WO2015052345A1

WO2015052345A1 - Compositions containing galectin-3 modulators for the treatment of bone disorders

Info

Publication number: WO2015052345A1
Application number: PCT/EP2014/071894
Authority: WO
Inventors: Johannes Grillari; Sylvia WEILNER
Original assignee: Universität Für Bodenkultur Wien
Priority date: 2013-10-11
Filing date: 2014-10-13
Publication date: 2015-04-16
Also published as: WO2015051850A1

Abstract

The invention relates to compositions and methods for the treatment, prophylaxis and diagnosis of disorders associated with aberrant bone mineral density. More particularly, the invention relates to increasing Galectin-3 levels in mesenchymal stem cells to increase osteogenesis. Compositions comprising Galectin-3 or nucleic acids encoding Galectin-3 may be administered systemically or locally and are useful for the treatment of diseases like osteoporosis and for accelerating bone healing. The invention also relates to methods for diagnosing bone diseases, wherein the level of Galectin-3 is measured.

Description

COMPOSITIONS CONTAINING GALECTIN-3 MODULATORS FOR THE

TREATMENT OF BONE DISORDERS

The present invention relates to the therapy, prophylaxis and diagnosis of disorders that are associated with aberrant bone mineral density.

Metabolism and remodeling of the bone structure are the result of coordinated actions of bone-resorbing osteoclasts and bone-forming osteoblasts. While upon activation, osteoclasts resorb a portion of bone and finally undergo apoptosis, newly generated osteoblasts form bone at the site of resorption. Since development of osteoclasts is controlled by pre-osteoblastic cells, resorption and formation of bones are tightly coordinated.

An imbalance between osteoclast and osteoblast activities can result in skeletal abnormalities like osteoporosis (OP), which is characterized by decreased bone density and micro-architectural deterioration of bone tissue. The osteoporotic syndrome encompasses primary disorders such as postmenopausal or age-related OP, and secondary conditions that accompany disease states or medications. Low bone mineral density and low bone mass are the most important risk factors for osteoporosis.

Overall, the fact that the repair capacity of fractures is reduced and disorders like osteopenia or osteoporosis increase with age, implicates that the bone

regeneration capacity is affected by the aging process. Decreased bone mineral density was also reported in patients with rheumatoid arthritis. (Hamalainen et al., Joint Bone Spine, Elsevier, Paris, FR vo. 74, no. 5, 2007).

Osteoporosis is estimated to affect 200 million women worldwide; in Europe, USA and Japan, osteoporosis affects an estimated 75 million people. Due to increased life expectancy, numbers are expected to increase.

Most of the currently available treatments aim at substantially increasing the bone density by using anti-resorptive strategies, i.e. they act by inhibiting bone resorption by osteoclasts. These therapies essentially include the use of

bisphosphonates, estrogens, selective estrogen receptor modulators (SERMs), calcitonin and monoclonal antibodies such as denosumab, a monoclonal antibody against receptor activator of NFKB-ligand (RANKL), a factor made by osteoblasts which stimulates osteoclast development. These therapies have been shown to have limited efficacy. The first, and to date only, approved agent that stimulates new bone formation in the treatment of osteoporosis in menopausal women is teriparatide, a portion of human parathyroid hormone, which is the primary regulator of calcium and phosphate metabolism in bone and kidney.

US2005/158321 discloses treatment of rheumatoid arthritis and inflammatory diseases including osteoporosis using an antagonist of galectin-3 activity.

Yin-Ji Li et al. (Laboratory Investigation, Nature Publishing Group, vol. 89, no 1 ,

2009, pp 26-37) report increased galectin-3 levels in ankle-joint extracts of rats with adjuvant-induced arthritis (AA rats) and describe amelioration of the severity of bone destruction by administering recombinant galectin-3 into the joint cavities of AA rats.

D'Amelio et al. (Bone 200807 US, vol. 43, no. 1 , 2008, pp 92-100) describes that T-cell activity contributes to the bone loss induced by estrogen-deficiency in postmenopausal women with osteoporosis. Inhibition of T cell activation and proliferation by mesenchymal stem cells (MSCs) was shown, and MSCs have been used in bone and cartilage regeneration in osteoporotic fracture and arthritis (Chanda et al., Journal of Cellular Biochemistry, vol. 1 1 1 , no. 1 1 1 , 2010, pp 249-257).

Sioud et al. (International Journal of Oncology, vol. 38, no.2, 201 1 , pp 385-390) report that MSC-mediated T-cell suppression occurs through galectin-1 and galectin- 3 secretion of MSCs. Galectin-3 was also identified as age-dependent protein in MSCs (Kasper et al., Stem Cells, vol. 27, no. 6, 2009, pp1288-1297).

Yamaza et al. (PlosOne, vol. 3, no. 7, 2008, e2615) disclose a stem cell based therapy for osteoporosis treatment.

It has been an object of the invention to provide new agents and therapies for the treatment of bone disorders that result in new bone formation, thus reducing fracture at a higher rate than the available anti-resorptive therapies.

To solve the problem underlying the invention, the inventors focused on the process of osteogenesis.

Osteogenesis, i.e. the differentiation of mesenchymal stem cells into

osteoblasts, is one of the basic mechanisms underlying the activities of osteoblasts, which are the key players in the formation of new bone.

In line with the finding that advanced age correlates with the incidence of reduced bone mineral density or bone mass disorders, it has been shown that the differentiation capacity of mesenchymal stem cells (MSCs) decreases with age, thereby contributing to slowed-down bone healing with age and to impaired bone remodeling leading to osteopenia or osteoporosis. The experiments of the present invention focus on the role of circulating microvesicles (MVs) and their effect on the osteogenic differentiation capacity of mesenchymal stem cells (MSCs). While searching for systemic factors that are deregulated in old age and influence osteogenic differentiation capacity of

mesenchymal stem cells (MSCs), it was surprisingly found that vesicular Galectin-3 is secreted by endothelial cells within CD63 negative and also CD63 positive MVs as well as in MVs isolated from human plasma of young people.

Galectin-3 (NG_017089.1 RefSeqGene, NR_003225.2, Isoforml : NM_002306.3 (SEQ ID NO. 1 ), Isoform 1 : Isoform 2: NP_002297.2 (SEQ ID NO. 2) , Isoform 2:

NM_001 177388.1 (SEQ ID NO. 3), NP_001 170859.1 (SEQ ID NO. 4); protein and cDNA sequence: GenBank accession No. : AB006780.1 ) is a ubiquitously expressed lectin. It belongs to the family of galectins, a class of proteins exhibiting a conserved carbohydrate-recognition domain (CRD) which facilitates a beta-galactoside binding activity due to its NWGR amino acid sequence (Leffler et al., 2004, Glycoconjugate journal ^, 433-440). In addition to the CRD, Galectin-3 has a collagen a-like and a short amino-terminal domain containing six predicted phosphorylation sites, whereof some are known to have also CRD-domain-independent functions. For example, phosphorylation of Serine 96 was shown to inhibit degradation of β-Catenin, an important mediator of Wnt-signalling (Song, S., et al, 2009, Cancer Res 69, 1343- 1349). Depending on the state and type of the cells, Galectin-3 can be found

intracellular^, in the extracellular matrix and the circulation. Recently, it has been shown that tyrosine phosphorylation by Calpain-4 is essential for Galectin-3 secretion (Menon et al., 201 1 , Biochem Biophys Res Commun 410, 91 -96). Galectin-3 has been shown to play a critical role in cellular processes such as pre-mRNA splicing, cell growth, cell cycle progression and apoptosis, as well as in systemic processes, inflammation, atherosclerosis, wound healing, prion infection and, most prominent, in tumour development and progression.

In the experiments of the present invention, it was tested if altered levels of Galectin-3, as observed in the plasma of young versus elderly individuals, might have an impact on the osteogenic differentiation capacity of mesenchymal stem cells.

It was confirmed that Galectin-3 levels are lower in elderly persons and that knock-down of Galectin-3 inhibits osteogenic differentiation of MSCs in vitro, while its overexpression before induction of osteogenesis accelerates the osteogenic differentiation process of MSCs even before Runx2 induces Galectin-3 expression during osteogenesis.

It can be concluded from the findings obtained in the experiments of the present invention that Galectin-3 will be useful in replacement therapies in patients with aberrant bone mineral density disorders, in particular in patients with reduced bone mass, e.g. osteoporotic patients, for restoring balanced osteogenesis.

In addition, it can be concluded that Galectin-3 levels in human plasma may serve as a biomarker indicating how permissive the systemic environment is to osteogenic differentiation.

In a first aspect, the present invention relates to a composition for the treatment and prophylaxis of disorders associated with aberrant bone mineral density or for accelerating bone healing, comprising, in a therapeutically effective amount, an agent that alters the level of Galectin-3 in mesenchymal stem cells, wherein said agent is selected from the group of

a) agents with the ability to increase the level of Galectin-3 in mesenchymal stem cells, selected from

i. Galectin-3 or fragments or variants or derivatives thereof, or ii. nucleic acid molecules encoding Galectin-3 or fragments or

variants thereof; or

iii. cells containing one or more nucleic acid molecules defined in ii. ; b) agents with the ability to decrease the level of Galectin-3 in mesenchymal stem cells, selected from

i. agents inhibiting Galectin-3 expression in mesenchymal stem

cells or

ii. agents targeting the Serine 96 phosphorylation site of Galectin-3 protein, or

iii. dominant negative alleles of Galectin-3.

For the purpose of embodiment a), Galectin-3 may be any isoform of the protein for example but not limited to isoform 1 comprising the nucleic acid sequence of SEQ ID NO. 1 or the amino acid sequence of SEQ ID NO. 2, isoform 2 comprising the nucleic acid sequence of SEQ ID NO. 3 or the amino acid sequence of SEQ ID NO. 4). Alternatively to the naturally occuring Galectin-3 protein, or a nucleic acid molecule encoding it, a variant Galectin-3 polypeptide or a fragment or a nucleic acid molecule encoding such variant or fragment may be used. Variant Galectin-3 polypeptides having substantial sequence similarity to the Galectin-3 protein, such as 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, specifically 99.5%, more specifically 99,9% sequence identity to a corresponding portion of Galectin-3, the corresponding portion being any contiguous sequence of any length, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more amino acids. In some embodiments, chemically similar amino acids may be substituted for amino acids in the Galectin-3 protein sequence (to provide conservative amino acid substitutions).

Furthermore, a Galectin-3 molecule modified by amino acids exchanges other than conservative substitutions may be useful, e.g. to enhance its activity. Optimization of the amino acid sequence may be achieved by methods known in the art, e.g. by site-directed mutagenesis. As an example, an optimized Galectin-3 mRNA sequence of SEQ ID No. 5 is provided. Furthermore polypeptides and/or peptides of Galectin-3 are provided comprising a sequence containing Serine 96 phosphorylation site of Galectin-3 (GRKKRRQRRRGGYPSSGQPSATGAY, SEQ ID NO. 10) or sequences containing Alanine (GRKKRRQRRRGGYPSSGQPAATGAY; SEQ ID NO. 1 1 ) or Aspartic acid (GRKKRRQRRRGGYPSSGQPDATGAY; SEQ ID NO. 12) to Serine 96 mutations.

In some embodiments, the agent with the ability to increase the level of

Galectin-3 in mesenchymal stem cells is a recombinant Galectin-3 protein encoded by an optimized Galectin-3 mRNA, specifically as of SEQ ID NO. 5. In some

embodiments, the agent is a functional fragment, a variant or derivative of Galectin-3, for example a Galectin-3 polypeptide or peptide comprising the amino acid sequence of SEQ ID NO. 10, SEQ ID NO. 1 1 or SEQ ID NO. 12. In some embodiments, the functional fragment of Galectin-3 is a peptide consisting of the amino acid sequence of SEQ ID NO. 10. In some embodiments, the Galectin-3 variant is a peptide consisting of the amino acid sequence of SEQ ID NO. 1 1 or SEQ ID NO. 12.

The term "functional variant, derivative or fragment" as used herein shall refer to any fragment or derivative or variant that has at least 10%, specifically at least 25%. Specifically at least 50%, specifically at least 90% Galectin-3 activity.

In certain embodiments, the therapeutically active agent is a Galectin-3 derivative in which Galectin-3 is coupled to a chemical moiety that affects an increase of its half-life, activity or uptake in bone. By way of example, Galectin-3 derivatives may be obtained by conjugation to polyethylene glycol (Iversen et al., Theranostics. 2013, 3(3):201 -9), or by N-glycosylation (Flintegaard et al., 2010, Endocrinology.

Nov;151 (1 1 ):5326-36). Alternatively, derivatization may be achieved by genetic modification that results in an N-terminal cyclic conformation (Cao et al., 2012,

Diabetes Res Clin Pract. Jun; 96(3):362-70).

In certain therapeutic methods of the invention, Intralipid®, an FDA-approved fat emulsion, may be injected before admistering Galectin-3 to effect the protein's half-life (Liu et al., 2013, Biochim Biophys Acta. Jun; 1830(6) :3447-53).

In a specific embodiment, the therapeutically active agent is a peptide

containing the phosphorylation site Serine 96 of Galectin-3. Without wishing to be bound by theory - since Galectin-3 phosphorylation may act as a scavenger of β-Catenin, excess Galectin-3 may compete with β-Catenin for phosphorylation, thereby protecting it from degradation so that it can exert its function in osteogenesis.

Galectin-3 variants, fragments or derivatives are useful within the scope of the present invention as long as their effect on differentiation of mesenchymal stem cells is equal to or greater than that of Galectin-3.

Preferably, a Galectin-3 peptide has a length of about 8 - 30 amino acids. In some embodiments, the Galectin-3 peptide has a length of 10 to 30 amino acids, 15 to 30 amino acids, 20 to 30 amino acids, 10 to 20 amino acids, 10 to 25 amino acids, or 15 to 25 amino acids. In some embodiemtns, the Galectin-3 peptide has a length of 15 to 25 amino acids.

According to specific embodiments of the invention, the Galectin-3 peptide has a length of 8, 9, 10, 1 1 , 12, 13, 14, 15, 1 6, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34 or 35 amino acids.

Galectin-3 variants or fragments may be routinely tested for usefulness in the present invention by transfecting MSCs or, as a model for MSCs, adipose-tissue derived stem cells (ASCs), with mammalian vector constructs containing the DNA sequence encoding the Galectin-3 protein or peptide of interest and determining its effect on osteogenic differentiation. MSCs and ASCs may be obtained by known methods, e.g. as described by Wolbank et al., 2007 (Tissue Eng 13, 1 173-1 183) and Wolbank et al., 2009 (Tissue Eng Part A 15, 1843-1854).

The usefulness of Galectin-3 variants or derivatives may also be tested by incubating the test cells with such variant or derivative of interest. The effect may be quantified, e.g. as described in the Examples, by Alizarin staining to determine the cells' degree of calcification, and additionally be confirmed by qPCR of the early osteogenic marker alkaline phosphatase (ALP) and the late osteogenic markers osteonectin (ON) and osteocalcin (OC).

With respect to embodiment a), the term "Galectin-3" as used herein refers both to the naturally occurring protein and its therapeutically or functionally active

variants/fragments/derivatives.

With respect to the present invention, the term "disorders associated with aberrant bone mineral density" ("bone mineral density disorders" or "bone density disorders" or "BMD disorders") refers both to conditions which are characterized, at least in part, by a decrease in bone mineral density (BMD), or bone mass respectively, that is associated with an aberrantly low level of Galectin-3, or, conversely, it refers to bone disorders associated with bone overgrowth and aberrantly high bone mineral density, in which bone formation and deposition exceed resorption.

For the purpose of the present invention, such disorders are due to an abnormal capacity of mesenchymal stem cells to differentiate into osteoblasts, such capacity including both the process of differentiation itself as well as its stimulation/activation.

A composition according to a) may be used to i) increase low bone density/low bone mass or to ii) accelerate bone healing, e.g. after fractures or iii) for the prevention of fractures in defined regions of the skeleton that are at high risk of fractures, e.g. the hip of a patient suffering from osteoporosis, iv) in dentistry/ periodontology when an increase of bone mass due to increased differentiation of mesenchymal stem cells is to be achieved. In some embodiments, treatment/administration with the composition comprising an agent with the ability to increase the level of Galectin-3 in mesenchymal stem cells increases bone mass and/or bone density by at least 5%, 10%, 20%, 30%, 40%, 50%, or 60%. In some embodiments, treatment/administration with the

composition comprising an agent with the ability to increase the level of Galectin-3 in mesenchymal stem cells increases bone mass and/or bone density by at least 10%. In some embodiments, treatment/administration with the composition comprising an agent with the ability to increase the level of Galectin-3 in mesenchymal stem cells increases bone mass and/or bone density by at least 25%.

In the case of i), Galectin-3 is the effective agent in a pharmaceutical

composition to be administered systemically/parenterally, e.g. by subcutaneous bolus injection.

In certain embodiments, when administered systemically, in order to enrich Galectin-3 in osteogenic cells and to avoid tissue or organ-unspecific side effects, it may be linked to a bone-targeting molecule. Examples for bone-targeting molecules are, without limitation, bisphosphonates, lipids, or acidic oligopeptides, as described by Low and Kopecek, 2012 (Adv Drug Deliv Rev. 64(12): 1 189-1204). Coupling

Galectin-3 to bone-targeting molecules may be achieved according to methods known in the art. Examples of such methods are conjugation of Galecin-3 itself, or its delivery vehicle, e.g. liposomes, nanoparticles or microspheres, respectively, to

bisphosphonates by a disulfide bridge (Doschak et al., 2009, Mol. Pharm. 6, 634-640) or to collagen-binding domains by fusing its cDNA to the N- or C-terminus of the protein (Ponnapakkam et al., 201 1 , Calcif. Tissue Int. 88 51 1-520). Alternatively, short peptides containing repetitive aspartate and/or glutamate sequences may be fused to the C- or N-terminus of the protein, such fusion constructs being obtainable by recombinant protein expression. Such constructs may additionally include a spacer such as the Fc region of human IgG to improve the targeting and/or to ensure the activity of the protein (Nishioka et al., 2006, Mol. Genet. Metab. 88 244-255).

Thus, in a further embodiment, the invention relates to Galectin-3, or a fragment or variant thereof, linked to a bone-targeting molecule.

According to yet another embodiment, Galectin-3 or a gene construct containing the Galectin-3 encoding DNA, is contained in a delivery vehicle. Examples of delivery vehicles for bone-targeting are cationic liposomes like dioleoyl trimethylammonium propane (DOTAP)-based cationic liposomes attached to six repetitive sequences of aspartate, serine, serine ((AspSerSer)(6)), as described by Zhang et al., 2012 (Nat Med 18(2): 307-14) for the delivery of siRNA to bone-forming surfaces.

In the case of ii) or iii), Galectin-3 is administered locally, either directly or as a component of a matrix (also known as "scaffold) or bolus or by implantation of

Galectin-3 overexpressing cells.

A composition according to a) may be used, but its use is not limited to, ghosal hematodiaphyseal dysplasia syndrome (GHDD), osteoporosis, osteogenesis imperfecta osteopenia, Paget's disease, osteomyelitis, hypercalcemia, osteonecrosis, hyperparathyroidism, lytic bone metastases, periodontitis, and bone loss due to immobilization.

As defined in US 20130195863, and also used herein, the term "osteoporosis" includes any form of osteoporosis. For example, osteoporosis includes primary osteoporosis, post-menopausal and age-related osteoporosis, endocrine osteoporosis (including hyperthyroidism, hyperparathyroidism, Gushing's syndrome, and acromegaly), hereditary and congenital forms of osteoporosis (including osteogenesis imperfecta, homocystinuria, Menkes' syndrome, Riley-Day syndrome), and

osteoporosis due to immobilization of extremities. The term also includes osteoporosis that is secondary to other disorders, including hemochromatosis, hyperprolactinemia, anorexia nervosa, thyrotoxicosis, diabetes mellitus, celiac disease, inflammatory bowel disease, primary biliary cirrhosis, rheumatoid arthritis, ankylosing spondylitis, multiple myeloma, lymphoproliferative diseases, and systemic mastocytosis. The term also includes osteoporosis secondary to surgery (e.g., gastrectomy) or to drug therapy, including chemotherapy, endocrine therapy, anticonvulsant therapy, immuno- suppressive therapy, and anticoagulant therapy. The term also includes osteoporosis secondary to glucocorticosteroid treatment for certain diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), asthma, temporal arthritis, vasculitis, chronic obstructive pulmonary disease, polymyalgia rheumatica,

polymyositis, and chronic interstitial lung disease. The term also includes osteoporosis secondary to glucocorticosteroid and/or immunomodulatory treatment to prevent organ rejection following organ transplant such as kidney, liver, lung, and heart transplants. The term also includes osteoporosis due to submission to microgravity, such as observed during space travel. The term also includes osteoporosis associated with malignant disease, such as breast cancer, prostate cancer.

A composition according to b) may be used for the therapy of disorders which aim at decreasing an aberrantly high bone density and bone overgrowth. Such disorders are caused by bone formation and deposition that exceed resorption, potentially resulting in pathologically increased bone mass and strength. Examples are sclerosteosis, Simpson-Golabi-Behmel syndrome (SGBS), Van Buchem Disease.

In order to determine whether a person is eligible for the treatment with a composition of the present invention, he or she may be first tested for BMD using approved physical methods. Examples are dual-energy X-ray absorptiometry (DXA or DEXA), quantitative computed tomography (QCT), qualitative ultrasound (QUS), single photon absorptiometry (SPA), dual photon absorptiometry (DPA), digital X-ray radiogrammetry (DXR) or single energy X-ray absorptiometry (SEXA). Such

measurements rely on the measurement of bone density, which is considered to be an indicator also of bone mass. Since the terms "bone density" and "bone mineral" density are often mostly interchangeably, they are also used, if not otherwise stated, synonymously for the purpose of the present invention. The currently used relevant measure when screening for osteoporosis is the T- score, which is a comparison of a patient's BMD to that of a healthy thirty-year-old. The criteria of the World Health Organization are: the normal T-score is > -1 .0; osteopenia is defined by a T-score of -1 .0 to -2.5; osteoporosis is defined by a T-score of < -2.5.

In the context of the present invention, the term "aberrant BMD" designates, if the T-score is the relevant parameter, a BMD level outside the T-score range of -1 ,0 - +0,5. For the purpose of the present invention, this term also encompasses a level of BMD that is to be increased during bone healing, when the bone repair capacity after fractures is reduced.

Subsequently to the step of measuring BMD, the person's Galectin-3 level in plasma is determined (either by a separate Galectin-3 test or by assessing Galectin-3 expression as a component of a diagnostic signature). If measuring BMD by a physical method, e.g. any of the methods mentioned above, is omitted, determining the

Galectin-3 level is used as the only test for diagnosing a disorder correlating an aberrant BMD. In this case, the Galectin-3 level is the parameter for eligibility for a Galectin-3-based therapy. Eligibility for a Galectin-3-based therapy is given if the Galectin-3 plasma level deviates from a value of young, healthy individuals, by more than 15%. In addition, eligibility is given in case that the patient has one or more Galectin-3 mutations, or deficiencies in the response to Galectin-3, e.g. mutations in the down-stream signaling events induced by Galectin-3, or impaired binding of Galectin-3 to receptors, or impaired cellular uptake of Galectin-3.

The therapeutically effective amount of Galectin-3, i.e. the amount effective at dosages and for periods of time necessary to achieve the desired therapeutic result, i.e. the desired bone mineral density, may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.

Response to Galectin-3-based therapy may be determined by standard methods, i.e. by determining BMD as described above.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result by increasing bone mineral density or preventing its decrease, thus preventing disorders like osteoporosis. A prophylactic dose may be used in subjects prior to or at an earlier stage of disease, and a prophylactically effective amount may be more or less than a therapeutically effective amount in some cases.

A composition of the invention containing the Galectin-3 protein, operably linked to a bone-targeting molecule, may be administered systemically, preferably

parenterally, e.g. intravenously or subcutaneously, or locally, e.g. in the form of bone implants, prostheses or internal patches around bones.

As mentioned above, the Galectin-3 protein/peptide may also be packaged into lipid vesicles, or mixed with a polymer like polyethylenimine linked to a bone targeting molecule.

In yet another embodiment, the Galectin-3 protein may be administered as a component of a so-called "protein activated matrix (PAM), i.e. a matrix impregnated with the protein of interest.

Matrices useful for drug delivery in bones, including controlled release composites, e.g. for delivering growth factors, are well known in the art and may be adapted for a therapeutic Galectin-3 protein. Examples are organic bone-derived matrices like demineralised bone matrix, autolyzed antigen-extracted allogenic bone; synthetic polymers like polylactic acid or polyglycolic acid homo-/heterodimer; natural polymers like collagen (types I and IV), non-collagenous proteins like fibrin and hydrogels. Example of inorganic matrices are natural bone mineral and thermoashed bone mineral, hydroxyapatite, tricalcium phosphate and other bioceramics, bioactive glass and coral (Kirker-Head, 2000, Adv Drug Deliv Rev 43: 65-92).

Detailed reviews of composites as well as methods for incorporating the therapeutic agent into such composites are provided by Lauzon et al., 2012 (Journal of

Controlled Release 1 62, 502-520), and by Soundrapandian et al., 2009 (AAPS

PharmSciTech. 10(4): 1 158-1 171 ).

Further examples of biomaterial matrices are described in US 20130195863. In yet another embodiment, to facilitate uptake of a Galectin-3 protein or peptide into the target cells, it may be operably linked to a CPP ("cell penetrating peptide").

Examples for CPPs, also known as protein transduction domains (PTDs), are transportan, pISI, Tat(48-60), pVEC, MAP and MTS, or the Oct4 transduction domain.

A CPP-linked Galectin-3 may be obtained by recombinant production of the respective

Galectin-3/CPP fusion protein/peptide.

For a formulation to be injected, the Galectin-3-containing composition contains components which are pharmaceutically acceptable. These may be in particular isotonic, sterile, saline solutions, for example but not limited to monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts, or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

In certain embodiments, the Galectin-3 therapeutic is a DNA molecule inserted in a vector that is administered according to methods for gene therapy known in the art. In some embodiments, the DNA molecule for gene therapy comprises the nucleic acid sequence of SEQ ID NO. 1 . In some embodiments, the DNA molecule for gene therapy comprises the nucleic acid sequence of SEQ ID NO. 3. In some embodiments, the DNA molecule for gene therapy comprises the nucleic acid sequence of SEQ ID NO. 5. Vectors may be prepared from different type of viruses, including adenoviruses, adeno-associated viruses (AAV), herpes viruses (HSV), lentiviruses and retroviruses. When the Galectin-3 therapeutic is used in a gene therapy using a virus, an

adenovirus vector is preferably used. As a useful adenovirus vector, a so-called "second generation adenovirus vector" (obtained from a first generation adenovirus vector lacking the E1 /E3 domain by deleting the E2 or E4) or a third generation adenovirus vector in which all viral coding sequences are deleted. DNA replication and packaging of such so-called "gutless" (or helper-dependent) adenoviral vectors depend on a helper virus to provide viral gene products to support the vector. Adenoviral and other viral vectors and the requirements they must fulfill to be suitable for bone- directed gene therapy, are described by Fischer et al., 201 1 , Journal of Cranio-Maxillo- Facial Surgery 39, 54-64).

Alternatively, the Galectin-3 therapeutic may be administered in the form of a naked DNA or mRNA molecule (e.g. a naked DNA or mRNA molecule comprising the sequence of SEQ ID NO. 1 , SEQ ID NO. 3 or SEQ ID NO. 5.

In general, as for application of the protein, the dosage of the Galectin-3 nucleic acid molecule, i.e. when inserted in a gene therapy vector or applied as naked DNA or RNA, may depend to a large extent on the condition and size of the subject being treated as well as the therapeutic formulation, frequency of treatment and the route of administration. Regimens for continuing therapy, including dose, formulation, and frequency may be guided by the initial response and clinical judgment.

To improve uptake of DNA in gene therapeutical applications of Galectin-3, physical and chemical methods may be useful. Examples of physical methods are electroporation (using short pulses of high voltage to carry DNA across the cell membrane), the so-called "gene gun" (using DNA coated with gold particles loaded into a "gun" achieves penetration into the cells), sonoporation (using ultrasonic frequencies) magnetofection (in which DNA is complexed to a magnetic particles). Examples of chemical methods are lipoplexes and polyplexes as well as dendrimers.

According to certain embodiments, to achieve bone-directed expression of

Galectin-3 in mesenchymal stem cells (osteoprogenitor cells), the vector construct may include an osteoprogenitor-specific promoter, as described for expression of BMP-2 (bone morphogenic protein 2) by Kumar et al., 2005, Biochimica et Biophysica Acta 1731 , 95 - 103. Known osteoprogenitor-specific promoters are the Runx-2/cbfa1 (RUNX) promoter, the osteopontin (OPN) promoter, the collagen type 1 a (COL) promoter and the osteocalcin (OCN) promoter. Since Galectin-3 expression is desirable during early stages of osteogenic differentiation (or activation of

differentiation, respectively), the promoter cloned upstream of the Galectin-3 encoding DNA sequence is the COL or the RUNX promoter, preferably the RUNX promoter.

In embodiments of local administration, e.g. for accelerating bone healing after a fracture, the therapeutic Galectin-3 encoding nucleic acid molecule may be delivered to the site of interest by means of viral or non-viral vectors or as naked DNA or RNA. As reviewed by Pelled et al., 2010 (Tissue Engineering: Part B, Volume 1 6, No.1 , 13- 20), localization of the therapeutic molecule within the fracture site may be assured either by physical placement at the target site or by gene release from a three- dimensional biomaterial implanted at or near the defect area. Useful physical placement methods include direct injection of the Galectin-3 protein, or the transgene respectively, into the fracture site. Preferably, in order for the DNA molecule to penetrate cells in situ, it is delivered by a virus or forced into cells' nuclei by an electric pulse or ultrasonic wave. Preferably, an adenoviral vector is used, as described for expressing bone morphogenetic protein (BMP) Egermann et al., 2006 (Hum Gene Ther. May;17(5):507-17).

Alternatively to using a vector, in vivo electroporation or sonoporation may be used to deliver the therapeutic locally. Using these methods, the Galectin-3 encoding nucleic acid molecule is directly injected into a fracture and an electric pulse or ultrasonic wave is applied to the site either trans- or percutaneously.

In a further embodiment, mesenchymal stem cells derived from any source, including but not limited to bone marrow, adipose tissue, umbilical tissue, urine, or placenta, genetically engineered to overexpress Galectin-3, as described in the

Examples, may be implanted at the defect site (Marie, 201 1 , Osteoporos Int 22:2023- 2026).

In an alternative embodiment, localizing Galectin-3 at the site of interest, e.g. the fracture site, e.g. by transgene expression, is achieved by first binding Galectin-3 DNA/RNA to a delivery system (e.g. by adsorption, entrapment or immobilization, or by covalent binding; Luginbuehl et al., 2004, Eur J Pharm Biopharm 58:197-208) and then implanting the gene-activated matrix (GAM) into the defect site, e.g. as described by Fang et al., 1996 (Proc Natl Acad Sci USA 93, 5753). To increase transfection efficiency, the DNA may be condensed by chemical vectors such as

polyethyleneimine, liposomes or calcium-phosphate precipitates.

Useful matrices (GAMs, "gene-activated matrices") have been described above in the context with matrices for the delivery of the Galectin-3 protein.

Also when the therapeutically active agent, either in the form of a

protein/peptide or in the form of a nucleic acid, is administered locally, either as such or incorporated in a matrix, it may advantageously be linked to a bone-targeting molecule. For the protein/peptide, this may be accomplished either by directly linking the protein/peptide to the bone-targeting molecule or by linking the delivery vehicle, e.g. a liposome, that contains the agent, with the bone-targeting molecule. In the case that a nucleic acid molecule is to be administered locally, incorporation of the bone- targeting molecule is achieved by linking it to the surface of the delivery vehicle. The same applies for a CPP.

In certain embodiments, a Galectin-3 therapeutic of the invention, either containing Galectin-3 or the nucleic acid molecule encoding it, may be combined with one or more other therapeutic agents, e.g. teriparatide, denosumab, blosozumab, romosozumab, or one or more bone growth factors or the respective encoding nucleic acid molecules, e.g. a BMP like BMP-2 and/or BMP-7, or RNAs, like e.g. RNAs antagonizing miR-31 .

In one aspect, the invention provides for a composition for the treatment and/or prophylaxis of disorders associated with aberrant bone mineral density (e.g. osteoporosis) comprising recombinant Galectin-3, fragments, variants or derivatives thereof, in a therapeutically effective amount. In some embodiments, Galectin-3 comprises the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4. In some embodiments, Galectin-3 is encoded by the nucleic acid sequence comprising SEQ ID NO. 1 or SEQ ID NO. 3. In some embodiments, Galectin-3 is encoded by the codon- optimized nucleic acid sequence comprising SEQ ID NO. 5. In some embodiments, the Galectin-3 fragment is a peptide comprising SEQ ID NO. 10. In some embodiments, the Galectin-3 variant is a peptide comprising SEQ ID NO. 1 1 or SEQ ID NO. 12.

In one aspect, the invention provides for a composition for accelerating bone healing comprising recombinant Galectin-3, fragments, variants or derivatives thereof, e.g.Galectin-3 comprising the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4; Galectin-3 encoded by the nucleic acid sequence comprising SEQ ID NO. 1 , SEQ ID NO. 3 or SEQ ID NO. 5; Galectin-3 peptide comprising SEQ ID NO. 10, SEQ ID NO. 1 1 or SEQ ID NO. 12 in a therapeutically effective amount.

In one aspect, compositions are provided for increasing bone mass and/or bone density (e.g. by at least 10%, by at least 25%) comprising recombinant Galectin-3, fragments, variants or derivatives thereof, e.g. Galectin-3 comprising the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4; Galectin-3 encoded by the nucleic acid sequence comprising SEQ ID NO. 1 , SEQ ID NO. 3 or SEQ ID NO. 5; Galectin-3 peptide comprising SEQ ID NO. 10, SEQ ID NO. 1 1 or SEQ ID NO. 12 in a

therapeutically effective amount

In a further embodiment said fragments, variants or derivatives of Galectin-3 may be linked to a bone-targeting molecule., for example but not limited to

bisphosphonates, lipids, or acidic oligopeptides or coupled to bone-targeting molecules achieved according to methods known in the art like conjugation of said fragments, variants or derivatives itself, or its delivery vehicle, e.g. liposomes, nanoparticles or microspheres, respectively, to bisphosphonates by a disulfide bridge or to collagen- binding domains by fusing its cDNA to the N- or C-terminus of the protein .

Alternatively, short peptides containing repetitive aspartate and/or glutamate

sequences or any other repetitive sequences may be fused to the C- or N-terminus of the fragment, derivative or variant. Such constructs may additionally include a spacer

In one aspect, the invention provides for a composition for the treatment and/or prophylaxis of disorders associated with aberrant bone mineral density (e.g.

osteoporosis) comprising a nucleic acid molecule e.g. a vector, naked DNA or RNA molecule, comprising a nucleic acid sequence encoding Galectin-3, fragments or variants thereof, in a therapeutically effective amount. In some embodiments, the nucleic acid molecule comprises the sequence of SEQ ID NO. 1 or SEQ ID NO. 3. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4. In some embodiments, the nucleic acid molecule comprises the codon-optimized nucleic acid sequence of SEQ ID NO. 5. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a Galectin-3 fragment comprising SEQ ID NO. 10. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a Galectin-3 variant comprising SEQ ID NO. 1 1 or SEQ ID NO. 12.

In one aspect, the invention provides for a composition for accelerating bone healing comprising a nucleic acid molecule (e.g. a vector, naked DNA or RNA molecule) comprising a nucleic acid sequence encoding Galectin-3, fragments or variants thereof e.g. a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4; a nucleic acid sequence comprising SEQ ID NO. 1 , SEQ ID NO. 3 or SEQ ID NO. 5; a nucleic acid sequence encoding a Galectin-3 peptide comprising SEQ ID NO. 10, SEQ ID NO. 1 1 or SEQ ID NO. 12 in a

therapeutically effective amount.

In one aspect, compositions are provided for increasing bone mass and/or bone density (e.g. by at least 10%, by at least 25%) comprising a nucleic acid molecule (e.g. a vector, naked DNA or RNA molecule) comprising a nucleic acid sequence encoding Galectin-3, fragments or variants thereof, e.g. a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4; a nucleic acid sequence comprising SEQ ID NO. 1 , SEQ ID NO. 3 or SEQ ID NO. 5; a nucleic acid sequence encoding a Galectin-3 peptide comprising SEQ ID NO. 10, SEQ ID NO. 1 1 or SEQ ID NO. 12 in a therapeutically effective amount.

In a composition of the invention according to embodiment b) ii., the inhibitor of Galectin-3 expression may be selected from any RNAi (RNA interference) molecule including siRNAs, miRNAs, LNAs, phosphorothioate RNAs, antisense

oligonucleotides, ribozymes and aptamers. Possible examples for siRNAs against Galectin-3 are siGal3_1 : 5'-GGAGAGUCAUUGUUUGCAA-3' (SEQ ID NO. 6), siGal3_2: 5'-GUACAAUCAUCGGGUUAAA-3' (SEQ ID NO. 7), siGal3_3: 5'- GGCCACUGAUUGUGCCUUA-3' (SEQ ID NO. 8), siGal3_4: 5'- GGUGAAGCCCAAUGCAAA-3' (SEQ ID NO. 9). In one aspect, a composition is provided for treatment and/or prophylaxis of a disorder associated with aberrantly high bone density and bone overgrowth (e.g., sclerosteosis, Simpson-Golabi-Beheml syndrome, Van Buchem Disease) comprising at least one siRNA selected from the group consisting of SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9 or comprising a fragment thereof of at least 10, 1 1 , 12, 13, 14, 15, 1 6, or 17 nucleic acids.

According to embodiment b) ii., the inhibitor is an agent targeting the active site (Serine 96) of the Galectin-3 protein, e.g. a protein/peptide that competes with phosphorylation or inhibits or prevents it.

According to embodiment b) iii., inhibition of Galectin-3 activity is achieved by overexpression of a dominant negative allele of Galectin-3. Dominant negative alleles of Galectin-3 may be selected upon testing truncation mutants of Galectin-3 that, upon overexpression in MSCs or during bone formation, inhibit or fail to accelerate osteogenic differentiation

In this case, an inhibitor of Galectin-3, linked to a bone-targeting molecule, is the effective agent in a pharmaceutical composition to be administered

systemically/parenterally/subcutaneously, or locally to the bone, either directly or in combination with one or more physical and chemical method to improve uptake, or as a component of a matrix (GAM, "gene activated matrix") or bolus.

As for the Galectin-3 protein or peptide, uptake of a Galectin-3 inhibitor into the target cell may be facilitated by operably linking it to a CPP ("cell penetrating peptide"), as described e.g. in WO2008033285 . Examples for CPPs, also known as protein transduction domains (PTDs), are transportan, pISI, Tat(48-60), pVEC, MAP and MTS, or the Oct4 protein transduction domain.

In a further aspect, the present invention relates to methods and assays for diagnosing in a subject a disorder associated with aberrant bone mineral density, wherein the Galectin-3 level in mesenchymal stem cells of said subject is determined in a sample and a Galectin-3 level deviating from that of a healthy young individual (by >15% or by < 15%) is indicative for an aberrant bone mineral density.

In preferred embodiments, the Galectin-3 protein level is determined by using an antibody binding specifically to Galectin-3. For the purpose of the present invention, neither the type of antibody nor the Galectin-3 epitope that it recognizes is critical. Preferably the antibody recognizes the full length of the Galectin-3 molecule, In particularly preferred embodiments, Galectin-3 levels are determined using an ELISA assay.

The term "ELISA" refers to enzyme-linked immunosorbent assay (or EIA).

Numerous ELISA methods and applications are known in the art and there are many ELISA test systems commercially available.

One example for an ELISA method is a "direct ELISA," wherein the Galectin-3 antigen in a sample is detected. In one embodiment of the direct ELISA, a sample containing Galectin-3 is exposed to a solid (i.e., stationary or immobilized) support (e.g., a microtiter plate well). Galectin-3 within the sample becomes immobilized to the stationary phase, and is detected directly using an enzyme-conjugated antibody specific for Galectin-3.

In an alternative embodiment, an "indirect ELISA" is used. In one embodiment, Galectin-3 is immobilized to a solid support (e.g., a microtiter plate well) as in the direct ELISA, but is detected indirectly by first adding the anti-Galectin-3 antibody, followed by the addition of a detection antibody specific for the anti-Galectin-3 antibody, also known as "species-specific" antibodies (e.g., a goat anti-rabbit antibody), which are available from various manufacturers known to those in the art.

In other embodiments, a "sandwich ELISA" is used, where Galectin-3 (e.g.

contained in a test sample) is immobilized on a solid support (e.g., a microtiter plate) via an antibody (i.e., a capture antibody) that is immobilized on the solid support and is able to bind to Galectin-3. Following the affixing of a suitable capture antibody to the immobilized phase, a sample is then added to the microtiter plate well, followed by washing. Galectin-3 present in the sample is bound to the capture antibody present on the support. In some embodiments, a sandwich ELISA is a "direct sandwich" ELISA, where the captured Galectin-3 antigen is detected directly by using an enzyme- conjugated antibody directed against the antigen. Alternatively, in other embodiments, a sandwich ELISA is an "indirect sandwich" ELISA, where the captured Galectin-3 antigen is detected indirectly by using an antibody directed against the antigen, which is then detected by another enzyme-conjugated antibody which binds the antigen- specific antibody, thus forming an antibody-antigen-antibody-antibody complex.

Suitable reporter reagents are then added to detect the third antibody. Alternatively, in some embodiments, any number of additional antibodies are added as necessary, in order to detect the antigen-antibody complex. In some preferred embodiments, these additional antibodies are labeled or tagged, so as to permit their visualization and/or quantitation.

As used herein, the term "capture antibody" refers to an antibody that is used in a sandwich ELISA to bind (i.e., capture) Galectin-3 in a sample prior to its detection. For example, in some embodiments, a polyclonal antibody against Galectin-3 serves as a capture antibody when immobilized in a microtiter plate well. This capture antibody binds Galectin-3 present in a sample added to the well. In one embodiment of the present invention, biotinylated capture antibodies are used in conjunction with avidin-coated solid support. Another antibody (i.e., the detection antibody) is then used to bind and detect the antigen-antibody complex, in effect forming a "sandwich" comprised of antibody-antigen-antibody (i.e., a sandwich ELISA).

As capture antibody, any specific Galectin-3-binding antibody may be used, e.g. polyclonal serum, or monoclonal antibodies. Monoclonal anti-Galectin-3 antibodies are commercially available, e.g. from Pierce (A3A12, B2C10).

In the diagnostic method of the present invention, the sample is a preparation containing plasma-derived MVs. The sample may be obtained according to methods known in the art as described by Lehmann et al., Cancer Res 68, 7864-7871 (2008), or using the method described in the Examples.

Alternatively, the Galectin-3 level may be determined directly in the plasma, i.e. without a preceding enrichment of MVs.

The invention furthermore comprises the following items:

1 . A composition for the treatment and prophylaxis of disorders associated with

aberrant bone mineral density or for accelerating bone healing, comprising, in a therapeutically effective amount, an agent that alters the level of Galectin-3 in mesenchymal stem cells, wherein said agent is selected from the group of a) agents with the ability to increase the level of Galectin-3 in mesenchymal stem cells, selected from

i. Galectin-3 or fragments or variants or derivatives thereof, or

ii. nucleic acid molecules encoding Galectin-3 or fragments or variants thereof; or

iii. cells containing one or more nucleic acid molecules defined in ii. ;

b) agents with the ability to decrease the level of Galectin-3 in mesenchymal stem cells, selected from

i. agents inhibiting Galectin-3 expression in mesenchymal stem cells, or ii. agents targeting the Serine 96 phosphorylation site of Galectin-3 protein, or iii. dominant negative alleles of Galectin-3.

2. The composition of item 1 , wherein Galectin-3, or a fragment or variant thereof, is linked to a bone-targeting molecule.

3. The composition of item 1 , wherein Galectin-3, or a fragment or variant thereof, is contained in a delivery vehicle that is linked to a bone-targeting molecule.

4. The composition of item 2 or 3, wherein said bone-targeting molecule is selected from the group of bisphosphonates, collagen-binding domains, lipids or acidic oligopeptides.

5. The composition of item 3, wherein said delivery vehicle is selected from

liposomes, nanoparticles or microspheres.

6. The composition of any one of items 1 to 5 for use in the prophylaxis and

treatment of disorders associated with a reduced bone mineral density.

7. The composition of any one of items 1 to 5 for use in the prophylaxis and

treatment of disorders associated with aberrantly increased bone mineral density.

8. The composition of any one of items 1 to 6 for or prophylaxis of osteopenia or osteoporosis.

9. The composition of any one of items 1 to 6 for local administration to accelerate bone healing, wherein said agent is incorporated in a matrix.

10. The composition of item 9, wherein said matrix is a demineralised bone matrix, an autolyzed antigen-extracted allogenic bone matrix, a polylactic acid or

polyglycolic acid homo- or heterodimer, a collagen matrix, fibrin or a hydrogel.

1 1 . The composition of item 1 , wherein said agent a) ii. is naked DNA or RNA.

12. The composition of item 1 , wherein said agent a) ii. is a DNA molecule inserted in a vector.

13. The composition of item 1 , wherein said agent a) iii. is a mesenchymal stem cell genetically engineered to overexpress Galectin-3.

14. Galectin-3, or a fragment or variant thereof, linked to a bone-targeting molecule. 15. The use of Galectin-3, or a fragment or variant thereof, for the treatment or

prophylaxis of disorders associated with decreased bone mineral density.

1 6. The use according to item 15, wherein said disorder is osteopenia or

osteoporosis. 7. A method for the treatment of disorders associated with decreased bone mineral density, said method comprising administering a composition, containing, as the active agent, an agent with the ability to increase the level of Galectin-3 in mesenchymal stem cells, wherein said agent is selected from

i. Galectin-3 or fragments or variants or derivatives thereof, or

ii. nucleic acid molecules encoding Galectin-3 or fragments or variants thereof; or iii. cells containing one or more nucleic acid molecules defined in ii..

18. A method for the treatment of disorders associated with increased bone mineral density, said method comprising administering a composition, containing, as the active agent, an agent with the ability to decrease the level of Galectin-3 in mesenchymal stem cells, wherein said agent is selected from

i. inhibitors of Galectin-3 expression in mesenchymal stem cells, or

ii. agents targeting the Serine 96 phosphorylation site of Galectin-3 protein, or iii. dominant negative alleles of Galectin-3.

19. The method of item 18, wherein said disorder is osteopetrosis.

20. A method of diagnosing in a subject a disorder associated with an aberrant bone mineral density, comprising determining said subject's Galectin-3 level

a) in microvesicles purified from plasma, or

b) directly in plasma.

21 . The method of item 20, wherein the Galectin-3 level is determined by an enzyme- linked immunosorbent assay (ELISA).

22. The method of item 20 or 21 , wherein a reduced Galectin-3 level is indicative of osteopenia or osteoporosis.

23. An assay to determine the Galectin-3 level for use in a method for the diagnosis of disorders associated with a decreased bone mineral density.

Brief description of the Figures:

Fig. 1 : Galectin-3 protein levels of microvesicles isolated from plasma of young and elderly donors

Fig. 2a: Sequence of Galectin-3 Isoform 1 (SEQ ID NO 1 )

Fig. 2b: Sequence of Galectin-3 Isoform 1 (SEQ ID NO 2)

Fig. 2c; Sequence of Galectin-3 Isoform 2 (SEQ ID NO 3)

Fig. 2d: Sequence of Galectin-3 Isoform 2 (SEQ ID NO 4)) Examples

In the Examples, the following materials and methods were used:

Cell culture

/^'. Human umbilical vein endothelial cell (HUVEC)

Endothelial cells were isolated from human umbilical veins as described

(Jaffe et al., 1973, Clin Invest 52, 2745-2756; Chang, 2005, Mutat Res 576, 39-53 HUVECs were cultivated in gelatin-precoated flasks in EGM (Lonza) at 37°C in a humidified atmosphere with 5% C0₂. Cells were passaged once or twice a week at a split ratio of 1 :2 to 1 :6 according to the growth rate. HUVECs were cultivated to senescence and stained for senescence- associated β-galactosidase (SA-β- gal) activity as described by Chang, et al., 2005, Exp Cell Res 309, 121 -136. For collection of supernatants, contact-inhibited (quiescent, PD19) and senescent (PD52 / 95% SA- -gal positive) cells were allowed to secrete into HUVEC medium, depending on the experiment, for 48 hours.

//^'. Human adipose-derived stem cells (ASCs)

Subcutaneous adipose tissue was obtained during outpatient tumescence liposuction under local anestesia. ASCs were isolated as described (Wolbank ei al., 2007, Tissue Eng 13, 1 173-1 183; Wolbank ei al., 2009, Tissue Eng Part A 15, 1843-1854) and cultured in DMEM-low glucose/HAM^'s F-12 supplemented with 4mM L-glutamine, 10% fetal calf serum (FCS, PAA) and 1 ng/ml_ recombinant human basic fibroblast growth factor (rhFGF, R&D Systems) at 37°C, 5% C0₂ and 95% air humidity. Cells were passaged once or twice a week at a split ratio of 1 :2 according to the growth rate.

All differentiation protocols were carried out in 24 well cell culture plates. For osteogenic differentiation, ASCs were seeded at a density of 2x10³ cell per well. 72 hours after seeding cells were incubated with osteogenic differentiation medium (DMEM-low glucose, 10% FCS, 4 mM L-glutamine, 10 nM

dexamethasone, 150 μΜ ascorbate-2-phosphat, 10 mM β-glycerolphosphate and 10 nM vitamine-D3) up to 4 weeks.

For Alizarin staining of calcified structures, cells were fixed for 1 hour in 70% ethanol at -20°C. After brief rinsing, cells were stained for 20 minutes with 40 mM Alizarin Red solution (Sigma) and washed with PBS. For quantification, Alizarin was extracted for 30 minutes using 200 μΙ 0.1 M HCL/0.5% SDS solution. b) Construction of Galectin-3 expression vector

Homo sapiens lectin, galactoside-binding, soluble, 3 (LGALS3 = Galectin-3), transcript variant 1 as transfection-ready DNA within the pCMV6-XL4 vector

(SC1 18706), was purchased from Origin. The restriction enzyme site NotI upstream and downstream of the gene of interest was used to cut out the insert. The obtained insert was cloned into the NotI restriction site of pcDNA 3.1 hgro (+), a mammalian expression vector containing a CMV promoter and conferring Hygromycin B resistance. c) Transfections

i) Human adipose-derived stem cells (ASCs)

ASCs were transfected using Neon® Transfection System (Life technologies). Cells were transfected according to the manufactures protocol. Briefly, 1 x10⁵

ASCs resuspended in 10 μΙ buffer R, were mixed with ^g of DNA or 10 pmol of siRNA and loaded into the Neon TM pipet tip. Subsequently cells were

electroporated using the recommended parameters: Pulse voltage: 1400 V; pulse width: 10 ms; pulse number: 3. After electroporation, cells were directly

transferred into a 6 well tissue culture vessel containing growth medium. ON-

TARGETplus Non-targeting Pool (20 nmol) (D-001810-1 0-20, Dharmacon), ON- TARGETplus SMARTpool, Human LGALS3, (5 nmol) (L-010606-00-0005, Dharmacon).

H) Human umbilical vein endothelial cells (HUVECs)

HUVECs were transfected using Neon® Transfection System (Life technologies).

Cells were transfected according to the manufactures protocol. Briefly; 5x10⁵ HUVECs resuspended in 100 μΙ buffer R were mixed with 5 μg of DNA and loaded into the Neon TM pipet tip. Subsequently, cells were electroporated using the recommended parameters: Pulse voltage: 1350 V; pulse width: 30 ms; pulse number: 1 . After electroporation, cells were directly transferred into a gelatin precoated culture flask containing growth medium. pmaxGFP vector (Amaxa). d) Assessment of apoptotic cell death

HUVECs were seeded in 12-well cell culture plates and were allowed to secrete into ASC or HUVEC medium for 48 hours. Thereafter, the cells were detached using 50 mM EDTA and stained with Annexin V- FITC and PI (Roche) according to the manufacturer^'s instructions. Analysis of the percentage of apoptotic and

necrotic/late-apoptotic cells were performed using a FACS-Calibur and the

CellQuest software (Becton Dickinson). e) Quantitative real-time PCR

Alizarin stainings were confirmed using different osteogenic differentiation marker genes. To this end, total ASCs RNA was isolated using Tri Reagent (Sigma) at different time points during osteogenesis. 7 days after differentiation start, the early osteogenic marker alkaline phosphate (ALP), 14 days after start of differentiation, the osteogenic marker osteonectin (ON) and 21 days after start of differentiation, the late osteogenic marker osteocalcin (OC) was measured Reverse transcription was performed using DyNAmo cDNA Synthesis Kit (Biozym) and qPCR was performed using the RotorGene2000 (Corbett).

Primer pair for quantifying ALP mRNA NM_000478.4 spans Exon 3 (319-438 nt) and 4 (439-554 nt). Primer pair for quantifying ON mRNA NM_0031 18

spans Exon 8 (789 -937 nt) and 9 (938-1086 nt). Primer pair for quantifying OC mRNA NM_001 199662 spans Exon 3 (296-396 nt) and 4 (397-592 nt). Primer pair for quantifying GAPDH mRNA NM_002046.4 span Exon 8 (700 -1 1 12 nt) and 9 (1 1 13-1383 nt). f) Microvesicle (MV) purification

MVs were purified by filtration and differential centrifugation as described by

Lehmann et al., Cancer Res 68, 7864-7871 (2008). In brief, cell culture supernatant was collected after a secretion period of 48 hours or plasma samples were thawed and diluted 1 :2 in PBS. The conditioned medium or the diluted plasma sample was centrifuged at 500 g for 15 minutes to sediment cells, at 14.000 g for 15 minutes to eliminate cell debris and filtered through a 0.22 μηπ filter. MVs were then sedimented by ultracentrifugation at 100.000 g for 60 minutes and the resultant pellet was washed with PBS. MV were used as fresh preparations for electron microscopy and differentiation experiments or conserved at -80°C for further analysis. For differentiation studies MV derived from 2x10⁴ HUVECs or 1 ml of human serum were resuspended in 50 μΙ ASC growth medium and added per well ASCs. g) Purification of CD63 positive exosomes

/^'. Preparation of immunoaffinity capture microbeads

CD63 monoclonal antibody immunoaffinity capture microbeads (Dynabeads® M- 270 Epoxy, Invitrogen) were prepared with the aid of Dynabeads® Antibody Coupling Kit (Invitrogen) according to the manufacturer's protocol. Briefly, 5 mg of Dynabeads were washed with 1 ml of C1 solution. The supernatant was removed by placing the tube on a magnet whereby beads were able to collect at the tube wall. 50 μΙ of monoclonal CD63 antibody (ab8219 Abeam) were mixed with 200 μΙ of C1 solution. Washed beads were first mixed with prepared antibody solution and 250 μΙ of C2 solution were added afterwards. Beads were incubated at 37°C on a roller over night.

The next day supernatant was removed by placing the tube on a magnet whereby beads were able to collect at the tube wall. Afterwards beads were washed with each 800 μΙ of HB, LB and finally SB buffer and stored at 4°C until use.

ii) Purification of CD63 positive exosomes by immunoaffinity capture microbeads MVs were isolated as described above and subsequently incubated with CD63 antibody-coupled Dynabeads for 2 h at 4°C on a roller. Afterwards the tube was placed on a magnet allowing the beads to collect at the wall. Supernatant containing MV depleted of CD63 positive exosomes was decanted. CD63 positive MVs were eluted through incubation of beads with 140 μΙ of citric acid, pH=3. The tube was placed on a magnet allowing the beads to collect at the wall.

Supernatant containing CD63 positive MVs was decanted and immediately mixed with 50 μΙ of 1 M NaOH for neutralization. CD63 positive MVs were conserved at - 80°C for further analysis. h) Electron microscopy

Purified MVs were left to settle on nickel coverslips (200 mesh, hexagonal,

Pioloform-coated Athene copper grids). After fixation with 4% paraformaldehyde, MVs were stained with 2% uranyl acetate for 30 seconds, coverslips were left to dry and visualized using a transmission electron microscopy (TEM), Philips model CM 12 electron microscope (Philips, Eindhoven, NL). For electron microscopy in-situ hybridization (EM-ISH), MV pellets were permeabilized with 0.1 % Triton-X for 5 minutes at room temperature. After washing with PBS, MVs were incubated for at least 4 hours with hybridization buffer as described 98. For each sample 1 pM of the LNA DIG-labeled single stranded probe (Exiqon, Denmark) was denaturated in denaturizing hybridization buffer (containing 50% formamide, 5x SSC, 5x Denhardt^'s solution, 0.1 % Tween, 0.25% CHAPS, 200μg ml-1 yeast RNA, 500μg ml-1 salmon sperm DNA) by incubation at 80°C for 5 minutes. Probes were placed on ice quickly. MVs were mixed with the probe and hybridized at 50°C over night. After hybridization, samples were washed stringently with 0.2 x SSC at 60°C for 1 hour. Thereafter, MV were incubated with Anti-DIG antibody (Roche) for 30 minutes and an additional hour with the second 5nm gold particle-labeled antibody (Sigma). After washing with PBS, MVs were embedded in Epon, approximately 80 nm sections on average, were cut using an Ultramicrotom (Ultracut, Reichelt) and then analyzed using transmission electron microscopy (TEM), Philips model CM 12 electron microscope (Philips, Eindhoven, NL). i) Western blot

Total proteins and proteins from MVs were extracted and separated on

polyacrylamide gels, before transfer to a PVDF membrane (BioRad). The

membrane was blocked in 3% skimmed milk, incubated with the CD63 antibody (sc- 15363 Santa Cruz), Galectin-3 antibody [A3A12] (ab2785 Abeam) or GAPDH antibody [FL-33] (sc-25778 Santa Cruz), followed by incubation with the

corresponding secondary antibody Alexa Fluor 680-conjugated anti-rabbit IgG (Molecular Probes) or Alexa Fluor 800-conjugated anti-mouse IgG (Molecular Probes). Signal intensities were analyzed by using the Odyssey infrared image system (LiCor). j) Statistics

Data were statistically analyzed using Student^'s t test, one-way ANOVA and one- way ANOVA followed by the Dunn^'s method as indicated. Analyses were performed with SigmaPlot 10.0 (SigmaPlot, Germany). The tests were two-sided with type 1 error probability of 0.05. Data are presented as mean values ± SEM. Example 1

Microvesicles isolated from plasma from elderly donors reduce osteogenic differentiation capacity of ASCs

In order to test whether osteogenic differentiation capacity of MSCs is influenced by ex vivo plasma-derived MVs and by the donors age, MVs smaller than 200 nm in diameter were isolated from donors younger than 25 or older than 55 years by different centrifugation steps. Electron microscopy was performed to confirm size and shape.

MSCs derived from adipose tissue were used as model system. ASCs were characterized in detail. In particular, the differentiation capacity towards the osteogenic and adipogenic lineage, the immunomodulatory properties as well as expression of typical and atypical surface markers were examined by phytohemagglutinin activation assay and flow cytometric analysis for the presence or absence of the surface markers CD14, CD34, CD45, CD73, CD90, HLA ABC, HLA DR and CD105.

Subsequently, ASCs were seeded one day before exposing them to plasma derived MVs for 72 hours. After 3 days osteogenic differentiation was induced as described { Wolbank et ai, 2009,Tissue Eng Part A 15, 1843-1854).

It could be shown that differentiation capacity was reduced to 30 % when cells were co-incubated with MVs isolated from the plasma of healthy elderly donors compared to ASCs exposed to MVs of young donors as quantified by Alizarin Red staining.

Example 2

Microvesicular Galectin-3 is elevated in healthy young donors

Since proteins have been reported to be packaged into MVs, the known microvesicular repertoire of proteins, as published by Mathivanan, S. & Simpson, R.J. ExoCarta: Proteomics 9, 4997-5000 (2009) was compared with a list of proteins known to be involved in the Wnt-siganling pathway, an important pathway for the induction of osteogenic differentiation. Galectin-3 resulted as the most prominent member.

MVs from plasma of healthy young (20-25 years) and healthy elderly female donors (older than 55 years) were isolated and Galectin-3 protein levels were analyzed by

Western blot. While no large differences in Galectin-3 levels in plasma of young donors were observed, reduced levels could be seen in the group of women older than 55 years (Figure: healthy elderly female donors (E); young healthy controls (Y). In order to exclude the possibility that Galectin-3 is not a component of MVs but co- sediments in protein aggregates due to ultracentrifugation, the pellet obtained by ultracentrifugation was used to purify CD63 positive MVs by an immuno-affinity capture assay, since CD63 is an established microvesicular marker. Successful separation of the CD63-containing fraction was confirmed by Western blot when CD63 positive MV fraction was loaded against CD63 negative MV fraction. Thus, it was confirmed that Galectin-3 is a component of ex-vivo plasma derived CD63 positive MVs.

Example 3

Impact of Galectin-3 level on osteogenic differentiation capacity

Since proteins are transferred to recipient cells by microvesicles, it was tested if altered levels of Galectin-3 in ASCs have an impact on osteogenic differentiation.

Osteogenic differentiation was induced 3 days after transient transfection of ASCs with a plasmid overexpressing Galectin-3. Elevated Galectin-3 levels were confirmed using Western blot. It was found that Galectin-3 alone was sufficient to significantly increase osteogenic differentiation ( ~3 fold), as quantitated by Alizarin staining as well as by qPCR of the early osteogenic marker ALP and the late osteogenic markers osteonectin (ON) and osteocalcin (OC). In agreement with this, osteogenic differentiation was decreased when ASCs were transfected with siRNA against Galectin-3. Knock-down of Galectin-3 was confirmed by Western blotting; it resulted in -30 % lower

osteogenesis as quantitated by Alizarin staining and confirmed by qPCR of ALP, ON and OC.

Example 4

Galectin-3 acts upstream of Runx-2 in increasing osteogenic differentiation capacity

Galectin-3 was already known to be expressed in the late stage of osteoblast maturation and that its expression is induced by the transcription factor Runx-2, a master regulator of ostogenic differentiation. In contrast to the work published before, it could be shown here that Galectin-3 acts upstream of Runx-2: Galectin-3

overexpressing ASCs, which were induced to undergo osteogenic differentiation, show elevated ALP m-RNA level as measured by qPCR and enhanced ALP enzymatic activity on day 7 while Runx-2 expression is not induced until day 12 as analyzed by qPCR. These observations suggest a surprising and unexpected mechanism, namely that Galectin-3 induces a positive feed forward loop, since its enhanced expression accelerates osteogenic differentiation and in association also Runx-2 expression, which in turn results in Galectin-3 expression. Example 5

Endothelial cells are a possible source of Galectin-3 containing plasma derived microvesicles

Since endothelial cells line the vasculature and secrete a large variety of factors to the circulation, it was examined whether they also secrete Galectin-3. Therefore MVs were isolated from the supernatant of human umbilical vein endothelial cells (HUVECs) after a 48 h secretion period by differential centrifugation. Electron microscopy confirmed the isolation of membrane vesicles smaller than 120 nm in diameter and positive for CD63, which is an established microvesicular marker. In addition, the presence of CD63 positive MVs in conditioned medium was confirmed by Western blot.

Subsequently, Western blot of CD63 positive MVs isolated from the cell culture supernatant revealed that Galectin-3 is indeed a component of CD63 positive MVs derived from endothelial cells.

In order to confirm that endothelially derived vesicles contain Galectin-3 in vivo as well, microvesicles positive for CD146, a specific endothelial surface marker, were purified from human plasma by immunoprecipitation. Subsequent analysis by ELISA

confirmed, that the majority of microvesicular Galectin-3 in human plasma is indeed a component of CD146 positive, endothelial derived microvesicles. Example 6

Endothelial microvesicles deliver genetic information to adipose-derived mesenchymal stem cells

In order to test whether endothelial MVs have the ability to interact with and transfer their cargo to MSCs, transiently transfected GFP-expressing HUVECs were prepared. 24 hours after transfection, HUVECS were washed twice and medium was changed in order to ensure the removal of remaining vector constructs in the

supernatant. After a secretion period of 48 hours, MVs were isolated from cell culture supernatant of transfected or untransfected HUVECs. Subsequently, ASCs were exposed to MVs for 3 days. Compared to MVs of untransfected cells, MVs isolated from GFP-transfected cells contained GFP mRNA as shown by qPCR and ASCs exposed to GFP-MVs showed GFP signals in a punctuate pattern within the cytoplasm compared to ASCs exposed to MVs of untransfected HUVECs, which showed no fluorescent signal No transfer of GFP was observed in the negative control. The obtained results indicate that a genetic transfer between endothelial derived MVs and ASCs is indeed possible.

Example 7

Microvesicles of senescent endothelial cells fail to induce osteogenic

differentiation capacity

Since senescent endothelial cells were shown to accumulate with age in vivo, it was tested whether MVs of senescent cells effect the differentiation capacity of ASCs differently when compared to MVs of early passage cells. After a secretion period of 48 hours, MVs smaller than 200 nm in diameter were isolated from cell culture

supernatant of HUVECs at an early population doubling level, at quiescence as well as from HUVECs at replicative senescence. HUVECS were passaged to 52 population doublings before irreversible growth arrest and morphological changes were observed. Replicative senescence of HUVECs was additionally confirmed by beta-galactosidase staining. During the secretion period, no differences in the number of apoptotic cells between the early population doubling and the replicative senescent culture was observed, thereby excluding that an observed effect might result from a larger share of apoptotic cells within the senescent cell culture.

Subsequently, ASCs were seeded 24 hours before exposing them to MVs isolated from cell culture supernatant of senescent or early passage quiescent HUVECs, and induced to undergo osteogenesis after a period of 72 hours. Osteogenic differentiation of ASCs incubated with MVs derived from replicative senescent cells was reduced to 50%, as quantified by Alizarin staining and by reduced ALP mRNA expression levels. Example 8

Vesicular Galectin-3 influences osteogenic differentiation capacity

In order to test if elevated microvesicular Galectin-3 levels indeed contribute to an enhanced osteoblastogenesis, HUVECs were transfected with Galectin-3 or the corresponding empty vector control. 24 hours after transfection, HUVECS were washed twice and medium was changed in order to ensure the removal of remaining vector constructs in the supernatant. After a secretion period of 48 hours, MVs were isolated from cell culture supernatant of transfected or untransfected HUVCEs.

Intracellular overexpression of Galectin-3 in endothelial cells was confirmed by Western blot. Subsequently, MVs of HUVECS transfected with Galectin-3 or with empty control vector were isolated and co-incubated with ASCs for 72 hours before osteogenic differentiation was induced. In addition enhanced Galectin-3 protein levels in vesicles isolated from plasmid transfected endothelial cells were confirmed by Western Blot in comparison to microvesicles isolated from control transfected cells. Exposure of MVs isolated from Galectin-3 overexpressing HUVECs to ASCs caused a doubling of calcium depositions as quantified by Alizarin Red staining, indicating that vesicular Galectin-3 level indeed impact on the osteogenic differentiation capacity of ASCs. Example 9

Functional implications of Serine at position 96 of Galectin-3

Serine 96 to Alanine (S96A) as well as Serine 96 to Aspartic acid (S96D) mutations were inserted by site directed mutagenesis into the Galectin-3 coding sequence and thus 3 different expression constructs were generated (Gal-3 wild type, S96A, and S96D). ASCs overexpressing S96A or S96D exhibited a markedly reduced osteogenic differentiation capacity compared to wild type Galectin-3 overexpressing cells indicating that the presence and accessibility of Serine 96 of Gal-3 is crucial for the pro-osteogenic effect of Galectin-3. In order to elucidate the molecular way of Galectin-3 action we found, that overexpression of wild type Galectin-3 leads to an accumulation of the osteogenesis inducing β-Catenin in the nucleus, while

overexpression of S96A or S96D mutants did not facilitate the translocation of β- Catenin into the nucleus. Since the S96 surrounding sequence of Galectin-3 resembles a phosphorylation site within beta-catenin that is phosphorylated by the kinase GSK3beta, our data suggest that Galectin-3 S96 might compete with beta catenin for GSK3 beta binding and phosphorylation.

Claims

1 . A composition for the treatment and prophylaxis of disorders associated with aberrant bone mineral density or for accelerating bone healing, comprising, in a therapeutically effective amount, an agent that alters the level of Galectin-3 in mesenchymal stem cells, wherein said agent is selected from the group of

a. agents with the ability to increase the level of Galectin-3 in mesenchymal stem cells, selected from

i. Galectin-3 or fragments or variants or derivatives thereof, or

ii. nucleic acid molecules encoding Galectin-3 or fragments or variants thereof; or iii. cells containing one or more nucleic acid molecules defined in ii. ;

b. agents with the ability to decrease the level of Galectin-3 in mesenchymal stem cells, selected from

2. The composition of claim 1 , wherein Galectin-3, or a fragment or variant thereof, is linked to a bone-targeting molecule.

3. The composition of claim 1 , wherein Galectin-3, or a fragment or variant thereof, is contained in a delivery vehicle that is linked to a bone-targeting molecule.

4. The composition of claim 2 or 3, wherein said bone-targeting molecule is selected from the group of bisphosphonates, collagen-binding domains, lipids or acidic oligopeptides.

5. The composition of claim 3, wherein said delivery vehicle is selected from liposomes, nanoparticles or microspheres.

6. The composition of any one of claims 1 to 5 for use in the prophylaxis and treatment of disorders associated with a reduced bone mineral density.

7. The composition of any one of claims 1 to 5 for or prophylaxis of osteopenia or osteoporosis.

8. The composition of any one of claims 1 to 5 for local administration to accelerate bone healing, wherein said agent is incorporated in a matrix.

9. The composition of claim 8, wherein said matrix is a demineralised bone matrix, an autolyzed antigen-extracted allogenic bone matrix, a polylactic acid or polyglycolic acid homo- or heterodimer, a collagen matrix, fibrin or a hydrogel.

10. The composition of claim 1 , wherein said agent a) ii. is naked DNA or RNA.

1 1 . The composition of claim 1 , wherein said agent a) ii. is a DNA molecule inserted in a vector.

12. The composition of claim 1 , wherein said agent a) iii. is a mesenchymal stem cell genetically engineered to overexpress Galectin-3.

13. Galectin-3, or a fragment or variant thereof, linked to a bone-targeting molecule.

14. The use of Galectin-3, or a fragment or variant thereof, for the treatment or prophylaxis of disorders associated with decreased bone mineral density.

15. The use according to claim 14, wherein said disorder is osteopenia or

osteoporosis.

1 6. A method for the treatment of disorders associated with decreased bone mineral density, said method comprising administering a composition, containing, as the active agent, an agent with the ability to increase the level of Galectin-3 in mesenchymal stem cells, wherein said agent is selected from

i. Galectin-3 or fragments or variants or derivatives thereof, or

17. A method for the treatment of disorders associated with increased bone mineral density, said method comprising administering a composition, containing, as the active agent, an agent with the ability to decrease the level of Galectin-3 in mesenchymal stem cells, wherein said agent is selected from

i. inhibitors of Galectin-3 expression in mesenchymal stem cells, or

18. The method of claim 17, wherein said disorder is osteopetrosis.

19. A method of diagnosing in a subject a disorder associated with an aberrant bone mineral density, comprising determining said subject's Galectin-3 level

a. in microvesicles purified from plasma, or

b. directly in plasma.

20. The method of claim 19, wherein the Galectin-3 level is determined by an enzyme-linked immunosorbent assay (ELISA).

21 . The method of claim 19 or 20, wherein a reduced Galectin-3 level is indicative of osteopenia or osteoporosis.

22. An assay to determine the Galectin-3 level for use in a method for the diagnosis of disorders associated with a decreased bone mineral density.