. In some embodiments, administration is in four injection sites. In some embodiments, administration is in five injection sites. In some embodiments, administration is in six injection sites.

[0158] Subcutaneous administration is particularly preferred for methods disclosed herein.VIII. Dosage

[0149] A FZD receptor polypeptide suitable for use with the disclosed methods and compositions comprising the same may be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, in some embodiments. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each subject and will depend upon a variety of factors, including the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the subject’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.

[0159] Upon improvement of a subject’s condition, a maintenance dose of the FZD receptor polypeptides disclosed herein may be administered, if necessary. As osteoclasts are continuously regenerated from bone marrow cells, and bone is continuously remodeled, ongoing treatment with a FZD receptor polypeptide will, in many cases, be required to maintain skeletal health. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of findings upon assessment of bone quality, changes in levels of bone related biomarkers, and / or associated symptoms to a level at which the improved condition isATTY DKT NO: ASHI-007WOretained when the bone quality, bone related biomarkers, and / or associated symptoms have been alleviated to a desired level. Changes in dosing may be based on a patient’s biomarker or clinical response. The patient’s biomarker may be selected from a change in one or more of: beta CTX, CTX / TRAP ratio, PINT, serum PTH, serum calcium, urine calcium, and urine calcium / creatinine ratio. Changes in dosing may be based on a patient’s change in BMD.

[0160] The FZD receptor polypeptides described herein can, for example, be administered at a dosage ranging from about 0.5 ng / kg to about 20 mg / kg of body weight, alternatively dosages between 0.05 mg and 1000 mg / dose, every 72 to 750 hours, or according to the requirements of the particular drug and the stage of therapy (e.g., early treatment versus maintenance phase) of the subject. The methods herein contemplate administration of an effective amount of the FZD receptor polypeptide or composition to achieve the desired or stated effect. Administration can be as a chronic or acute therapy.

[0161] In some embodiments, the effective amount is at least about 15 mg / kg. In some embodiments, the effective amount is about 15 mg / kg or less. For example, the effective amount may be about 15 mg / kg, such as about 15 mg / kg administered twice weekly by subcutaneous administration.

[0162] In some embodiments, the effective amount is at least about 45 mg / kg. In some embodiments, the effective amount is about 45 mg / kg or less. For example, the effective amount may be about 45 mg / kg, such as about 45 mg / kg administered twice weekly by subcutaneous administration.

[0163] Administration of the FZD receptor polypeptides disclosed herein could be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter or by direct intralesional injection. This may be administered once or more times weekly, once or more times monthly, and once or more times annually.

[0164] The description above describes multiple aspects and embodiments of the disclosure. The patent application specifically contemplates all combinations and permutations of the aspects and embodiments.EXAMPLES

[0165] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.ATTY DKT NO: ASHI-007WOExample 1 - Evaluation of ipafricept in vivo in a murine model of ADO2 osteopetrosis

[0166] The objective of the proposed study is to evaluate the effect of ipafricept, a recombinant fusion protein (or polypeptide) containing the extracellular domain of FZD8 that can inhibit Wnt signaling by competing for ligands, on the osteopetrotic bone phenotypes in adult AD02 mice starting at 11 to 12 weeks of age. A murine model of AD02 osteopetrosis has been well characterized in the art (see Alam et al., Chloroquine increases osteoclast activity in vitro but does not improve the osteopetrotic bone phenotype of AD02 mice, Bone 2014 December; 153: 116160; Alam et al., Interferon Gamma, but not Calcitriol Improves the Osteopetrotic Phenotypes in AD02 Mice, J Bone Miner Res. 2015 Nov;30(l 1) :2005-l 3. Epub 2015 May 14; see Alam et al., Bone marrow transplantation as a therapy for autosomal dominant osteopetrosis type 2 in mice, FASEB J. 2022 Sep;36(9):e22471). Briefly, the model uses mice (129 background strain) bearing a knock-in mutation of the major mutation found in C1CN7 (C1CN7G213R / +) in human ostepetrosis. These heterozygous adult mice bear manifestations of mild to moderate disease, including higher bone mass and increased numbers of poorly resorbing osteoclasts. In the experimental design, a breeding strategy will be used to generate an adequate number of female AD02 mice to support an efficacy study (approximately 8 mice per group). An additional group of age-matched wild type mice will also be used as a reference group for normal bone biology. Mice will be housed in polycarbonate cages in a vivarium maintained on a 12-h light and 12-h dark cycle and will be fed a regular diet and water ad libitum. The procedures performed throughout the experiment will be in accordance with the ethical standards and guidelines recommended for the use of experimental animals in research.

[0167] Based on previous published studies in this model (see Alam et al., Bone marrow transplantation as a therapy for autosomal dominant osteopetrosis type 2 in mice, FASEB J.2022 Sep;36(9):e22471), 12 weeks should offer an adequate window of time to detect any beneficial activity of ipafricept in the AD02 mouse model. If ipafricept is highly active, a signal might be apparent earlier, and thus a 4 week timepoint will also be assessed. FZD8 is highly conserved between human and mouse (>95% amino acid identity), thus commercially - available human ipafricept will be used (e.g. available from MedChem Express for research use). Two doses of ipafricept will be evaluated, 15 mg / kg and 45 mg / kg bodyweight administered by intraperitoneal (IP) route twice a week. Both these doses are anticipated to provide a high degree of target coverage. An ADO2 control group will be dosed with vehicle (PBS buffer). Quantitative anlayses of bone density and bone quality will be performed in vivoATTY DKT NO: ASHI-007WO(in live mice) at 4 weeks and 12 weeks using dual energy X-ray absorptiometry (DXA; PIXImus II mouse densitometer; Lunar Corp., Madison, WI, USA) and micro-computed tomography (microCT; Skyscan 1176, Bruker, MA) with an isotropic voxel size of 9 pm3, with settings for each in-vivo scan including X-ray peak potential 50 kVp, X-ray intensity 500 uA, and image resolution of 4000 x 2672 pixels.

[0168] For analysis using in vivo DXA, the whole body, femur, and lumbar vertebrae 3 through 5 (L3-5) will be scanned. The global window for the whole body will be defined as the whole-body image minus calvarium, mandible and teeth. After completion of the scan of each bone, mutually exclusive region of interest (ROI) boxes will be drawn around the bone from which femur and spine (lumbar vertebrae 3 through 5) aBMD (areal bone mineral density, g / cm2) measurements will be obtained. For analysis using in vivo microCT, the growth plate location will be identified from the scout-view and trabecular bone measurements consisting of 2 mm will be completed from about 0.5 mm below the growth plate. Finally, 3D and 2D morphometric evaluations will be performed for the trabecular bone from each scan, and bone volume as a fraction of total volume (BV / TV) and micro-structural parameters (such as trabecular number Tb.N, trabecular spacing Tb.Sp and trabecular thickness Tb.Th). Micro CT analysis of cortical bone will be conducted on a mid-shaft region of the femur. The strength and quality of cortical bone will be assessed via calculation of the polar moment of inertia (Ip) of the femur, from microCT data.

[0169] Analyses will include quantitative assessment of changes bone density, morphometry and quality between ADO2 mice treated with a vehicle control vs ADO2 mice treated with ipafricept, where it is anticipated that many of these metrics will be elevated in ADO2 mice and might be reduced with ipafricept treatment at 4 weeks and / or 12 weeks. In addition, the degree of restoration of the bone phenotype will be assessed by comparison of ADO2 mice treated with ipafricept to age-matched healthy control mice (WT CTRL). Data to be assessed include DXA and microCT data generated for bone mineral density (aBMD), trabecular bone (micro CT analysis of BV / TV in the region of trabecular bone of the femur and DXA analysis of the spine), cortical bone (DXA analysis of the femur, micro CT analysis of the femur at midshaft), and microstructural parameters such as the number of trabeculae (Tb.N) in trabecular bone.

[0170] The ability of ipafricept to promote osteoclast activity in vivo in the context of ADO2 would be supported by trends and / or statistically significant changes in DXA and microCT data generated for the above parameters. If the activity of ipafricept is substantial, stasticallyATTY DKT NO: ASHI-007WOsignificant results could be obtained in as little as 4 weeks. An optimal result would include ability to reduce the bone density and improve structural morphometry in AD02 mice to a level similar to that observed in healthy controls, while preserving bone strength as assessed by the polar moment of intertia. These results would indicate that treatment with ipafricept has adequately activated the mutant osteoclasts and addressed the imbalance in bone homeostasis (potentially via action on both osteoclasts and osteoblasts) to restore an appropriate balance between bone resorption and synthesis. Both doses of ipafricept are anticipated to provide substantial (close to complete) target coverage and thus a clear dose response is not necessarily anticipated, although some trends may be apparent. Longitudinal analysis of AD02 mice treated with ipafricept may provide insights into the ability of ipafricept to continue to activate mutant osteoclasts. For example, if analyses reveal that ipafricept is highly active after 4 weeks of dosing, analysis of bone parameters at 12 weeks will provide additional insights into any potential tachphylaxis or adaptation, given cross-talk between osteoclasts and osteoblasts, or if ipafricept continues to demonstrate activity after more prolonged exposure.

[0171] As FZD8 is highly conserved between human and mouse (95% amino acid identity), and effector function is not a concern for this mechanism of action which involves binding ligands to prevent their engagement with FZD receptors, ipafricept (human FZD8, IgGl) was used in the AD02 mouse model. Ipafricept (IP A) was purchased from Med Chem Express. The material provided had an accompanying certificate of analysis reporting a high-quality product, with a concentration of 5.21 mg / mL, purity of 98.73% monomer by size exclusion high performance liquid chromatography, and < 0.012 EU / mg. Doses were chosen to provide a high level of target coverage in this study.Table 6. Design of Efficacy Study in ADO2 Mice

[0172] The study design is described above and summarized in Table 6. The breeding scheme yielded the numbers of animals shown in the right column of Table 6. Dosing was initiated in adult mice of 12 weeks of age (mean 12 weeks ± 1 week). Statistical analyses were performedATTY DKT NO: ASHI-007WOusing one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test in GraphPad Prism. There was no mortality or unscheduled euthanasia in the study.

[0173] Four weeks after study initiation, significant increases in bone mineral density (BMD) were apparent by both DXA (FIG. 1) and microCT (FIG. 2), in both cortical and trabecular bone, in ADO2 mice as compared to age-matched wild type mice, consistent with the published osteopetrotic disease phenotype (Alam I, Gray AK, Chu K, et al. Generation of the first autosomal dominant osteopetrosis type II (AD02) disease models. Bone. 2014;59:66-75). Analysis using DXA revealed significant decreases in BMD for both doses of ipafricept in whole body areal BMD (aBMD), femur and spine, as compared to AD02 mice treated with vehicle (FIG. 1). There were no significant differences between AD02 mice treated with ipafricept and age-matched control wild-type mice for whole body aBMD and spine aBMD, whereas treatment of ADO2 mice with ipafricept at 45 mg / kg resulted in a significant decrease in aBMD of the femur at 4 weeks (FIG. 1), suggesting over-treatment. This change in BMD is consistent with an increase in the activity of the abundant but hypofunctional osteoclasts with ipafricept treatment, resulting in rescue of the osteopetrotic phenotype and restoration of bone homeostasis.

[0174] Similar changes in BMD were apparent by analysis using microCT at 4 weeks (FIG.2). Significant increases, reflecting the disease phenotype, were apparent in AD02 mice as compared to age-matched wild type controls for trabecular bone volume / total volume (B V / TV), cortical bone in the femur (Cortical B V), number of trabeculae (Tb.N) and trabecular spacing (Tb.S). Both trabecular and cortical bone volume and number of trabeculae were significantly reduced in AD02 mice with treatment using ipafricept, at either dose level. In the trabecular compartment, the number of trabeculae was also significantly reduced as compared to age-matched wild type control mice, with corresponding increases in trabecular spacing (Tb.S). Trends for a decrease were also apparent in BV / TV. These findings reflect active, ongoing bone remodeling associated with osteoclast activity and indicate that these doses of ipafricept are likely higher than optimal.

[0175] Analyses using DXA and microCT at 12 weeks after study initiation indicated that bone remodeling was ongoing (FIG. 1 and FIG. 2), with no evidence of tachyphylaxis or loss of drug activity, and that bone quality was compromised in some parameters, as treatment with ipafricept resulted in decreases that exceeded the age-matched wild type controls.

[0176] To further assess cortical bone quality and robusticity, an analysis of the polar moment of inertia (Ip) was undertaken at the femur midshaft using microCT (FIG. 3). The Ip assessesATTY DKT NO: ASHI-007WQdistribution of bone mass around its longitudinal axis, quantifying its resistance to torsional stress, and has been found to give better precision in measurements of bone strength than 3 point bending tests of mouse femur and tibia (Jamsa T, Jalovaara P, Peng Z, Vaiinanen HK, Tuukkanen J. Comparison of three-point bending test and peripheral quantitative computed tomography analysis in the evaluation of the strength of mouse femur and tibia. Bone.1998;23(2): 155-161)). There were no significant differences in Ip across groups at week 4, indicating preservation of bone quality while rescuing the osteopetrotic phenotype with ipafricept treatment (FIG. 3). At week 12, significant decreases in Ip compared to age-matched wild type controls is apparent, consistent with ongoing drug activity and overtreatment at these doses for this time frame. Overall, these data support the use of ipafricept to rescue the osteopetrotic phenotype in this mouse model of AD02, and indicate that lower doses are likely to be efficacious, with preservation of bone quality, for longer-term dosing.INCORPORATION BY REFERENCE

[0177] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0178] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

ATTY DKT NO: ASHI-007WOCLAIMS WHAT IS CLAIMED IS:

1. A method of treating a bone disorder by modulation of WNT signaling pathway in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a frizzled (FZD) receptor polypeptide.

2. A method of treating a bone disorder by inhibition of a WNT signaling pathway in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of an inhibitory frizzled (FZD) receptor polypeptide.

3. The method of claim 1 or claim 2, wherein the bone disorder is a bone resorption-related disorder and is caused by an imbalance of osteoblast and osteoclast activity.

4. The method of claim 1 or claim 2, wherein the bone disorder is a bone resorption-related disorder and is characterized by an imbalance of osteoblast and osteoclast activity.

5. The method of any of claims 1-4, wherein the bone disorder is osteopetrosis.

6. The method of any of claims 1 -5, wherein the bone disorder is autosomal dominant osteopetrosis type II (AD02).

7. The method of any one of claims 1-5, wherein the bone disorder is autosomal recessive osteopetrosis (ARO).

8. The method of claim 7, wherein ARO comprises attenuated ARO.

9. The method of any one of claims 1-5, wherein the bone disorder comprises intermediate autosomal osteopetrosis.

10. A method of improving homeostasis of bone turnover in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a frizzled (FZD) receptor polypeptide.

11. The method of claim 10, wherein the improvement of homeostasis of bone turnover comprises a restoration of balance between bone resoiption and bone formation asATTY DKT NO: ASHI-007WOdetermined by improvement in bone metrics as assessed by dual energy X-ray absorptiometry (DXA) and / or computed tomography scan (CT).

12. A method of modulating homeostasis of bone turnover in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitory frizzled (FZD) receptor polypeptide.

13. The method of claim 12, wherein the modulation of homeostasis of bone turnover comprises a restoration of balance between bone resoiption and bone formation as determined by improvement in bone metrics as assessed by dual energy X-ray absorptiometry (DXA) and / or computed tomography scan (CT).

14. A method of modulating osteoclast activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a frizzled (FZD) receptor polypeptide.

15. The method of claim 14, wherein the method comprises administering to the subject a therapeutically effective amount of an inhibitory frizzled (FZD) receptor polypeptide.

16. The method of claim 14 or 15, wherein the modulation of osteoclast activity comprises an increase in osteoclast function and / or activity, osteoclast activation, and stimulation of osteoclast differentiation.

17. The method of claim 14 or 15, wherein the modulation of osteoclast activity comprises an increase in one or more of osteoclast function, osteoclast activity, osteoclast activation, and stimulation of osteoclast differentiation.

18. The method of any of claims 14-17, wherein the modulation of osteoclast activity is at neutral pH.

19. The method of any one of claims 1-18, wherein the subject, at baseline, is characterized by bone quality as measured by at least one of bone mineral density (BMD) and morphometry.

20. The method of any one of claims 1-19, wherein the subject, at baseline, is characterized by an increase in BMD.ATTY DKT NO: ASHI-007WO21. The method of any one of claims 1-1 , wherein the subject, at baseline, is characterized by an elevated BMD relative to a healthy reference range.

22. The method of any of claims 19-21, wherein the BMD is assessed by dual energy X-ray absorptiometry (DXA), computed tomography scan (CT), and / or radiography.

23. The method of any one of claims 1-22, wherein the subject, at baseline, is characterized by the level of one or more biochemical biomarkers.

24. The method of claim 23, wherein the one or more biochemical biomarkers comprises one or more bone resorption biomarkers and / or one or more bone formation biomarkers.

25. The method of claim 24, wherein the one or more bone resorption biomarkers is selected from the group consisting of C-terminal telopeptide of type 1 collagen (CTX), Tartrate-resistant acid phosphatase (TRAP), N-terminal telopeptide (NTX), the ratio of CTX / TRAP, the ratio of NTX / TRAP, and osteocalcin.

26. The method of claim 25, wherein the one or more bone formation biomarkers is selected from the group consisting of procollagen type 1 N-terminal propeptide (P1NP), bone-specific alkaline phosphatase, and osteocalcin.

27. The method of any one of claims 1-26, wherein the subject has been diagnosed with autosomal dominant osteopetrosis type II (AD02).

28. The method of any one of claims 1-27, wherein the subject has been diagnosed with autosomal dominant osteopetrosis type II (AD02) and has an autosomal dominant mutation in chloride voltage-gated channel 7 (CLCN7), TCIRG1, or PLEKHM1.

29. The method of claim 27 or 28, wherein the diagnosis of AD02 is characterized by one or more of BMD, morphometry, and radiography of the subject.

30. The method of any one of claims 1-26, wherein the subject has been diagnosed with autosomal recessive osteopetrosis (ARO).

31. The method of any one of claims 1-26, wherein the subject has been diagnosed with autosomal recessive osteopetrosis (ARO) and has an autosomal recessive mutation in one orATTY DKT NO: ASHI-007WOmore genes selected from the group consisting of TCIRG1, CLCN7, 0STM1, PLEKHM1, SNX10, and CAII.

32. The method of any one of claims 1-26, wherein the subject has been diagnosed with ARO and has an autosomal recessive mutation in TNFSF11 or TNFRSF11 A.

33. The method of any one of claims 30-32, wherein the diagnosis of ARO is characterized by one or more of BMD, morphometry, and radiography of the subject.

34. The method of any one of claims 20-26, wherein the diagnosis of osteopetrosis is accompanied by and associated with a reduced level of one or more bone-resorption biomarkers in the subject as compared to the level of one or more bone-resorption biomarkers in a healthy reference subject.

35. The method of any one of claims 27-34, wherein the diagnosis of osteopetrosis is accompanied by and associated with (a) a reduced level of one or more bone-resorption biomarkers in the subject as compared to the level of said one or more bone-resorption biomarkers in a healthy reference range, or (b) a reduced ratio of CTX / TRAP relative to a healthy reference range.

36. The method of any one of claims 1-35, wherein the subject, at baseline, experiences one or more of the following: increased likelihood of bone fracture, increased likelihood of brittle bones, osteonecrosis, osteomyelitis, stunted growth, skeletal deformity, pancytopenia, macrocephaly, hepatosplenomegaly, abnormal cortical bone morphology, excessive sclerosis of the vertebral endplates, bone pain, cranial neuropathies and narrowed medullary cavities, as compared to an untreated subject having a bone disorder.

37. The method of any one of claims 1-35, wherein the subject, at baseline, experiences one or more of the following: increased likelihood of bone fracture, increased likelihood of brittle bones, osteonecrosis, osteomyelitis, stunted growth, skeletal deformity, pancytopenia, macrocephaly, hepatosplenomegaly, abnormal cortical bone morphology, excessive sclerosis of the vertebral endplates, bone pain, cranial neuropathies and narrowed medullary cavities, as compared to a subject not having a bone disorder.ATTY DKT NO: ASHI-007WO38. The method of any one of claims 1 -37, wherein the administration of the FZD receptor polypeptide results in improvement in bone quality as measured by at least one of bone mineral density (BMD) and morphometry.

39. The method of any one of claims 1-38, wherein the administration of the FZD receptor polypeptide results in an increase in level of one or more bone-resorption biomarkers in the subject as compared to the baseline level of the one or more bone-resorption biomarkers.

40. The method of claim 39, wherein the bone resorption markers may be selected from the group consisting of C-terminal telopeptide of type 1 collagen (CTX), Tartrate-resistant acid phosphatase (TRAP), N-terminal telopeptide (NTX), the ratio of CTX / TRAP, the ratio of NTX / TRAP, and osteocalcin.

41. The method of any one of claims 1-40, wherein the FZD receptor polypeptide comprises a fragment of an extracellular domain of a human FZD receptor and a human Fc domain.

42. The method of claim 41, wherein the fragment of the extracellular domain of the human FZD receptor comprises a Fri domain of the human FZD receptor.

43. The method of claim 42, wherein the Fri domain of the human FZD receptor comprises the Fri domain of human FZD8.

44. The method of claim 43, wherein the Fri domain of human FZD8 comprises the amino acid sequence of SEQ ID NO: 18.

45. The method of claim 42, wherein the Fri domain of the human FZD receptor comprises the Fri domain of human FZD4.

46. The method of claim 45, wherein the Fri domain of human FZD4 comprises the amino acid sequence of SEQ ID NO: 14.

47. The method of claim 42, wherein the Fri domain of the human FZD receptor comprises the Fri domain of human FZD5.ATTY DKT NO: ASHI-007WO48. The method of claim 47, wherein the Fri domain of human FZD5 comprises the amino acid sequence of SEQ ID NO: 15.

49. The method of any one of claims 41-48, wherein the human Fc is human IgGl Fc comprising the amino acid sequence of SEQ ID NO: 34.

50. The method of claim 41, wherein the FZD receptor polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 60-67.

51. The method of claim 50, wherein the FZD receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 60.

52. The method of any one of claims 1-51, wherein the subject is a human subject.

53. The method of any one of claims 1-52, wherein the subject is an infant, child, or an adult.

54. The method of any of claims 1-53, wherein the FZD receptor polypeptide is administered by subcutaneous route.

55. The method of any of claims 1 -53, wherein the FZD receptor polypeptide is administered by intravenous route.

56. The method of any of the previous claims, wherein the dose of FZD receptor polypeptide is changed based on a patient’s biomarker or clinical response.

57. The method of claim 56, wherein the patient’s biomarker is selected from a change in one or more of: beta CTX, CTX / TRAP ratio, P1NP, serum PTH, serum calcium, urine calcium, and urine calcium / creatinine ratio.

58. The method of claim 56, wherein the dose of FZD receptor polypeptide is changed based on a patient’s change in BMD.

59. A therapeutically effective amount of a frizzled (FZD) receptor polypeptide, for use in a method of therapy, wherein the method of therapy comprises the method of any one of claims 1-58.

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