Compositions and methods for treating bone loss

Abstract

GREM2 compositions and formulations thereof for treating or preventing one or more symptoms of a disease associated with loss of bone mineral density are described. Recombinant GREM2 has been demonstrated to be capable of reducing bone resorption, lowering bone transition markers, and increasing bone strength. Methods of using the GREM2 compositions and formulations are also provided. Preferably, the pharmaceutical formulation containing GREM2 is administered by a parenteral route to prevent or alleviate one or more symptoms of osteoporosis, bone mass reduction, bone fracture and / or bone cancer.

Description

TECHNICAL FIELD

[0001] The disclosed invention relates generally to compositions and methods for treating conditions and diseases associated with bone loss. BACKGROUND

[0002] Osteoporosis is a common bone disease characterized by low bone mineral density (BMD), deterioration of bone microarchitecture, and susceptibility to fractures (Sözen, T. et al., Eur J Rheumatol, 4(1): 46-56, (2017)). According to the World Health Organization, osteoporosis is defined as a BMD t-score equal to or less than -2.5. It is estimated that more than 200 million people worldwide have osteoporosis, while it is estimated that more than 40% of the population present with low bone mass, i.e., osteopenia (Sözen, T. et al., Eur J Rheumatol, 4(1): 46-56, (2017); Varacallo M et al., StatPearls, (2022)). Given that osteoporosis is an age-related condition, the prevalence of osteoporosis is expected to continue to increase as the aging population expands worldwide.

[0003] Currently, different pharmacological agents are available for the treatment of osteoporosis. For example, first-line drugs for the treatment of osteoporosis or osteopenia include bisphosphonates, such as alendronate and zoledronate. However, long-term use of these drugs can lead to several adverse effects, such as atypical fractures and osteonecrosis of the jaw (ONJ) (Salari, P. and M. Abdollahi, J Pharm Sci, 2012. 15 (2): 305-17). In addition to bisphosphonates, denosumab is a commonly used agent for the treatment of osteoporosis. However, atypical fractures and ONJ are also known to be associated with long-term use of denosumab. Anabolic agents teriparatide, abaloparatide, and romosozumab are potentially associated with serious drug adverse effects, such as osteosarcoma and adverse cardiac events. Therefore, there is an urgent need for new drugs to safely and effectively treat these diseases.

[0004] It is an object of the present invention to provide compositions for the treatment and / or prevention of bone loss.

[0005] It is another object of the present invention to provide methods for the treatment and / or prevention of bone loss in a subject. SUMMARY

[0006] The secreted BMP antagonist Gremlin-2 (GREM2) has been found to be a negative regulator of osteoclastogenesis and bone resorption. In addition, GREM2 has been found to be associated with preservation of bone mineral density and reduction of markers of bone loss in vivo.

[0007] Compositions and formulations containing soluble GREM2 protein or nucleic acids encoding soluble protein are disclosed. The compositions and formulations are suitable for use in treating a disease, disorder, or condition associated with bone loss. In some forms, the composition comprises one or more soluble proteins containing all or a functional portion of amino acids 22 to 168 of a Gremlin-2 (GREM2) protein or a functional variant thereof. In some forms, the composition has an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a functional fragment or variant thereof. For example, in some forms, the GREM2 protein has about 70% to about 99% sequence similarity to SEQ ID NO: 1 or SEQ ID NO: 2. In some forms, the GREM2 protein has about 70%, 75%, 80%, 85%, 90%, 92.5%, 95%, 99% sequence similarity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] In some forms, the soluble protein contains a functional portion of amino acids 22 to 168 of a Gremlin-2 (GREM2) protein or a variant thereof. In some forms, the functional activity of the GREM2 variant is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100%, such as 105%, 110%, or more, of the GREM2 protein set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Generally, a variant of either of SEQ ID NO: 1 and SEQ ID NO: 2 is considered to have a function of directly or indirectly preventing or reducing bone loss in a subject in need thereof. For example, in some forms, the GREM2 variant has a function of increasing bone mineral density and reducing levels of bone turnover markers, such as Ctx and NTx markers.

[0009] In some forms, the functional portion or functional variant contains the antagonism of BMP2 by the native GREM2 protein. In some forms, a variant of any one of SEQ ID NO: 1 and SEQ ID NO: 2 can have the function of directly or indirectly reducing BMP2 activity in a cell if the variant has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100% (such as 105%, 110% or more, etc.) of the functional effect of the non-variant or native sequence from which it is derived. The variant of GREM2 should generally retain the consensus sequence CX6QX6CX6NX2CXGXCXSX3PX (8-13) CX2CXPX8TLXCX (15-18) CXC (SEQ ID NO: 4). Functional GREM2 variants can retain the ability to bind bone morphogenetic protein 2 (BMP-2). Such binding is the basis for the BMP antagonism of GREM2 (Hsu et al., Molecular Cell 1 :673-684 (1998)).

[0010] In some forms, the functional portion or functional variant of a GREM2 protein prevents or reduces bone loss by modulating the function of one or more other proteins. For example, in some forms, a variant of any one of SEQ ID NO: 1 and SEQ ID NO: 2 can have the function of directly or indirectly modulating the activity of one or more other proteins in a cell if the variant has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100% (such as 105%, 110% or more, etc.) of the functional effect of the non-variant or native sequence from which it is derived. Exemplary proteins whose function can be modified by GREM2 proteins and variants thereof include, but are not limited to, GDF8 (growth differentiation factor-8), TGF-b (transforming growth factor beta), and SMADs (mothers against decapentaplegic suppressor), such as SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, and / or SMAD8, etc.

[0011] In some forms, the composition comprises one or more isolated nucleic acids encoding a soluble protein containing a functional GREM2 protein or functional variant thereof as disclosed herein, preferably in a vector for delivery and expression in a cell, preferably a mammalian cell. Increasing GREM2 expression is effective to increase bone mineral density and reduce bone turnover marker (e.g., Ctx and NTx marker) levels.

[0012] Pharmaceutical formulations comprising a GREM2 protein or nucleic acid and a pharmaceutically acceptable carrier are also provided. In some forms, the pharmaceutical is lyophilized, or is in liquid or powder form. Kits comprising a peptide are also described. Typically, the kits comprise one or more single unit doses of the peptide and instructions for administering the dose to treat one or more symptoms associated with bone loss in a subject.

[0013] Methods of using the disclosed compositions and formulations, including but not limited to GREM2 proteins, nucleic acids, and small molecules, are provided. Therapeutic methods comprising cells and other therapeutic agents, including GREM2 peptides, nucleic acids, and / or small molecules, are described. Generally, the methods involve treating a subject (e.g., a human) having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition comprising a genetically modified GREM2 peptide, nucleic acid, and / or small molecule. In some embodiments, the methods administer to a subject (e.g., a human) having a disease, disorder, or condition a pharmaceutical composition expressing a recombinant GREM2 protein in an amount effective to treat the disease, disorder, or condition. For example, in some embodiments, the methods treat a disease or disorder associated with elevated BMP expression by administering to a subject an effective amount of a pharmaceutical composition comprising a recombinant GREM2 peptide and / or protein.

[0014] In some forms, the diseases and / or disorders to be treated include, but are not limited to, pre-osteoporosis, osteoporosis, osteopenia, bone fracture, and bone cancer. The disclosed compositions and methods can also be used to treat bone loss associated with various diseases. Such diseases include, for example, rheumatoid arthritis, type 1 diabetes, hyperthyroidism, hyperparathyroidism, celiac sprue, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis, among others). Particular conditions that can be treated using the disclosed compositions and methods include dysplasias in which bone growth or bone development is abnormal, as well as osteopenia, osteoporosis, and bone loss of various causes.Representative examples of such disorders include chondrodysplasia, craniometaphyseal dysplasia, enchondromatosis, fibrous dysplasia, osteopetrosis, hypophosphatemic rickets, Marfan syndrome, multiple hereditary exostoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteosclerosis, pseudogout, and pyogenic osteomyelitis, periodontal disease, antiepileptic drug-induced bone loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism syndrome, weightlessness-induced bone loss, male osteoporosis, postmenopausal bone loss, osteoarthritis, renal osteodystrophy, infiltrative disorders of bone, oral bone loss, osteonecrosis of the jaw, juvenile Paget's disease, melorheumatosis, metabolic bone disease, mastocytosis, sickle cell anemia / sickling disease, organ transplant-related bone loss, kidney transplant-related bone loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis, thalassemia, mucopolysaccharidosis, Fabry disease, Turner syndrome, Down syndrome, Cushing's syndrome, leprosy, Perthes disease, adolescent idiopathic scoliosis, infantile onset multisystem inflammatory disease, Winchester syndrome, Menkes disease, Wilson's disease, ischemic bone disease (such as Legg-Calve-Perthes disease, regional migratory osteoporosis), anemic states, steroid-induced disorders, glucocorticoid-induced bone loss, heparin-induced bone loss, bone marrow disorders, scurvy, malnutrition, calcium deficiency, idiopathic or congenital osteopenia or osteoporosis, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory disorders, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, menorrhagia, amenorrhea, pregnancy, diabetes, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly, hypogonadism, immobilization or disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis, chondropathy, joint replacement-related bone loss, HIV-related bone loss, growth hormone deficiency-related bone loss, cystic fibrosis-related bone loss, fibrous dysplasia, chemotherapy-related bone loss, tumor-induced bone loss, cancer-related bone loss, hormone ablative bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa, facial bone loss-related diseases, skull bone loss-related diseases, jaw bone loss-related diseases, head bone loss-related diseases, and space travel-related bone loss. Further disorders involve aging-related bone loss, including aging-related facial bone loss, aging-related skull bone loss, aging-related jaw bone loss, and aging-related head bone loss.

[0015] In some forms, the GREM2 compositions and formulations are administered to a subject in need thereof to reduce one or more symptoms associated with bone loss, e.g., reduced bone mineral density, reduced bone strength, and reduced bone volume / tibia volume ratio (BV / TV). In some forms, the disease, disorder, or condition can be associated with elevated or specific expression of a protein, e.g., a bone morphogenetic protein. For example, the GREM2 compositions can be administered to a subject in need thereof to reduce expression of one or more bone turnover markers.

[0016] Additional advantages of the disclosed methods and compositions will be set forth in part in the description that follows, and in part will be obvious from the description, or can be learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and attained by the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application, as claimed. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and together with the description, serve to explain the principles of the disclosed methods and compositions.

[0018] Figure 1A and 1B shows TRAP staining of osteoclasts using different concentrations of GREM2. Figure 1A are photomicrographs of representative TRAP staining results of different concentrations of GREM2 treatment. Figure 1B is a scatter plot showing the quantification of TRAP staining of RAW264.7 treated with or without GREM2. ** p < 0.01, *** p < 0.001, Student’s t test compared to control. All values are given as mean ± SEM.

[0019] Figure 2A and 2B shows results of the calcium phosphate surface resorption assay. Figure 2A are photomicrographs of the osteoclast resorption assay of RAW264.7 treated with no rankl, rankl, and both rankl and GREM2. Figure 2B is a bar graph of total resorption area (%).

[0020] Figure 3A and 3B is a scatter plot showing BMD of the GREM2 group and control group of healthy mice 4 weeks post-treatment ( Figure 3A ) and bone volume to tibia volume ratio (BV / TV) scatter plot ( Figure 3B ) of healthy mice 4 weeks post-treatment. In Figure 3AIn particular, the difference between the GREM2 group and the control group was ~14% in Figure 3B In particular, the difference was ~28%.

[0021] Figure 4A and 4B are scatter plots of BMD (bone mineral density) ( Figure 4A ) and BV / TV (bone volume / tissue volume) ( Figure 4B ) for the GREM2 group and the control group of ovariectomized (OVX) mice at 4 weeks post-treatment. DETAILED DESCRIPTION

[0022] The compositions and methods disclosed herein are based on the identification of Gremlin-2 (GREM2), an antagonist of bone morphogenetic proteins (BMPs), as a negative regulator of osteoclastogenesis and bone resorption. Gremlin 2 (GREM2) is a member of the DAN family of bone morphogenetic protein (BMP) antagonists and is a secreted BMP antagonist (Cheung et al., The Journal of Clinical Endocrinology & Metabolism, 2013. 98(9):E1557-E1561). In a gene expression profiling study, GREM2 was one of six genes that were significantly upregulated at least 2-fold during osteoblast differentiation in MC3T3 cells (Zamurovic, N. et al., J Biol Chem, 2004. 279(36):37704-15). The other five genes were: TGF-β1 (transforming growth factor-beta 1), TGF-β3 (transforming growth factor-beta 3), PDGF (alpha platelet-derived growth factor, alpha), IGFBP10 (insulin-like growth factor binding protein 10), and activin (inhibin beta-A). These other genes are other members of the TGF-beta superfamily that are involved in the BMP signaling pathway but are not BMP antagonists.

[0023] GREM2 expression increases during early osteoblast differentiation and remains unchanged throughout all other stages of bone differentiation and mineralization (Ideno, H. et al., Experimental Cell Research, 315(3): 474-484 (2009)). Despite the increase in GREM2 expression during osteoblast differentiation, GREM2 overexpression inhibited bone differentiation in murine preosteoblasts and human bone marrow-derived mesenchymal stem cells (Wang et al., J Cell Biochem, 118(2): 286-297 (2017)).

[0024] Mechanism studies in non-limiting examples show that increased expression of GREM2 is positively correlated with increased bone mineral density (BMD) and that GREM2 is a negative regulator of osteoclastogenesis and bone resorption. In addition, examples demonstrate that serum levels of GREM2 are correlated with increased BMD and decreased levels of bone turnover markers in humans. Finally, examples demonstrate that GREM2 treatment increases BMD and bone volume to tibia volume ratio (BV / TV) in both healthy mice and ovariectomized mice. Accordingly, compositions and formulations containing GREM2 are disclosed. The compositions are effective for treating and / or preventing conditions and diseases associated with bone loss, such as osteoporosis and osteopenia.

[0025] I. DEFINITIONS

[0026] As used herein, the term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide, RNA (e.g., including but not limited to mRNA, tRNA, and rRNA), or a precursor. The polypeptide, RNA, or precursor can be encoded by a full-length coding sequence, or by any portion thereof. The term also encompasses the coding region of a structural gene, and plus and minus

[0027] As used herein, "mammal" includes both human and non-human, and includes, but is not limited to, humans, non-human primates, canids, felines, murines, bovids, equids, and suids.

[0028] As used herein, a "vector" is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted and propagated. The vectors described herein can be expression vectors.

[0029] As used herein, an "expression vector" is a vector that includes one or more expression control sequences.

[0030] As used herein, an "expression control sequence" is a DNA sequence that controls and regulates transcription and / or translation of another DNA sequence.

[0031] As used herein, the term "treatment" includes alleviating symptoms associated with a particular disorder or condition and / or preventing or eliminating the symptoms.

[0032] "Operably linked" refers to a juxtaposition wherein the components so described are configured to perform their usual function. For example, a control sequence or promoter operably linked to a coding sequence is capable of influencing the expression of the coding sequence, and an organelle localization sequence operably linked to a protein will direct the localization of the attached protein to a particular organelle.

[0033] As used herein, "transformation" and "transfection" include the introduction of a nucleic acid (e.g., a vector) into a cell by a variety of techniques known in the art.

[0034] "Effective amount" and "therapeutically effective amount" are used interchangeably, when applied to nanoparticles, therapeutic agents, and pharmaceutical compositions described herein, to mean the amount necessary to effect a desirable therapeutic result. For example, an effective amount is a level effective to treat, cure, or reduce symptoms of a disease for which the composition and / or therapeutic agent or pharmaceutical composition is administered.

[0035] The term "pharmaceutically acceptable" or "biologically compatible" means, within the scope of sound medical judgment, compositions, polymers and other materials and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, and the like. The phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, solvent or encapsulating material, involved in carrying or transporting any subject composition from one organ, or portion of the body, to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not injurious to the patient. The term "pharmaceutically acceptable salt" is art-recognized and includes relatively non-toxic, inorganic and organic acid addition salts of the compounds. Examples of pharmaceutically acceptable salts include salts derived from inorganic acids such as hydrochloric and sulfuric acids, and the like, and salts derived from organic acids such as ethanesulfonic, benzenesulfonic, and p-toluenesulfonic acids, and the like. Examples of suitable inorganic bases for salt formation include ammonium, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc hydroxides, carbonates, and bicarbonates. Salts can also be formed with suitable organic bases including non-toxic and strong enough to form such salts. For illustrative purposes, the class of such organic bases can include mono-, di-, and trialkylamines such as methylamine, dimethylamine, and trimethylamine, and the like; mono-, di-, or trihydroxyalkylamines such as mono-, di-, and triethanolamine; amino acids such as arginine and lysine, and the like; guanidine; N-methylglucamine; N-methylreducoglucamine; L-glutamine; N-methylpiperizine; morpholine; ethylenediamine; N-benzylphenethylamine; and the like.

[0036] The term "pharmaceutically acceptable salt" is art-recognized and includes relatively non-toxic, inorganic and organic acid addition salts of the compounds. Examples of pharmaceutically acceptable salts include salts derived from inorganic acids such as hydrochloric and sulfuric acids, and the like, and salts derived from organic acids such as ethanesulfonic, benzenesulfonic, and p-toluenesulfonic acids, and the like. Examples of suitable inorganic bases for salt formation include ammonium, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc hydroxides, carbonates, and bicarbonates. Salts can also be formed with suitable organic bases including non-toxic and strong enough to form such salts. For illustrative purposes, the class of such organic bases can include mono-, di-, and trialkylamines such as methylamine, dimethylamine, and trimethylamine, and the like; mono-, di-, or trihydroxyalkylamines such as mono-, di-, and triethanolamine; amino acids such as arginine and lysine, and the like; guanidine; N-methylglucamine; N-methylreducoglucamine; L-glutamine; N-methylpiperizine; morpholine; ethylenediamine; N-benzylphenethylamine; and the like.

[0037] The term "inhibit" or "reduce" in the context of inhibition means a decrease or reduction in activity and amount. This can be a complete inhibition or reduction, or a partial inhibition or reduction, of activity or amount. Inhibition or reduction can be in comparison to a control or standard level. Inhibition can be measured as a % value, e.g., from 1% up to 100%, such as 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. For example, a composition comprising a therapeutic agent can inhibit or reduce one or more markers of a disease or disorder in a subject by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% compared to the activity and / or amount of the same marker in a subject who has not received the composition or who has not been treated with the composition. In some forms, inhibition and reduction can be in comparison to levels of mRNA, protein, cells, tissues, and organs.

[0038] The term "treat" or "delay development" in the context of a disease or disorder means to alleviate, reduce, or otherwise prevent the disease, disorder, or condition from occurring or progressing in an animal that can be predisposed to the disease, disorder, and / or condition but has not yet been diagnosed with the disease; to inhibit the disease, disorder, or condition, e.g., to impede its progress; and to relieve the disease, disorder, or condition, e.g., to cause regression of the disease, disorder, and / or condition. Treating a disease or condition includes improving at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating pain in a subject with an analgesic, even if such agent cannot treat the cause of the pain. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or improving the disease state, and remission and improving the prognosis. A subject is successfully "treated" if one or more symptoms associated with a coronavirus infection are reduced or eliminated, including but not limited to, reducing and / or inhibiting syncytia formation and lung injury, improving the quality of life of those afflicted, reducing the dose of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging the life of an individual.

[0039] The term "biodegradable" generally refers to a material that will degrade or erode under physiological conditions into smaller units or chemical species that can be metabolized, eliminated, or excreted by the subject. The time of degradation is a function of composition and morphology.

[0040] The term "fragment" means a portion of a polypeptide or nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0041] The terms "isolated," "purified," or "biologically pure" mean a material that is not contained in its natural state in a different degree. "Isolated" refers to a degree of separation from natural sources or the surrounding environment. "Purified" refers to a higher degree of separation than isolated.

[0042] "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications yet retains a substantial property. A typical polypeptide variant differs in amino acid sequence from another reference polypeptide. Generally, the differences are limited so that the reference polypeptide and the variant are substantially similar overall, and identical in many regions. The variant and reference polypeptide can differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). The substituted or inserted amino acid residues can or can not be naturally encoded by the genetic code. Variants of a polypeptide can be naturally occurring, such as allelic variants, or they can be variants that are not found in nature. The term "polypeptide" includes proteins and fragments thereof. "Protein" or "polypeptide" or "peptide" means any chain of more than two natural or non-natural amino acids that make up all or a portion of a naturally occurring or non-naturally occurring polypeptide or peptide, whether or not there are post-translational modifications (e.g., glycosylation or phosphorylation), as described herein. Polypeptides are disclosed herein as sequences of amino acid residues. Those sequences are written from left to right in the direction from the amino to the carboxyl terminal end. According to standard naming conventions, amino acid residue sequences are named by either the three-letter code or the one-letter code, as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0043] As used herein, a protein is "soluble" when the protein lacks any transmembrane domains or protein domains that anchor or integrate the polypeptide into a membrane in which such polypeptide is expressed. Soluble proteins lack additional hydrophobic sequences and are translocated by a translocator (e.g., a protein channel) to complete their folding and modification in the lumen of the endoplasmic reticulum.

[0044] The term "dosage regimen" refers to drug administration with respect to formulation, route of administration, drug dosage, dosing interval, and duration of treatment.

[0045] Unless otherwise indicated herein, the recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated in the specification as if it were individually recited herein. Unless otherwise indicated herein, the use of the alternative (e.g., "or", "either") is intended to be the inclusive, and not the exclusive use (e.g., "or" is intended to mean any of the items listed or any combination of the items listed). Unless otherwise indicated herein, the use of the term "including" is intended to be the inclusive, and not the exclusive use (e.g., "including" is intended to mean that there are additional items that are not listed).

[0046] II. Compositions

[0047] Compositions that increase GREM2 expression are provided for use in the disclosed methods. The compositions generally comprise one or more GREM2 proteins or nucleic acids or small molecules that encode GREM2 or increase its expression. In some forms, the compositions comprise a soluble protein comprising all or a functional portion of amino acids 22 to 168 of Gremlin-2 (GREM2) protein or a functional variant thereof. In some forms, the compositions comprise a nucleic acid encoding a soluble protein comprising all or a functional portion of amino acids 22 to 168 of Gremlin-2 (GREM2) protein. The compositions are effective to intervene or inhibit the physiological action of bone morphogenetic protein-2 (BMP2).

[0048] A. GREM2 Proteins

[0049] Compositions of GREM2 proteins are provided for administration to a subject. The human Gremlin-2 (GREM2) amino acid sequence (UniProtKB - Q9H772_HUMAN) is as follows:

[0050] MFWKLSLSLFLVAVLVKVAEARKNRPAGAIPSPYKDGSSNNSERWQHQIKEVLASSQEALVVTERKYLKSDWCKTQPLRQTVSEEGCRSRTILNRFCYGQCNSFYIPRHVKKEEESFQSCAFCKPQRVTSVLVELECPGLDPPFRLKKIQKVKQCRCMSVNLSDSDKQ (SEQ ID NO: 1).

[0051] In general, the composition contains soluble proteins, wherein the soluble proteins comprise one or more GREM2 polypeptides. In some forms, the one or more GREM2 polypeptides include any naturally occurring polypeptide of a DAN (differentially screened, selectively expressed, neural crest abnormality) family member and any variants thereof (variants, fragments, etc.) that retain useful activity (e.g., fusion proteins and peptidomimetic forms). For example, a GREM2 polypeptide has a sequence that is at least about 70% identical to the sequence of a GREM2 polypeptide, e.g., has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% sequence similarity. Polypeptides having less than 70% and more than 99% identity derived from any known GREM2 sequence are included. Preferably, the GREM2 protein prevents or reduces bone loss. Also preferably, the GREM2 protein promotes bone growth and bone mineralization. In some forms, the GREM2 polypeptide can antagonize the function of BMP2 directly or indirectly, e.g., by binding to BMP2 or by binding to a BMP receptor. Examples of GREM2 polypeptides include human Gremlin-2 (GREM2) (SEQ ID NO: 1) and soluble proteins containing functional fragments of Gremlin-2 protein, e.g., amino acids 22 to 168 of Gremlin-2 (GREM2) protein (SEQ ID NO: 2).

[0052] In some forms, the soluble protein comprises a GREM2 protein variant. In some forms, the composition comprises a GREM2 variant having about 70% to about 99% sequence similarity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some forms, the composition comprises a GREM2 variant that is at least about 70%, 75%, 80%, 85%, 90%, up to 95% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. A typical polypeptide variant differs in amino acid sequence from another, reference polypeptide. Generally, the differences are limited so that the reference polypeptide and the variant are closely similar in overall sequence and in many regions are identical. The variant and the reference polypeptide can differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). The substituted or inserted amino acid residues can or can not be those encoded by the genetic code. Variants of a polypeptide can be naturally occurring, such as allelic variants, or they can be variants that are not found in nature.

[0053] Modifications and alterations of the polypeptide constructs disclosed herein can be made and still obtain a molecule with similar characteristics to the polypeptide (e.g., conservative amino acid substitutions). For example, certain amino acids within a sequence can be substituted for other amino acids without appreciable loss of activity. Since it is the functional properties of a polypeptide that are of ultimate concern, certain amino acid sequences can be substituted for other amino acids in a polypeptide sequence, and the resulting polypeptide can still obtain similar properties.

[0054] In making such changes, the hydropathic indices of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is understood in the art. It is accepted that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0055] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, and antigens, among others. It is known in the art that the amino acids can be substituted for other amino acids in such a way as to obtain a functionally equivalent polypeptide. In making such changes, amino acids with hydropathic indices of +2 or less are substituted for other amino acids with hydropathic indices of +2 or less, and those with hydropathic indices of +1 or less are particularly preferred, and those with hydropathic indices of +0.5 or less are even more particularly preferred.

[0056] Substitutions of like amino acids can also be made on the basis of hydrophilicity, particularly where the biologically functional equivalent polypeptide or peptide thereby created is intended for immunological use. The following amino acid residues are designated as hydrophilic: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically functional equivalent, particularly an immunologically functional equivalent polypeptide. In such changes, amino acids having a hydrophilicity value of ±2 are preferably substituted for one another, amino acids having a hydrophilicity value of ±1 are particularly preferred, and those having a hydrophilicity value of ±0.5 are even more particularly preferred.

[0057] Amino acid substitutions are typically based on the relative similarity of the code- sub stituent amino acid side chains, for example, their hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions that take various of the foregoing characteristics into consideration are well-known to those of skill in the art, and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Val), (Leu: He, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: He, Leu). Polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to a polypeptide of interest.

[0058] "Identity" and "similarity" can be readily calculated by known methods, such as those described in: Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo and Lipman, SIAM J Applied Math, 48: 1073 (1988).

[0059] The preferred method for determining identity is designed to give the largest match between the test sequence and the reference sequence. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined using analysis software (i.e., the sequence analysis software package of the Genetics Computer Group, University of Wisconsin, Madison, Wis.) that incorporates the algorithm of Needelman and Wunsch (J. Mol. Biol., 48: 443-453, 1970) (e.g., NBLAST, and XBLAST). Default parameters are used to determine the identity of the polypeptides of the disclosure.

[0060] For example, a polypeptide sequence can be identical, i.e., 100% identical, to a reference sequence, or it can include up to a certain integer number of amino acid alterations as compared to the reference sequence, such that the identity is less than 100%. Such alterations are determined by comparing the reference polypeptide sequence to the polypeptide sequence whose identity is being determined, and are typically made up of at least one amino acid deletion, substitution (including conservative and non-conservative substitutions), or insertion, where the alteration can occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence, or anywhere between those terminal positions, or individual alterations can occur at scattered locations throughout the reference sequence, or in one or more contiguous groups within the reference sequence. For a given identity, the number of amino acid alterations is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percentage value of the corresponding percentage of identity (divided by 100), and then subtracting that product from the total number of amino acids in the reference polypeptide.

[0061] In some forms, the GREM2 protein is a recombinant protein derived from GREM2 or a fragment of GREM2. SEQ ID NO: 2 is an exemplary recombinant GREM2 protein:

[0062] RKNRPAGAIPSPYKDGSSNNSERWQHQIKEVLASSQEALVVTERKYLKSDWCKTQPLRQ TVSEEGCRSRTILNRFCYGQCNSFYIPRHVKKEEESFQSCAFCKPQRVTSVLVELECPGLDPPFRLKKIQKVKQCRCMSVNLSDSDKQ (SEQ ID NO: 2)

[0063] In some forms, the functional activity of the GREM2 variant is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100%, such as 105%, 110%, or more, of the functional activity of the GREM2 protein of SEQ ID NO: 1 or SEQ ID NO: 2. In general, variants of either of SEQ ID NO: 1 and SEQ ID NO: 2 are considered to have the function of directly or indirectly preventing or reducing bone loss in a subject in need thereof. For example, in some forms, the GREM2 variant has the function of increasing bone mineral density and reducing levels of bone turnover markers, such as CTx and NTx markers.

[0064] In some forms, the soluble protein contains a functional portion of amino acids 22 to 168 of a Gremlin-2 (GREM2) protein or a variant thereof. In some forms, the functional portion or functional variant contains the antagonistic effect of the native GREM2 protein on BMP2. In some forms, a variant of any one of SEQ ID NO: 1 and SEQ ID NO: 2 is considered to have a function to directly or indirectly reduce BMP2 activity in a cell if it has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100% (such as 105%, 110% or more, etc.) of the functional effect of the non-variant or native sequence from which it is derived. Variants of GREM2 should generally retain the consensus sequence CX6QX6CX6NX2CXGXCXSX3PX (8-13) CX2CXPX8TLXCX (15-18) CXC (SEQ ID NO: 4). Functional GREM2 variants can bind to bone morphogenetic protein 2 (BMP-2). Such binding is the basis for the BMP antagonistic effect of GREM2 (Hsu et al., Molecular Cell 1 :673-684 (1998)).

[0065] In some forms, the functional portion or functional variant of a GREM2 protein prevents or reduces bone loss by modulating the function of one or more other proteins. For example, in some forms, a variant of any one of SEQ ID NO: 1 and SEQ ID NO: 2 can have a function to directly or indirectly modulate the activity of one or more other proteins in a cell if it has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more than 100% (such as 105%, 110% or more, etc.) of the functional effect of the non-variant or native sequence from which it is derived. Exemplary proteins whose function can be modified by GREM2 proteins and variants thereof include, but are not limited to, GDF8 (growth differentiation factor-8), TGF-β (transforming growth factor beta), and SMADs (mothers against decapentaplegic inhibitors), such as SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, and / or SMAD8, etc.

[0066] As used herein, “native protein” refers broadly to a molecule having an amino acid sequence that can be isolated from an organism and that has not been modified by recombinant DNA technology or other methods. Thus, “native protein” includes naturally occurring alleles and variants of the protein.

[0067] Typically, GREM2 protein variants include molecules or sequences that are modified from the native protein but still retain the pharmacological activity of interest. Thus, the term "protein variant" includes molecules or sequences in which a non-native residue replaces a native residue, a non-native residue is added, or a non-native residue is deleted. A native residue can be removed for any reason that the native residue provides a structural feature or biological activity that is not required for the pharmacological activity of interest of the fusion molecule of the application. Thus, the term "protein variant" includes molecules or sequences that lack one or more native protein sites or residues that affect or participate in any number of cellular processes, including but not limited to: (1) intracellular signaling, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with other proteins (e.g., dimerization domains), (6) binding to receptors or other proteins that do not affect the pharmacological activity of interest, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0068] Functionally active fragments of a GREM2 protein can be obtained by screening recombinantly produced polypeptides from corresponding fragments of nucleic acids encoding a GREM2 protein. In addition, fragments can be chemically synthesized using techniques known in the art, such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. Fragments can be prepared (recombinantly or by chemical synthesis) and tested to identify such peptide fragments that can function as antagonists (inhibitors) of BMP-mediated signaling.

[0069] Functional variants can be produced by modifying the structure of a GREM2 protein for purposes such as enhancing therapeutic efficacy or stability (e.g., ex vivo shelf life and resistance to proteolysis in vivo). Such modified GREM2 proteins are considered functional equivalents of the naturally occurring GREM2 protein if they retain BMP antagonistic effects. Modified GREM2 proteins can also be prepared by, for example, amino acid substitution, deletion, or addition. For example, a leucine single substitution to isoleucine or valine, an aspartic acid single substitution to glutamic acid, a threonine single substitution to serine, or an amino acid similar substitution to a structurally related amino acid (e.g., a conservative substitution). There is reason to expect that the (mutation) will not significantly affect the biological activity of the resulting molecule. A conservative substitution is a substitution occurring within a family of amino acids that are related in structure by having in common polar or nonpolar, charged or uncharged, hydrophilic or hydrophobic properties. Whether a change in the amino acid sequence of a GREM2 protein will result in a functional equivalent can be readily determined by assessing the ability of the variant GREM2 protein to produce a response in a similar manner to the wild-type GREM2 protein.

[0070] Exemplary variants include one or more peptides having an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to one or more of the corresponding amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 2. For example, a variant can include one or more peptides having an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to one or more of the corresponding amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2. In some forms, a variant includes one or more peptides having an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 1. In other forms, a variant includes one or more peptides having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1. In other forms, a variant includes one or more peptides having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2.

[0071] The disclosed soluble proteins containing a GREM2 protein or functional variant thereof can have a length of up to 150, 200, 250, 300, 400, 500, 1000, or 2000 residues. In particular forms, the soluble protein can have a length of at least 50, 60, 70, 80, 90, 100, 120, or 140 residues. In further embodiments, the soluble protein can have a length of 50 to 200 residues, 50 to 100 residues, 50 to 90 residues, 50 to 80 residues, 50 to 70 residues, or 50 to 60 residues. As used herein, the term "residue" refers to an amino acid or an amino acid analog.

[0072] B. Nucleic Acids

[0073] The disclosed compositions can include an isolated nucleic acid encoding any of the GREM2 proteins, e.g., soluble GREM2 proteins including fragments and functional variants disclosed herein. In some forms, the nucleic acid is a recombinant nucleic acid. In some forms, the nucleic acid can be single-stranded or double-stranded. Such nucleic acids can be DNA molecules or RNA molecules. These nucleic acids can be used, e.g., in methods of making GREM2 proteins or as direct therapeutic agents (e.g., in gene therapy methods).

[0074] In some forms, the nucleic acid encodes a GREM2 protein or functional fragment thereof. Increased expression of GREM2 effectively antagonizes BMP2 activity, thereby increasing bone mineral density and reducing levels of bone turnover markers (e.g., CTx and NTx markers).

[0075] The GPRM2 nucleic acid sequence contains 4176 bp, as detailed below (NCBI Reference Number: NM_022469.4):

[0076]

[0077] The term "isolated nucleic acid" refers to a nucleic acid that is separated from other nucleic acid molecules that are present in the mammalian genome, including nucleic acids that normally flank the nucleic acid on one or both sides in the mammalian genome. An isolated nucleic acid can be, for example, a DNA molecule, provided that one of the nucleic acid sequences normally found immediately flanking the DNA molecule in a naturally occurring genome has been removed or deleted. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA, or a genomic DNA fragment produced by PCR or restriction analysis), as well as a recombinant DNA that is incorporated into a vector, a self-replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or is integrated into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid, such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid, and the like. A nucleic acid that is present in a number of hundreds to millions of other nucleic acids, for example, in a cDNA library or a genomic library, or in a gel slice containing a restriction digest of genomic DNA, is not considered to be an isolated nucleic acid.

[0078] In some forms, the nucleic acid encoding a GREM2 protein includes a nucleic acid variant of SEQ ID NO: 3. Variant nucleotide sequences include sequences that differ due to one or more nucleotide substitutions, additions or deletions, such as allelic variants. In some forms, the nucleic acid is about 70% to about 95% identical to SEQ ID NO: 3. In some forms, the nucleic acid is at least 70%, 80%, 85%, 90%, 95% identical to SEQ ID NO: 3. Isolated nucleic acid sequences or recombinant nucleic acid sequences that are 97%, 98%, 99%, or 100% identical are also contemplated.

[0079] In some forms, also described herein are isolated nucleic acids that differ from SEQ ID NO: 3 due to the degeneracy of the genetic code. For example, a number of amino acids are specified by more than one triplet. The substitution of one codon for another codon that specifies the same amino acid, or a synonymous codon (e.g., CAU and CAC are synonymous codons for histidine) can result in "silent" mutations that do not affect the amino acid sequence of the protein. However, it is believed that there are DNA sequence polymorphisms among mammalian cells that result in changes in the amino acid sequence of the protein. Those skilled in the art will recognize that one or more nucleotide variations in a nucleic acid encoding a particular protein, up to about 3% to 5% of the nucleotides, are due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are contemplated.

[0080] The nucleic acids can be in the sense or antisense orientation, or can be complementary to a reference sequence encoding a GREM2 polypeptide or protein. Thus, nucleic acids encoding SEQ ID NO: 1 and SEQ ID NO: 2, fragments and variants thereof, are provided in sense and antisense, and single- and double-stranded forms. The nucleic acids can be DNA, RNA, or nucleic acid analogs. The nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modifications can improve, for example, stability of the nucleic acids, hybridization, or solubility. Modifications at the base moiety can include replacement of deoxythymidine with deoxyuridine, and replacement of deoxycytidine with 5-methyl-2'-deoxycytidine or 5-bromo-2'-deoxycytidine. Modifications at the sugar moiety can include modification of the 2' hydroxyl of ribose to form a 2'-O-methyl or 2'-O-allyl sugar. The deoxyribose phosphate backbone can be modified to yield morpholino nucleic acids in which each base moiety is linked to a six-membered morpholine ring, or peptide nucleic acids in which the deoxyphosphate backbone is replaced with a pseudopeptide backbone and four base moieties are retained. See, e.g., Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7: 187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4: 5-23. In addition, the deoxyphosphate backbone can be replaced with, e.g., a phosphorothioate or phosphorodithioate backbone, phosphoramidite or alkylphosphotriester backbone. In preferred embodiments, the nucleic acid molecule is messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo.

[0081] C. Vectors Expressing or Encoding GREM2 Proteins

[0082] In some embodiments, the nucleic acid encoding a GREM2 protein is present in a vector. The nucleic acid in the vector can be operably linked to one or more expression control sequences. For example, the control sequences can be integrated into the genetic construct such that the expression control sequences effectively control expression of the coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence that consists of a region of DNA usually within 100 nucleotides upstream of the start of transcription (usually near the start site of RNA polymerase II). To place a coding sequence under the control of a promoter, the translational start site of the polypeptide’s translation reading frame must be positioned between one and about fifty nucleotides downstream of the promoter. Hamann et al., J. Biol. Eng., 13:7 (2019) demonstrated that gene expression in hBMSCs driven by the cytomegalovirus (CMV) promoter resulted in 10-fold higher resulting transgene expression than transfection with plasmids containing the elongation factor 1 alpha (EF1a) or Rous sarcoma virus (RSV) promoters.

[0083] Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when positioned at different distances from the transcriptional start site. Enhancers can also be positioned downstream of the transcriptional start site. A coding sequence is “operably linked” and “under the control” of an expression control sequence in a cell when the RNA polymerase is able to transcribe the coding sequence into mRNA, which can then be translated into the protein encoded by the coding sequence.

[0084] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, tobacco mosaic viruses, herpes viruses, cytomegaloviruses, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA). Recent transfection studies investigated mini-circle DNA (mcDNA), a nucleic acid derived from pDNA by recombinationally removing bacterial sequences. mcDNA can be used to introduce L1 RNA into host cells using methods known in the art (Mun et al., Biomaterials, 2016; 101 :310-320).

[0085] Whenever a method comprises combining or contacting compositions or components or reagents, the method is performed to produce a number of different mixtures. For example, if a method comprises 3 mixing steps, if those steps are performed separately, a unique mixture is formed after each of those steps. In addition, regardless of how the steps are performed, a mixture is formed upon completion of all steps. The disclosure encompasses these mixtures obtained by practicing the disclosed methods and mixtures comprising any of the disclosed reagents, compositions, or components, e.g., disclosed herein.

[0086] D. Pharmaceutical Formulations

[0087] The GREM2 compositions described herein can be configured for administration to a subject in need thereof. Generally, the formulation is suitable for directly or indirectly increasing bone mineral density and reducing bone marker turnover in cells by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%. In some forms, the formulation is also suitable for directly or indirectly reducing the activity of BMP2 in cells by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%.

[0088] Exemplary GREM2 composition formulations include liquids and dry powders. In some forms, the amount of GREM2 protein, nucleic acid, or small molecule is about 1 wt% to about 100 wt% (inclusive), about 1 wt% to about 80 wt%, about 1 wt% to about 50 wt%, preferably about 1 wt% to about 40 wt%, more preferably about 1 wt% to about 20 wt%, most preferably about 1 wt% to about 10 wt%. The above ranges include all values from 1% to 100%.

[0089] 1. Parenteral formulations

[0090] The compositions described herein (i.e., vectors encoding GREM2) can be formulated for parenteral administration. For example, parenteral administration can include intravenous, intradermal, intraperitoneal, intramuscular, subcutaneous, by injection, by infusion, etc. to a patient.

[0091] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Generally, such compositions can be prepared as injectables, e.g., solutions or suspensions; solid forms suitable for reconstitution into solutions or suspensions prior to injection; emulsions such as water-in-oil (w / o), oil-in-water (o / w), and microemulsions thereof, liposomes, or emulsomes.

[0092] The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils (such as vegetable oils, e.g., peanut oil, corn oil, sesame oil, etc.)), and combinations thereof. Appropriate flowability can be maintained, for example, by using coatings (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and / or by using surfactants. In many cases, it will be preferable to include an isotonic agent, such as sugar or sodium chloride.

[0093] The active compound can be prepared in water or other solvents or dispersion media as a solution and dispersion of a free acid or base or a pharmaceutically acceptable salt thereof, suitably mixed with one or more pharmaceutically acceptable excipients, including but not limited to surfactants, dispersants, emulsifiers, pH adjusters, viscosity modifiers, and combinations thereof.

[0094] Suitable surfactants can be anionic, cationic, amphoteric, or nonionic. Suitable anionic surfactants include, but are not limited to, surfactants containing carboxylate, sulfonate, and sulfate ions. Examples of anionic surfactants include sodium, potassium, and ammonium salts of long-chain alkyl sulfonic acids and alkyl aryl sulfonic acids, such as sodium dodecylbenzene sulfonate; sodium dialkyl sulfosuccinate, such as sodium dodecylbenzene sulfonate; sodium dialkyl sulfosuccinate, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates, such as sodium dodecyl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds, such as benzalkonium chloride, benzyl chloride, hexadecyltrimethylammonium bromide, stearyl dimethyl benzyl ammonium chloride, polyoxyethylene, and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglycerol-4-oleate, sorbitol acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.

[0095] The formulation may contain preservatives to prevent microbial growth. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain antioxidants to prevent degradation of the active ingredient.

[0096] Formulations are typically buffered to a pH of 3 to 8 for parenteral administration after reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0097] Water-soluble polymers are often used in parenteral administration formulations. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0098] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. A sterile powder for reconstitution can be prepared by vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution. The powder can be reconstituted by the addition of water or another sterile medium just prior to use. Methods for preparing porous particles are well known in the art.

[0099] i. Controlled release formulations

[0100] The parenteral formulations described herein can be formulated for controlled release, including immediate release, delayed release, extended release, pulsatile release, and combinations thereof.

[0101] a. Nanoparticles and microparticles

[0102] For parenteral administration, the GREM2 composition and optionally one or more other active agents can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release of the compound and / or one or more other active agents. In embodiments in which the formulation comprises two or more agents, the agents can be formulated for the same type of controlled release (e.g., delayed release, extended release, immediate release, or pulsatile release), or the agents can be independently formulated for different types of release (e.g., immediate release and delayed release, immediate release and extended release, delayed release and extended release, delayed release and pulsatile release, etc.).

[0103] For example, the GREM2 composition and / or one or more other active agents can be incorporated into polymeric microparticles that provide controlled release of the drug. Release of the agent is controlled by diffusion of the agent from the microparticles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethyl cellulose and other natural or synthetic cellulose derivatives.

[0104] Alternatively, the GREM2 composition can be incorporated into microparticles made from a material that is insoluble or slowly soluble in aqueous solution, but is capable of being degraded within the gastrointestinal tract by means including enzymatic degradation, the surfactant action of bile acids, and / or mechanical erosion. As used herein, the term "slowly soluble in water" refers to a material that is not soluble in water within 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances, and mixtures thereof. Suitable fats and fatty substances include fatty alcohols (such as lauryl, myristyl, stearyl, cetyl, or cetearyl alcohols, etc.), fatty acids and derivatives thereof, including but not limited to fatty acid esters, fatty acid glycerols (mono-, di-, and triglycerides), and hydrogenated fats. Specific examples include, but are not limited to, hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oil available under the trade name Sterotex®, stearic acid, cocoa butter, and stearyl alcohol. Suitable waxes and wax-like materials include natural or synthetic waxes, hydrocarbons, and ordinary waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffin wax, and candelilla wax. As used herein, wax-like materials refer to any material that is generally solid at room temperature and has a melting point of about 30°C to 300°C.

[0105] In some cases, it can be desirable to alter the rate at which water penetrates into the microparticles. To this end, a rate-controlling agent (wicking agent) can be formulated with the fats or waxes listed above. Examples of rate-controlling materials include certain starch derivatives (such as waxy maltodextrin and tumble-dried corn starch), cellulose derivatives (such as hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose, and carboxymethylcellulose), alginic acid, lactose, and talc. In addition, a pharmaceutically acceptable surfactant (such as lecithin) can be added to facilitate degradation of such microparticles.

[0106] Water-insoluble proteins such as zein can also be used as materials to form microparticles containing a pharmaceutical agent. In addition, water-soluble proteins, polysaccharides, and combinations thereof can be formulated with a pharmaceutical agent into microparticles, which are then cross-linked to form an insoluble network. For example, cyclodextrins can be complexed with individual pharmaceutical agent molecules, which are then cross-linked.

[0107] b. Methods of making nanoparticles and microparticles

[0108] The GREM2 compositions can be encapsulated or incorporated into a carrier material by known pharmaceutical formulation techniques to produce medicated microparticles. For formulations of fats, waxes or wax-like materials, the carrier material is typically heated above its melting temperature and the medicament is added to form a mixture comprising medicament particles suspended in the carrier material, medicament dissolved in the carrier material or a mixture of both. The microparticles can then be formulated by a variety of methods including, but not limited to, coacervation, extrusion, spray chilling or water dispersion processes. In a preferred process, the wax is heated above its melting temperature, the medicament is added and the molten wax-medicament mixture is allowed to solidify as the mixture cools with continuous stirring. Alternatively, the molten wax-medicament mixture can be extruded and spheronized to form granules or beads. These processes are known in the art. For certain carrier materials, it can be desirable to use solvent evaporation techniques to produce medicated microparticles. In this case, the medicament and carrier material are co-dissolved in a mutual solvent which can then be used to produce microparticles by a variety of techniques including, but not limited to, forming an emulsion in water or other suitable medium, spray drying or by evaporating the solvent from a bulk solution and milling the resulting material.

[0109] In certain embodiments, the medicament in particulate form is uniformly dispersed in a material that is insoluble or slowly soluble in water. To minimize the size of the medicament particles in the composition, the medicament powder itself can be milled to produce fine particles prior to formulation. Jet milling processes known in the pharmaceutical arts can be used for this purpose. In certain embodiments, the medicament in particulate form is uniformly dispersed in a wax or wax-like substance by heating the wax or wax-like substance above its melting point and adding the medicament particles while stirring the mixture. In this case, a pharmaceutically acceptable surfactant can be added to the mixture to facilitate dispersion of the medicament particles.

[0110] The particles can also be coated with one or more modified release coatings. Solid esters of fatty acids, which are hydrolyzed by lipases, can be sprayed onto the microparticles or medicament particles. Zein is an example of a naturally water-insoluble protein. It can be coated onto the medicated microparticles or medicament particles by spray coating or wet granulation techniques. In addition to naturally water-insoluble materials, some substrates of digestive enzymes can also be treated with a cross-linking procedure to form an insoluble network. A number of methods of cross-linking proteins have been reported that are initiated by chemical and physical means. One of the most common methods to achieve cross-linking is the use of chemical cross-linking agents. Examples of chemical cross-linking agents include aldehydes (glutaraldehyde and formaldehyde), epoxides, carbodiimides, and genipin. In addition to these cross-linking agents, oxidized sugars and natural sugars have also been used for cross-linking of gelatin. Cross-linking can also be achieved using enzymatic methods; for example, transglutaminase has been approved as a GRAS substance for cross-linking of seafood products. Finally, cross-linking can be initiated by physical means such as heat treatment, ultraviolet irradiation, and gamma irradiation, among others.

[0111] To form a cross-linked protein coating around the drug-containing microparticles or drug particles, a water-soluble protein can be sprayed onto the microparticles and then cross-linked by one of the methods described above. Alternatively, the drug-containing microparticles can be microencapsulated within a protein by coacervation (e.g., by addition of a salt) and then cross-linked. Some proteins suitable for this purpose include gelatin, albumin, casein, and gluten.

[0112] Polysaccharides can also be cross-linked to form a water-insoluble network. For many polysaccharides, this can be achieved by reaction with calcium salts or polyvalent cations that cross-link the primary polymer chains. Pectin, alginate, dextran, amylose, and guar gum cross-link in the presence of polyvalent cations. Complexes can also form between oppositely charged polysaccharides; pectin and chitosan, for example, can form complexes through electrostatic interactions.

[0113] 2. Enteric Formulations

[0114] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be produced using compression or molding techniques known in the art. Gelatin or non-gelatin capsules can be produced using techniques known in the art to enclose liquid, solid, and semi-solid fill materials in either hard or soft capsule shells.

[0115] Oral mRNA delivery using capsules is described in Abramson et al., Matter, 5(3):975-987 (2022) (incorporated herein by reference), which uses branched hybrid poly(beta-amino ester) mRNA nanoparticles.

[0116] Formulations can be prepared using pharmaceutically acceptable carriers. “Carrier” as generally used herein includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrants, bulking agents, stabilizers, and combinations thereof.

[0117] Carriers also include all components of a coating composition, which can include plasticizers, pigments, colorants, stabilizers, and glidants.

[0118] Examples of suitable coating materials include, but are not limited to: cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, and hydroxypropyl methyl cellulose acetate succinate; polyvinyl acetate phthalate; acrylic polymers and copolymers; and methacrylic acid resins (marketed under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany)); zein, shellac, and polysaccharides.

[0119] In addition, the coating material can also contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilizers, pore-forming agents, and surfactants, among others.

[0120] "Diluents", also known as "fillers", are generally necessary to increase the bulk of a solid dosage form, in order to provide a practical size for tablet compression or bead and granule formation. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starch, pregelatinized starch, silicon dioxide, titanium dioxide, magnesium aluminometasilicate, and powdered sugar.

[0121] "Binders" are used to impart cohesiveness to a solid dosage formulation, ensuring that the tablet or bead or granule remains intact after the dosage form has been formed. Suitable binder materials include, but are not limited to, starches, pregelatinized starches, gelatin, sugars (including sucrose, glucose, dextrose, lactose, and sorbitol), polyethylene glycols, waxes, natural and synthetic gums (e.g., acacia, tragacanth, sodium alginate), celluloses (including hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose, and magnesium aluminum silicate), and synthetic polymers (e.g., acrylic and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / poly methacrylic acid, and polyvinylpyrrolidone).

[0122] "Lubricants" are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, polyethylene glycol, talc, and mineral oil.

[0123] "Disintegrants" are used to facilitate the disintegration or "break-up" of the dosage form after administration, and generally include, but are not limited to, starches, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, pregelatinized starch, clays, celluloses, alginine, gums, or cross-linked polymers, such as cross-linked PVP (Polyplasdone® XL produced by GAF Chemical Corp.).

[0124] "Stabilizers" are used to inhibit or retard decomposition reactions of the drug, including, for example, oxidation reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; vitamin E, tocopherols and their salts; sulfites (e.g., sodium metabisulfite), cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).

[0125] Oral dosage forms such as capsules, tablets, solutions, and suspensions can be formulated for controlled release. For example, one or more compounds and optionally one or more other active agents can be formulated as nanoparticles, microparticles, and combinations thereof, and encapsulated in soft or hard gelatin or non-gelatin capsules, or dispersed in a dispersion medium to form an oral suspension or syrup. The particles can be formed from the agent and a controlled release polymer or matrix. Alternatively, the agent particles can be coated with one or more controlled release coatings prior to incorporation into the final dosage form.

[0126] In another embodiment, one or more compounds and optionally one or more other active agents are dispersed in a matrix material that gels or emulsifies upon contact with an aqueous medium, such as a physiological fluid. For gels, the matrix swells and coats the active agent, which is released slowly over time by diffusion and / or degradation of the matrix material. Such matrices can be formulated into tablets or as fillers for hard and soft capsules.

[0127] In another embodiment, one or more compounds and optionally one or more other active agents are formulated into a solid oral dosage form, such as a tablet or capsule, and the solid dosage form is coated with one or more controlled release coatings, such as a delayed release coating or extended release coating. One or more layers of the coating can also contain a compound and / or other active agent.

[0128] III. Methods of Treatment

[0129] Methods of using the disclosed compositions and formulations, including but not limited to GREM2 proteins, nucleic acids, and small molecules, are provided.

[0130] Methods of treatment comprising cells and other therapeutic agents, including GREM2 peptides, nucleic acids, and / or small molecules, are described.

[0131] Exemplary methods involve treating a subject (e.g., a human) having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition comprising a genetically modified GREM2 peptide, nucleic acid, and / or small molecule. In some embodiments, the method administers a pharmaceutical composition expressing a recombinant GREM2 protein to a subject (e.g., a human) having a disease, disorder, or condition in an amount effective to treat the disease, disorder, or condition. For example, in some embodiments, the method treats a disease or disorder associated with bone loss by administering to the subject an effective amount of a pharmaceutical composition comprising a recombinant GREM2 peptide and / or protein. In another exemplary form, the method treats a disease or disorder associated with elevated BMP expression by administering to the subject an effective amount of a pharmaceutical composition comprising a recombinant GREM2 peptide and / or protein.

[0132] In some forms, in vivo gene therapy can be employed whereby genetic material encoding a GREM2 peptide and / or a molecule capable of upregulating GREM2 is directly transferred into the patient.

[0133] In these embodiments (i.e., in vivo gene therapy), the genetic material is introduced into the patient by a viral-derived vector or by non-viral techniques. In vivo nucleic acid therapy can be achieved by direct transfer of functionally active DNA into the in vivo somatic tissues or organs of a mammal. Nucleic acids can be administered in vivo by viral means. A therapeutic gene expression cassette typically consists of a promoter driving gene transcription, the transgene of interest, and a termination signal ending gene transcription. Such expression cassettes can be embedded into a plasmid (circularized double-stranded DNA molecule) as a delivery vehicle. Plasmid DNA (pDNA) can be directly injected in vivo by various injection techniques, where hydrodynamic injection achieves the highest gene transfer efficiency in major organs by rapidly injecting a large volume of pDNA solution and transiently inducing pores in the cell membrane. To help the negatively charged pDNA molecules penetrate the hydrophobic cell membrane, chemicals including cationic lipids and cationic polymers have been used to condense pDNA into lipoplexes and polyplexes, respectively.

[0134] A GREM2 peptide or nucleic acid molecule encoding GREM2 can be packaged into a retroviral vector using a packaging cell line that produces replication-defective retroviruses, as is well known in the art. Other viral vectors can also be used, including recombinant adenoviruses and vaccinia viruses, which can be rendered non-replicating. Nucleic acids can also be delivered by other carriers, including liposomes, polymeric microparticles and nanoparticles, and polycations such as desialidated proteins / polylysine, etc. Various techniques and methods for in vivo gene delivery using the disclosed vectors and carriers are known in the art (reviewed in Wang et al., Discov. Med., 18(97):67-77 (2014)). A major advance in DNA vector design is minicircle DNA (mcDNA), which differs from pDNA in lacking a CpG-rich backbone sequence derived from bacteria. When administered in vivo, mcDNA mediates safer, higher, and more durable transgene expression compared to conventional pDNA.

[0135] A. effective amount

[0136] Formulations containing one or more GREM2 proteins or nucleic acids and one or more therapeutic, prophylactic, and / or diagnostic agents generally comprise an effective amount of a mixture of one or more GREM2 proteins or nucleic acids and one or more therapeutic, prophylactic, and / or diagnostic agents. Effective amounts of the combination GREM2 proteins or nucleic acids are provided herein. It will be appreciated that in some forms, an effective amount of one or more GREM2 proteins or nucleic acids and one or more therapeutic, prophylactic, and / or diagnostic agents is different from the amount of the one or more therapeutic, prophylactic, and / or diagnostic agents that would be effective to achieve the same result in the absence of the GREM2 protein or nucleic acid.

[0137] When used to treat bone loss in a subject, the amount of GREM2 protein or nucleic acid present in a pharmaceutical dosage unit or otherwise administered to a subject can be an amount effective to reduce production of bone morphogenetic protein-2 (BMP2), inhibit its activation, or inhibit its signaling. In some forms, the compositions and formulations are suitable for use in reducing the activity of BMP2 in a cell by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, directly or indirectly. In some forms, the amount of GREM2 protein or nucleic acid present in a pharmaceutical dosage unit or otherwise administered to a subject can be an amount effective to increase bone mineral density and / or bone strength. Preferably, the amount of GREM2 protein or nucleic acid present in a pharmaceutical dosage unit is administered in an amount effective to increase bone mineral density by 5% to 15% one year after administration.

[0138] In some forms, the amount of GREM2 protein or nucleic acid present in a pharmaceutical dosage unit or otherwise administered to a subject can be an amount effective to reduce expression of one or more bone turnover markers. Bone turnover biomarkers (BTMs) are byproducts of the bone remodeling process that can be measured in urine or serum and are indicative of the rate of bone turnover. In some forms, the bone turnover marker is a bone resorption marker. Non-limiting examples of bone resorption markers include N-terminal telopeptide of type I collagen (NTX), C-terminal telopeptide of type I collagen (CTX), hydroxyproline, total pyridinoline, free deoxypyridinoline (DPD), bone sialoprotein (BSP), and tartrate-resistant acid phosphatase 5b. In some forms, the bone turnover marker is a bone formation marker. Examples of bone formation markers include, but are not limited to, total alkaline phosphatase, bone-specific alkaline phosphatase, osteocalcin, and type I serum pro-collagen (C-terminal / N-terminal): C1NP or P1NP.

[0139] In some forms, a pharmaceutical composition containing a GREM2 protein or nucleic acid is administered to a subject in need thereof to deliver a GREM2 protein or nucleic acid in an amount of between about 0.1 mg to about 1,000 mg, inclusive, preferably between about 0.5 mg to about 100 mg, inclusive, more preferably between about 1 mg to about 15 mg, inclusive, e.g., 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, and / or 15 mg. The dose can be between about 0.01 mg / kg to about 100 mg / kg, inclusive, preferably between about 0.05 mg / kg to about 10.0 mg / kg, inclusive, more preferably between about 0.1 mg / kg to about 1.5 mg / kg, inclusive, e.g., 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, and / or 1.5 mg / kg.

[0140] B. Dosage Regimens

[0141] A dosage regimen for a pharmaceutical composition containing one or more GREM2 proteins or nucleic acids and, optionally, one or more therapeutic, prophylactic, and / or diagnostic agents can comprise one or more administrations of the pharmaceutical composition.

[0142] In some forms, the pharmaceutical composition is administered at least 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 30 hours, or more than 30 hours, up to 36 or 48 hours, before or after the patient is detected to have a disease or a symptom associated with a disease. In other forms, the pharmaceutical composition is administered at least 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 30 hours, or more than 30 hours, up to 36 or 48 hours, before or after the administration of a separate therapeutic, prophylactic, or diagnostic agent. In certain forms, the additive or more than additive effect of administering a pharmaceutical composition containing one or more GREM2 proteins or nucleic acids in combination with one or more therapeutic and / or prophylactic agents is evident one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, or more than three weeks after administration.

[0143] An effective amount of the pharmaceutical composition and optionally one or more therapeutic and / or prophylactic agents can be administered as a single unit dose (e.g., as a dose unit), or as sub-therapeutic doses administered within a limited time interval. Such unit doses can be administered daily for a limited period of time, such as up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days, or up to 20 days, or up to 25 days are all specifically contemplated. Such unit doses can be administered at other intervals, such as twice a week, three times a week, weekly, every two weeks, or monthly, for up to six months or a year.

[0144] In some forms, the pharmaceutical composition is administered to a subject in need thereof for an unlimited period of time. For example, a subject having a bone loss-related disorder or disease (e.g., osteoporosis) can require treatment for an unlimited period of time to prevent, delay, or slow the onset of and / or reduce the severity of symptoms associated with the disorder or disease. In these forms, the pharmaceutical composition can be administered for a period of time that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, up to and including the lifetime of the subject being treated.

[0145] C. Disease being treated

[0146] Methods of treating a disease and / or disorder in a subject in need thereof are provided. The subject to be treated can have a disease, disorder, or condition such as, but not limited to, pre-osteoporosis, osteoporosis, osteopenia, bone fracture, and bone cancer. The disclosed compositions and methods can also be used to treat bone loss associated with various diseases. Such diseases include, for example, rheumatoid arthritis, type 1 diabetes, hyperthyroidism, hyperparathyroidism, celiac sprue, inflammatory bowel disease (such as Crohn’s disease and ulcerative colitis).

[0147] “Osteoporosis” is defined in the art as a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Any bone can be affected by osteoporosis, although the hip, spine, and wrist are common sites of fracture or break in subjects having or at risk for osteoporosis. Osteoporosis is defined as a bone mineral density (BMD) value more than 2.5 SD below the mean for young adults, i.e., a T-score of -2.5 SD. Severe osteoporosis (established osteoporosis) uses the same threshold, but with one or more prior fragility fractures. The preferred site for BMD measurement for diagnostic purposes is at the hip (at the total hip or femoral neck). The same threshold values used for women are appropriate for men, as the age-adjusted fracture risk is more or less the same for any given BMD.

[0148] "Osteopenia" is a pre-osteoporotic condition characterized by a mild thinning of bone mass that is less severe than osteoporosis. Osteopenia results when bone formation is insufficient to offset normal bone loss. Osteopenia is generally considered the first step on the path to osteoporosis and is defined as a bone mineral density (BMD) value between 1 SD and 2.5 SD below the young adult mean, with a T-score of -1 SD to -2.5 SD. Bone mineralization reduction can also be referred to as osteopenia, whether or not osteoporosis is present.

[0149] "Pre-osteoporosis" includes both osteopenia and other conditions that result in a high risk of future osteopenia, osteoporosis, and bone fracture. Nonetheless, a subject who is considered to be at low or no risk of developing osteoporosis or osteopenia according to current BMD measurements can still be at risk for pre-osteoporosis or bone fracture because the BMD measurement assesses bone status at the time of measurement, not the rate of bone metabolism or the propensity for future BMD reduction. Most bone fractures occur in subjects who have not been previously diagnosed with osteoporosis or pre-osteoporosis. There is a substantial need for better risk assessment and stratification tools for those who have not yet developed osteoporosis or osteopenia, but are expected to progress to a symptomatic disease state that can be measured by BMD at a rate higher than normal.

[0150] In some forms, the method includes administering a GREM2 composition to a subject in need thereof for the treatment of bone fractures and cartilage defects, e.g., in humans and other animals. In some forms, the method includes administering a GREM2 composition to a subject in need thereof for prophylactic use in reducing closed and open fractures, as well as improving prosthetic augmentation. Osteogenic agent-induced new bone formation is useful in repairing congenital, trauma-induced, or tumor resection-induced craniofacial defects, and can also be used in cosmetic surgery. In certain cases, the GREM2 composition can provide an environment that attracts osteoblasts, can stimulate the growth of osteoblasts and osteoblast precursor cells. It can also induce differentiation.

[0151] In some forms, the method comprises administering a Grem2 composition to a subject in need thereof for the treatment of bone loss-related diseases or disorders including, but not limited to, hyperparathyroidism-, chronic kidney disease such as bone mineral disorder, sex hormone deprivation or ablation (androgens and / or estrogens), rheumatoid arthritis, severe burns such as bone resorption and low bone formation, hyperparathyroidism, hypercalcemia, hypocalcemia, hypophosphatemia, osteodystrophy (tumor-induced), hyperphosphatemia, vitamin D deficiency, hyperparathyroidism (including familial hyperparathyroidism) and pseudohypoparathyroidism, tumor bone metastasis. In some forms, the method comprises administering a Grem2 composition to a subject in need thereof for the treatment of bone loss-related diseases or disorders including, but not limited to, bone loss due to tumors or chemotherapy, e.g., bone and bone marrow tumors, ischemic bone disorders, periodontal disease and oral bone loss, Cushing's disease, Paget's disease, thyrotoxicosis, chronic diarrhea or malabsorption, tubular acidosis or anorexia nervosa.

[0152] Specific conditions that can be treated using the disclosed compositions and methods include dysplasias in which bone growth or bone development is abnormal, as well as osteopenia, osteoporosis, and bone loss of various origins. Representative examples of such conditions include chondrodysplasia, craniometaphyseal dysplasia, enchondromatosis, fibrous dysplasia, osteopetrosis, hypophosphatemic rickets, Marfan syndrome, multiple hereditary exostoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions, pseudarthrosis, and pyogenic osteomyelitis, periodontal disease, antiepileptic drug-induced bone loss, primary and secondary hyperparathyroidism, familial hyperparathyroidism syndrome, weightlessness-induced bone loss, male osteoporosis, postmenopausal bone loss, osteoarthritis, renal osteodystrophy, infiltrative disorders of bone, oral bone loss, osteonecrosis of the jaw, juvenile Paget's disease, melorheumatosis, metabolic bone disease, mastocytosis, sickle cell anemia / sickling disease, organ transplant-related bone loss, kidney transplant-related bone loss, systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenile arthritis, thalassemia, mucopolysaccharidosis, Fabry disease, Turner syndrome, Down syndrome, Crouzon syndrome, leprosy, Perthes disease, adolescent idiopathic scoliosis, infantile onset multisystem inflammatory disease, Winchester syndrome, Menkes disease, Wilson disease, ischemic bone disease (such as Legg-Calve-Perthes disease, regional migratory osteoporosis), anemic states, steroid-induced conditions, glucocorticoid-induced bone loss, heparin-induced bone loss, bone marrow disorders, scurvy, malnutrition, calcium deficiency, idiopathic osteopenia or osteoporosis, congenital osteopenia or osteoporosis, alcoholism, chronic liver disease, postmenopausal state, chronic inflammatory conditions, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy, diabetes, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, acromegaly, hypogonadism, immobility or disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis, chondropathy, joint replacement-related bone loss, HIV-related bone loss, growth hormone deficiency-related bone loss, cystic fibrosis-related bone loss, fibrous dysplasia, chemotherapy-related bone loss, tumor-induced bone loss, cancer-related bone loss, hormone ablative bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa, facial bone loss-related diseases, skull bone loss-related diseases, jaw bone loss-related diseases, head bone loss-related diseases, and space travel-related bone loss. Further conditions involve aging-related bone loss, including aging-related facial bone loss, aging-related skull bone loss, aging-related jaw bone loss, and aging-related head bone loss.

[0153] In some forms, the method comprises administering a GREM2 composition to a subject in need thereof for the treatment of non-union bone fractures, slow-healing bone fractures, fetal and neonatal dysplasia (e.g., hypocalcemia, hypercalcemia, calcium receptor deficiency, and vitamin). It can also be applied to conditions characterized by failure of bone formation or healing, including D deficiency, osteonecrosis, including osteonecrosis of the jaw, and osteogenesis imperfecta.

[0154] In some forms, the GREM2 compositions and formulations are administered to a subject in need thereof to reduce one or more symptoms associated with bone loss, e.g., reduced bone mineral density, reduced bone strength, reduced bone volume / tibia volume ratio (BV / TV), and increased risk of bone fracture. In addition to direct measurement of BMD, several conventional risk factors for osteoporosis and bone fracture are often assessed prior to or concurrently with diagnosing osteoporosis or assessing pre-osteoporotic conditions. Such risk factors include, but are not limited to, gender, where women have a greater chance of developing osteoporosis or osteopenia due to less bone tissue and changes that occur during menopause; age, where the skeleton becomes thinner and weaker with age; small body size; race, where white and Asian women are at the highest risk, African American and Hispanic women have a lower but significant risk; family history, where the risk of fracture is thought to be due in part to genetics. Subjects with a history of bone fractures in their parents have also been reported to have osteopenia and can be at risk for bone fractures.

[0155] Other important risk factors include abnormally low sex hormone levels, manifested as abnormal menstrual period absence (amenorrhea), low estrogen levels such as found in postmenopausal women (including but not limited to low levels of any one or more of the major estrogens, estradiol, estrone, and estriol, and intermediates, precursors and estrogen derivatives of androgens), and low testosterone levels such as found in older men. Subjects with anorexia nervosa are also at elevated risk of osteoporosis. Diets low in calcium and vitamin D can also result in higher rates of bone mass loss. Subjects on long-term glucocorticoid use and some anticonvulsants also result in bone density loss and fractures. Subjects exhibiting these risk factors are often found to have osteoporosis or pre-osteoporotic conditions when assessed by BMD. Also at risk for osteoporosis or osteopenia are subjects who are lifestyle inactive or who are bedridden for long periods, subjects who smoke or who drink excessively. A number of risk rules and indices have been constructed that integrate these variables into a clinically useful measure of absolute or relative risk, such as the Osteoporosis Risk Assessment Tool (ORAI), the Osteoporosis Self-Assessment Tool (OST), and the like; such multivariate approaches tend to have fairly high sensitivity for osteoporosis, but low specificity. For example, the OST has been reported to identify more than 90% of women with osteoporosis (and 100% of women with osteoporosis over the age of 65), but more than half of the women identified by the tool as needing a BMD test are found to actually not have osteoporosis at the time of testing (see Bone Health and Osteoporosis: A Report of the Surgeon General (2004) Chapter 10, also Woolf & Pfleger, Burden of Major Musculoskeletal Conditions, Bulletin of the World Health Organization (2003) 81 : 646-656).

[0156] In some forms, the disease, disorder, or condition can be associated with elevated or specific expression of a protein, e.g., a bone morphogenetic protein. For example, a GREM2 composition can be administered to a subject in need thereof to reduce expression of one or more bone turnover markers. Bone turnover biomarkers (BTMs) are byproducts of the bone remodeling process that can be measured in urine or serum and are indicative of the rate of bone turnover. In some forms, the bone turnover marker is a bone resorption marker. Non-limiting examples of bone resorption markers include N-terminal telopeptide of type I collagen, C-terminal telopeptide of type I collagen (CTX), hydroxyproline, total pyridinoline, free deoxypyridinoline (DPD), bone sialoprotein (BSP), and tartrate-resistant acid phosphatase 5b. In some forms, the bone turnover marker is a bone formation marker. Examples of bone formation markers include, but are not limited to, total alkaline phosphatase, bone-specific alkaline phosphatase, osteocalcin, and type I serum pro-collagen bone collagen (C-terminal / N-terminal): C1NP or P1NP.

[0157] D. Subject to be treated

[0158] A subject in need of treatment is a subject having a bone loss-related disease or a subject expressing one or more symptoms of a bone loss-related disease. Exemplary bone loss-related diseases include osteopenia, osteoporosis, pre-osteoporosis, or bone cancer. In some forms, the subject is a mammal, including but not limited to murine, simian, human, mammalian farm and livestock, mammalian sport animals, and mammalian pets. Preferably, the subject is a human.

[0159] Methods of assessing and detecting bone loss in a subject are known to one of ordinary skill in the art. An apparently healthy subject is a subject who shows no signs or symptoms of disease.

[0160] The effects of a pharmaceutical composition comprising one or more GREM2 proteins or nucleic acids can be compared to a control. Suitable controls are known in the art and include, for example, untreated subjects or placebo-treated subjects. A typical control is a comparison of the condition or symptoms of a subject before and after administration of a pharmaceutical composition comprising one or more GREM2 proteins or nucleic acids. The condition or symptoms can be a biochemical, molecular, physiological, or pathological readout. For example, the effects of a composition on a particular symptom, pharmacological, or physiological indicator can be compared to an untreated subject or the condition of a subject prior to treatment. In some forms, the symptoms, pharmacological, or physiological indicators of a subject are measured prior to treatment and again one or more times after treatment begins. In some forms, the control is a reference level, or an average value determined based on measuring the symptoms, pharmacological, or physiological indicators of one or more subjects that do not have the disease or disorder to be treated (e.g., healthy subjects). In some forms, the effects of treatment are compared to conventional treatments known in the art. Suitable control subjects are unvaccinated subjects, or subjects that receive the same amount of therapeutic, prophylactic, and / or diagnostic agents in the absence of a pharmaceutical composition comprising one or more GREM2 proteins or nucleic acids.

[0161] E. Route of administration

[0162] A pharmaceutical composition comprising one or more GREM2 proteins or nucleic acids is typically administered to a subject in an effective amount. As used herein, the term "effective amount" means a dosage sufficient to inhibit or prevent symptoms of one or more infections or diseases or otherwise provide the desired pharmacological and / or physiological effect. The precise dosage will vary according to a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the specific stage of the disease, and the treatment affected, etc.

[0163] In some forms, the disclosed pharmaceutical compositions containing a GREM2 peptide and / or small molecule are administered parenterally. The phrases "parenterally administered" and "parenteral administration" are art-recognized terms and include administration of a compound by injection or infusion, such as by intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intratracheal, intranasal, intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal, transtracheal, subcutaneous, subdermal, intraarticular, subcapsular, subarachnoid, intraspinal, and intrastemal injection and infusion. The disclosed pharmaceutical compositions containing one or more GREM2 proteins or nucleic acids can be administered parenterally, e.g., by subdural, intramuscular, intravenous, intrathecal, intraventricular, intraarterial, intraamniotic, intraperitoneal, or subcutaneous routes. In preferred forms, the disclosed pharmaceutical compositions containing one or more GREM2 proteins or nucleic acids are administered by intraperitoneal, intravenous, intramuscular, subcutaneous, and intradermal administration. The most preferred forms of administration are subcutaneous, intravenous, and intramuscular administration.

[0164] IV. Kits

[0165] Also disclosed are medical kits. A medical kit can include, for example, a supply of a pharmaceutical composition containing one or more GREM2 peptides and / or small molecules, and optionally one or more therapeutic, prophylactic, or diagnostic agents, either alone or together in the same mixture. The active agents can be provided separately (e.g., lyophilized), or in the form of a pharmaceutical composition. The active agents can be in unit dosage form, or in the form of a stock solution that should be diluted prior to administration. In some forms, the kit includes a supply of a pharmaceutically acceptable carrier. The kit can also include a device for administering the active agents or compositions, such as a syringe. The kit can include printed instructions for administering the compounds in the uses described above.

[0166] The disclosed compositions and methods can be further understood by the following numbered paragraphs.

[0167] 1. A method of treating a subject having a bone loss-related disease, disorder, or condition, the method comprising administering to the subject a composition comprising:

[0168] (a) an effective amount of a soluble protein comprising all or a functional portion of amino acids 22 to 168 of a Gremlin-2 (GREM2) protein or a functional variant thereof, wherein the functions of the functional portion and functional variant include all or at least 20% of the natural GREM2 protein antagonism of bone morphogenetic protein-2 (BMP2); or

[0169] (b) a nucleic acid encoding the soluble protein of (a), wherein the nucleic acid comprises a sequence that mediates expression of the soluble protein in a cell of the subject following the administration.

[0170] 2. The method of paragraph 1, wherein the soluble protein comprises the amino acid sequence of SEQ ID NO: 2 or a functional variant thereof having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NO: 2.

[0171] 3. The method of paragraph 1 or 2, wherein the amino acid sequence of the soluble protein comprises at least 70% and less than 100% sequence identity to any one of SEQ ID NO: 2.

[0172] 4. The method of any one of paragraphs 1 to 3, wherein the functional variant binds to BMP2 with at least 60% of the affinity of a GREM2 protein.

[0173] 5. The method of any one of paragraphs 1 to 4, wherein the functional variant comprises the sequence CX6QX6CX6NX2CXGXCXSX3PX (8-13) CX2CXPX8TLXCX (15-18) CXC (SEQ ID NO: 4).

[0174] 6. The method of any one of paragraphs 1 to 5, wherein the nucleic acid is RNA or DNA.

[0175] 7. The method of paragraph 6, wherein the nucleic acid is a modified or unmodified oligonucleotide; selected from the group comprising an antisense oligonucleotide, a short interfering RNA, a microRNA, a peptide nucleic acid, a locked nucleic acid, an aptamer, and a spiegelmer.

[0176] 8. The method of any one of paragraphs 1 to 7, wherein the nucleic acid is an mRNA.

[0177] 9. The method of any one of paragraphs 1 to 8, wherein the nucleic acid comprises one or more expression control sequences.

[0178] 10. The method of any one of paragraphs 1 to 9, wherein the nucleic acid is or is encoded by a vector or transposon.

[0179] 11. The method of paragraph 10, wherein the vector is a viral vector.

[0180] 12. The method of paragraph 11, wherein the viral vector is selected from the group consisting of a lentiviral vector, an adeno-associated viral (AAV) vector, or an adenoviral vector, or a herpes simplex virus (HSV) vector, or a vesicular stomatitis virus (VSV) vector, or a human bocavirus vector (hBoV), or a chimeric vector comprising a combination of any two or more of an adeno-associated viral (AAV) vector, a herpes simplex virus (HSV) vector, a vesicular stomatitis virus (VSV) vector, or a human bocavirus vector (hBoV).

[0181] 13. The method of paragraph 10, wherein the vector is a nucleic acid expression vector selected from the group consisting of a plasmid, a cosmid, and a replicon.

[0182] 14. The method of any one of paragraphs 1 to 13, wherein the composition is administered at intervals selected from the group consisting of once a day, twice a day, once a week, twice a week, three times a week, four times a week, once every two weeks, once every three weeks, about once a month, once every two months, once every three months, once every four months, once every six months, once every eight months, once every nine months, and once a year.

[0183] 15. The method of any one of paragraphs 1 to 14, wherein the composition is administered once or more times per week for a period of time of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more.

[0184] 16. The method of any one of paragraphs 1 to 15, wherein the subject is a human.

[0185] 17. The method of any one of paragraphs 1 to 16, wherein the composition is administered to a human subject at a dose of between 0.01 mg / kg of subject body weight to 100 mg / kg of subject body weight, inclusive.

[0186] 18. The method of any one of paragraphs 1 to 17, wherein the composition is administered in an amount effective to reduce bone morphogenetic protein (BMP) expression in the subject.

[0187] 19. The method of any one of paragraphs 1 to 18, wherein the composition is administered in an amount effective to reduce one or more bone turnover markers in the subject.

[0188] 20. The method of any one of paragraphs 1 to 19, wherein the disease or disorder to be treated is selected from the group consisting of pre-osteoporosis, osteoporosis, osteopenia, bone fracture, and / or bone cancer.

[0189] 21. The method of any one of paragraphs 1 to 20, wherein the composition is administered orally, by injection, or by infusion.

[0190] 22. The method of paragraph 21, comprising subcutaneously, intravenously, intramuscularly, intraperitoneally, or intradermally administering the composition to the subject.

[0191] 23. The method of any one of paragraphs 1 to 22, wherein the composition further comprises a pharmaceutically acceptable buffer, carrier, diluent, or excipient.

[0192] The application will be further understood with reference to the following non-limiting examples.

[0193] Examples

[0194] Materials and Methods

[0195] Transcriptome-wide association study (TWAS), colocalization analysis, and Mendelian randomization analysis based on summary data.

[0196] Using publicly available data, the relationship of GREM2 gene expression to BMD was assessed. TWAS was constructed using S-PrediXcan (Gamazon, E.R., et al. A gene-based association method for mapping traits using reference transcriptome data. Nature Genetics, 2015. 47(9): p. 1091-1098). Using expression quantitative trait loci (eQTL) from 49 different tissues from GTEx v7 (The Genotype-Tissue Expression (GTEx) project. Nat Genet, 2013. 45(6): p. 580-5) as exposure and heel estimated BMD

[10] as outcome, genes were identified that had expression associated with BMD. To assess causality, a colocalization analysis was performed (Giambartolomei, C., et al. Bayesian Test for Colocalisation between Pairs of Genetic Association Studies Using Summary Statistics. PLOS Genetics, 2014. 10(5): p. e1004383), and a summary data-based Mendelian randomization analysis (SMR) of the resulting genes was generated (Zhu, M., et al. Integrating genome-wide association and transcriptome prediction model identifies novel target genes for osteoporosis. Osteoporosis International, 2021. 32(12): p. 2493-2503). Causal association was considered to exist if the significance p-value was significant in both the S-PrediXcan and SMR analyses. Causal association also had to have a non-significant association with the posterior colocalization probability (p coloc) and the HEIDI test, which is a complementary analysis to SMR.

[0197] Trap staining and calcium phosphate resorption assay.

[0198] Murine RAW 264.7 macrophages (ATCC TIB-71) were maintained in DMEM (ATCC 30-2002) supplemented with 10% FBS (Gibco) and penicillin / streptomycin 100 units / ml (Gibco). Cells were incubated at 37°C in a 5% CO2atmosphere. RAW264.7 cells were cultured in 96-well plates (SPL Life Sciences #30096) for TRAP staining and RNA extraction, respectively. DMEM supplemented with 10% FBS and penicillin / streptomycin was used as basal medium for RAW264.7 cell differentiation. To generate osteoclasts, RAW 264.7 cells were seeded at 6000 cells / cm2for 24 hours. The culture medium was then changed to 10 ng / ml RANKL (R&D Systems) with or without G REM2 protein (R&D Systems; #2069-PR-050 / CF; recombinant mouse) to initiate differentiation. The medium was changed every 3 days. Cell cultures were harvested on day 5 and cells were fixed and stained for TRAP activity using the TRAP staining kit (Sigma Aldrich #387A-1KT) according to the manufacturer’s instructions. After plates were dried overnight at room temperature, TRAP-positive multinucleated (nuclei >3) cells were counted as osteoclasts using an optical microscope (Nikon Eclipse TS100).

[0199] Calcium phosphate surface resorption assays were performed on Osteo Assay Surface 96-well plates (Corning #3988). For the analysis of pit formation surfaces, the culture medium was aspirated from the wells on day 5 and 100 μL of 10% bleach solution was added. Cells were incubated with the bleach solution for 5 minutes at room temperature. The wells were washed twice with distilled water and dried overnight at room temperature. Images of each well were recorded at 100x magnification using the stitching function of the color imaging microscope (Olympus DP74). Stitched microscope images were post-processed and analyzed with Adobe PhotoShop 2020 CC and the resorbed area was calculated with Image J software. The effect of G REM2 treatment on osteoclast resorptive activity was detected using the in vitro pit formation assay.

[0200] Human cross-sectional study

[0201] A cross-sectional study of 1104 subjects from the Hong Kong Osteoporosis Study (Cheung et al. (2018). Serum GREM2 was stratified into gender-specific quartiles, and the association between serum GREM2 quartiles and bone mineral density at the lumbar spine, total hip, and femoral neck was assessed using analysis of covariance (ANCOVA). The association between serum GREM2 and bone resorption markers (CTx and NTx) and bone formation markers (P1NP and osteocalcin) was also assessed. The associations were adjusted for age, gender, height, weight, alcohol drinking status, smoking status, and physical activity.

[0202] Preparation of recombinant mouse GREM2

[0203] 1. Cell stock

[0204] The GREM2 expression plasmid was transfected into BL21(DE3) competent cells according to the manufacturer's instructions. Monoclonal E. coli carrying the GREM2 expression plasmid were selected by AMP agar plates, followed by growing them in LB + AMP + 2% glucose at 37°C, 220 rpm overnight. The glycerol stock of the monoclonal was prepared by mixing the overnight culture and sterile 50% glycerol at a 1 : 1 ratio, and stored at -80°C.

[0205] 2. Mouse GREM2 inclusion body expression

[0206] Mouse GREM2 expression seed culture was prepared from the glycerol stock in 25 mL LB + ampicillin (100 ug / ml) + 2% glucose and grown overnight at 37°C, 220 rpm shaking. The next day, 24 mL of seed culture was added to 1 L of 2xYT medium containing ampicillin (100 μg / ml) in a flask with a baffle. The cell culture was induced with 0.5 mM IPTG at 0.8 to 1.0 OD for 4 or 6 hours. The cell pellet was harvested by centrifugation at 4696 xg for 15 minutes at 4°C. The cell pellet was stored at -80°C until further processing.

[0207] 3. Inclusion body purification

[0208] Two buffers were used for inclusion body purification: cell lysis buffer (50 mM Tris, 5 mM EDTA, pH 8.0) and IB wash buffer (50 mM Tris, 5 mM EDTA, 0.5 M NaCl, 0.5% Triton X-100, pH 8.0). Approximately 80 g of cell pellet was resuspended in 320 mL of cell lysis buffer (approximately 25% by wet cell weight) in ice via sonication (700 W at 40% power) until homogenized. Inclusion bodies were collected by centrifugation at 5000 xg for 30 min at 4°C, and the supernatant was discarded. The inclusion bodies were then resuspended by sonication and washed five times with IB wash buffer, followed by washing with cell lysis buffer.

[0209] 4. Dissolution and clarification of mouse GREM2 inclusion bodies

[0210] Dissolve approximately 6 g of wet IB in 60 mL of denaturing buffer (100 mM Tris, 8 M urea, 1 mM EDTA, 10 mM MTT, pH 8.5). Incubate at room temperature for 4 hours. Clarify the dissolved GREM2 by centrifugation at 4°C for 10 min at 4696 xg, then further centrifuge the supernatant at 4°C for 10 min at 20000 xg.

[0211] 5. Cation exchange chromatography purification of GREM2 from degenerated mouse cells

[0212] 50 mL of clarified degenerated mouse GREM2 protein was loaded onto a 5 mL HiTrap SP HP column (Cytiva # 17115201) equilibrated with buffer A (20 mM MES, 6 M urea, pH 6.0). The column was washed with 25 mL of buffer A and then eluted with a linear 0-100% gradient buffer B (20 mM MES, 6 M urea, 1 M NaCl, pH 6.0). The protein was eluted with a 5 mL fraction in a 50 mL volume. The purity of the target protein bands was checked by SDS-PAGE, and then selected for protein refolding.

[0213] 6. Mouse GREM2 protein refolding

[0214] The denatured GREM2 protein was diluted dropwise in refolding buffer (50 mM Tris, 0.5 M arginine, 1 mM EDTA, 5 mM GSH, 1 mM GSSG, 0.5 mM cysteine, pH 8.5) with stirring at room temperature to a final concentration of approximately 0.1 mg / ml. The refolding mixture was then incubated at room temperature for 3 days.

[0215] 7. Cation exchange (CNX) chromatography purification of refolded mouse GREM2

[0216] The refold mixture was diluted 2-fold with ultrapure water, then filtered with a 0.2 pm bottle top filter, then loaded onto a 5 mL HiTrap SP HP column (Cytiva #17115201) equilibrated with Buffer Al (0.25 M arginine, 50 mM MES, pH 6.0). The column was washed with 45 mL Buffer Al, then eluted with a linear 0-100% gradient of Buffer Bl (1 M arginine, 50 mM MES, pH 6.0). The protein was eluted in 5 mL fractions over a 50 mL volume. The purity of the target protein band was checked by SDS-PAGE.

[0217] 8. Dialysis of purified mouse GREM2

[0218] The CNX chromatography purified protein at >95% purity was pooled together. The protein solution was then dialyzed three times against 50 volumes of dialysis buffer (20 mM MES, pH 6.0) through a SnakeSkin pleated dialysis tubing (7000 MWCO) to remove arginine. The protein solution after dialysis was filtered sterilized through a 0.2 pm syringe filter and stored at -20 °C.

[0219] 9. SDS-PAGE assay

[0220] The protein concentration of the final GREM2 protein was estimated by BCA assay. The gel electrophoresis sample was prepared by diluting the GREM2 protein into the final buffer MES buffer at pH 6.0 with 0.5 M arginine and DTT reducing SDS sample buffer. 5 pg of protein was loaded into each lane of an 18% SDS-PAGE gel and run at 100 V for 6 hours. An unstained protein ladder was used as a size reference.

[0221] 10. BMP-2 inhibition dual luciferase activity QC assay

[0222] BMP response reporter osteoblast cell line (BRITER) (Cat. No. EBI001) was purchased from Kerafast Ltd, dual luciferase detection kit was purchased from Sigma-Aldrich (Cat. No. SCT152). BRITER cell line was cultured in DMEM high glucose (culture medium) containing 10% FBS and 1% penicillin / streptomycin, and serum starved in DMEM high glucose containing 0.3% FBS and 1% penicillin / streptomycin (serum starved medium).

[0223] BRITER cells were grown in 96-well plates to confluence. The media was washed away by rinsing the cells once with 200 μΐ^per well of lx PBS. The cells were then incubated with serum-starved media for 2 hours at 37 °C, 5% C02. Different concentrations of GREM2 protein were pre-incubated with 1 nm BMP-2 in serum-starved media for approximately 1 hour of incubation time. The pre-incubation time was approximately 45 minutes to 1 hour, followed by the addition of the serum-starved BRITER cell line. The BRITER cell line was then incubated with BMP-2 or GREM2 / BMP-2 mixture for 2 hours. Finally, the cells in the 96-well plate were rinsed once with lx PBS and the cells were lysed with 50 μΐ^per well of passive lysis buffer by gentle pipetting. Luciferase assay was performed according to the manufacturer's instructions.

[0224] GREM2 treatment of healthy female C57BL / 6J mice.

[0225] Ten-week-old female C57BL / 6J mice (Jackson Laboratory) were ordered and acclimated for 2 weeks. A total of 12 mice were randomly assigned to either a treatment group (N=6) or a control group (N=6). Recombinant mouse GREM2 protein (0.33 μg / g) was delivered to the treatment group mice by subcutaneous injection three times per week for 4 weeks (week 0 to week 4). The control group mice were similarly treated with vehicle buffer in place of the GREM2 protein. At weeks 0 and 4, the mice were examined for BMD and trabecular bone volume to total volume fraction (BV / TV) by micro-CT scanning under anesthesia. The mean BMD and BV / TV of the GREM2 treated mice and the control group were compared using a one-tailed t-test.

[0226] GREM2 treatment of ovariectomized (OVX) female C57BL / 6J mice.

[0227] Eight-week-old female C57BL / 6J mice (Jackson Laboratory) were ordered and acclimated for 2 weeks. At 10 weeks of age, the mice were ovariectomized to produce OVX mice that mimic postmenopausal osteoporosis. The mice were allowed to recover for 2 weeks. Then, a total of 12 mice were randomly assigned to either a treatment group (N=6) or a control group (N=6). Recombinant mouse GREM2 protein (0.33 μg / g) was delivered to the treatment group mice by subcutaneous injection three times per week for 4 weeks (week 0 to week 4). The control group mice were similarly treated with vehicle buffer in place of the GREM2 protein. At weeks 0 and 4, the mice were examined for BMD and trabecular bone volume to total volume fraction (BV / TV) by micro-CT scanning under anesthesia. The mean BMD and BV / TV of the GREM2 treated mice and the control group were compared using a one-tailed t-test.

[0228] Results

[0229] GREM2 expression is causally related to BMD.

[0230] Table 1 shows the results of the TWAS analysis. GREM2 expression was found to be significantly positively associated with BMD in S-PrediXcan and SMR. In addition, non-significant associations were also observed in coloc and HEIDI. These results suggest that higher expression of the GREM2 gene is causally related to BMD.

[0231] Table 1: Association between GREM2 expression and BMD

[0232] Gene b_PrediXcan p_PrediXcan p_coloc b_SMR_multi p_SMR_multi p_HEIDI GREM2 0.229 1.19E-141 1 0.212122 9.51E-10 0.082167

[0233] GREM2 is a negative regulator of osteoclastogenesis and bone resorption.

[0234] By GREM2 treatment, TRAP-positive multinucleated osteoclasts were significantly reduced in a dose-dependent manner Figure 1A and 1B . A 27.3% and 55.2% reduction in the number of osteoclasts was observed in cells treated with 1 pg / mL and 5 pg / mL GREM2, respectively, compared to controls (RANKL-treated cells without GREM2 treatment).

[0235] After RANKL treatment, osteoclasts form resorption pits on the calcium phosphate surface. With 5 pg / mL GREM2 treatment, the percentage of resorption area was significantly reduced by 18.0% Figure 2A and 2B .

[0236] Serum GREM2 levels were associated with increased BMD and decreased bone turnover marker levels in humans.

[0237] Using the lowest quartile as a reference, the highest quartile of serum GREM2 was observed to be significantly associated with higher BMD at all sites measured, as well as reduced bone formation (P1NP and OC) and resorption (CTx and NTx) markers (Table 2).

[0238] Table 2: Association between the highest quartile of serum GREM2 and BMD or bone turnover markers, using the lowest quartile as a reference.

[0239] Results Estimate S.E. 95% CI lower limit 95% CI upper limit P value Lumbar spine 0.042 0.013 0.016 0.067 0.001* Total hip BMD 0.022 0.01 0.003 0.041 0.020* Femoral neck BMD 0.019 0.01 0.001 0.038 0.042* P1NP -0.191 0.094 -0.375 -0.007 0.042* OC -0.193 0.093 -0.375 -0.011 0.038* CTx -0.287 0.092 -0.468 -0.106 0.002* NTx -0.345 0.087 -0.517 -0.174 <0.001*

[0240] GREM2 treatment of healthy mice resulted in increased BMD and BV / TV when compared to controls.

[0241] After 4 weeks of treatment with recombinant GREM2 in healthy mice, it was found that BMD and BV / TV were significantly higher in the proximal tibia in the treatment group when compared to the control group. The mean difference in BMD was -0.018 g / cm 3 (p = 0.007), and the mean difference in BV / TV was -3.466 (p = 0.006) Figure 3A and 3B .

[0242] GREM2 treatment in OVX mice resulted in increased BMD and BV / TV compared to the control group.

[0243] After 4 weeks of treatment with recombinant GREM2 in OVX mice, it was found that BMD and BV / TV were significantly higher in the proximal tibia in the treatment group when compared to the control group. The mean difference in BMD was -0.012 g / cm 3 (p = 0.042), and the mean difference in BV / TV was -1.495 (p = 0.033) Figure 4A and 4B .

[0244] Conclusion

[0245] In this study, the potential of GREM2 to prevent and treat osteoporosis was evaluated. It was found that GREM2 gene expression was causally related to BMD in humans, and circulating GREM2 was also positively correlated with BMD in cross-sectional studies. It was also found that circulating GREM2 was negatively correlated with both bone resorption and bone formation markers, suggesting that the increase in BMD can be partly through the inhibition of bone turnover. This was supported by in vitro experiments that showed that GREM2 treatment reduced osteoclastogenesis and bone resorption in osteoclasts in a dose-dependent manner. Since age-related and postmenopausal osteoporosis are characterized by increased bone turnover, the inhibition of bone turnover can improve age-related and postmenopausal bone loss.

[0246] In vivo studies confirmed that recombinant GREM2 treatment resulted in increased BMD in healthy mice and OVX mice, strengthening GREM2 as a potential treatment for age-related and postmenopausal osteoporosis. Taken together, these results suggest that GREM2 is a novel protein that can be used to prevent and treat osteoporosis and bone loss. Furthermore, serum GREM2 can be a blood-based biomarker for osteoporosis and bone turnover.

[0247] Those of skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method of treating a subject suffering from a bone loss-related disease, disorder, or condition, the method comprising administering to the subject a composition comprising: (a) An effective amount of a soluble protein comprising all or a functional portion of amino acids 22 to 168 of Gremlin-2 (GREM2) protein or a functional variant thereof, wherein the function of the functional portion and the functional variant comprises all or at least 20% of the antagonistic effect of the native GREM2 protein against bone morphogenetic protein-2 (BMP2); or (b) The nucleic acid encoding the soluble protein of (a), wherein the nucleic acid contains a sequence that mediates the expression of the soluble protein in the subject's cells after administration.

2. The method of claim 1, wherein the soluble protein comprises the amino acid sequence SEQ ID NO: 2 or a functional variant thereof, the functional variant having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% of the same amino acid sequence as any one of SEQ ID NO:

2.

3. The method of claim 1 or 2, wherein the amino acid sequence of the soluble protein has at least 70% and less than 100% sequence identity with any one of SEQ ID NO:

2.

4. The method of any one of claims 1 to 3, wherein the functional variant binds to BMP2 with at least 60% affinity for the GREM2 protein.

5. The method of any one of claims 1 to 4, wherein the functional variant comprises the sequence CX6QX6CX6NX2CXGXCXSX3PX (8-13) CX2CXPX8TLXCX (15-18) CXC (SEQ ID NO: 4).

6. The method of any one of claims 1 to 5, wherein the nucleic acid is RNA or DNA.

7. The method of claim 6, wherein the nucleic acid is a modified or unmodified oligonucleotide; selected from the group consisting of antisense oligonucleotides, short interfering RNA, microRNA, peptide nucleic acid, locked nucleic acid, aptamer, and spiegelmer.

8. The method of any one of claims 1 to 7, wherein the nucleic acid is mRNA.

9. The method of any one of claims 1 to 8, wherein the nucleic acid comprises one or more expression control sequences.

10. The method of any one of claims 1 to 9, wherein the nucleic acid is a vector or transposon, or is encoded by a vector or transposon.

11. The method of claim 10, wherein the vector is a viral vector.

12. The method of claim 11, wherein the viral vector is selected from the group consisting of: lentiviral vectors, adeno-associated virus (AAV) vectors, or adenovirus vectors, or herpes simplex virus (HSV) vectors, or herpetic stomatitis virus (VSV) vectors, or human bocavirus vectors (hBoV), or chimeric vectors comprising any two or more of the following: adeno-associated virus (AAV) vectors, herpes simplex virus (HSV) vectors, herpetic stomatitis virus (VSV) vectors, or human bocavirus vectors (hBoV).

13. The method of claim 10, wherein the vector is a nucleic acid expression vector selected from the group consisting of plasmids, granules, and replicons.

14. The method of any one of claims 1 to 13, wherein the composition is applied at intervals selected from the group consisting of: once a day, twice a day, once a week, twice a week, three times a week, four times a week, once every two weeks, once every three weeks, about once a month, once every two months, once every three months, once every four months, once every six months, once every eight months, once every nine months, and once a year.

15. The method of any one of claims 1 to 14, wherein the composition is applied once or more per week for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer.

16. The method of any one of claims 1 to 15, wherein the subject is a human.

17. The method of any one of claims 1 to 16, wherein the composition is administered to a human subject at a dose between 0.01 mg / kg of subject body weight and 100 mg / kg of subject body weight, including the extreme values.

18. The method of any one of claims 1 to 17, wherein the composition is administered in an amount that effectively reduces the expression of bone morphogenetic protein (BMP) in the subject.

19. The method of any one of claims 1 to 18, wherein the composition is administered to effectively reduce the amount of one or more bone turnover markers in the subject.

20. The method of any one of claims 1 to 19, wherein the disease or disorder to be treated is selected from the group consisting of: pre-osteoporosis, osteoporosis, osteopenia, fracture and / or bone cancer.

21. The method of any one of claims 1 to 20, wherein the composition is administered orally, by injection, or by infusion.

22. The method of claim 21, comprising administering the composition subcutaneously, intravenously, intramuscularly, intraperitoneally, or intradermally to the subject.

23. The method of any one of claims 1 to 22, wherein the composition further comprises a pharmaceutically acceptable buffer, carrier, diluent, or excipient.

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