WO2015051850A1 - Compositions contenant des modulateurs de galectine-3 pour le traitement de troubles osseux - Google Patents

Compositions contenant des modulateurs de galectine-3 pour le traitement de troubles osseux Download PDF

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WO2015051850A1
WO2015051850A1 PCT/EP2013/071264 EP2013071264W WO2015051850A1 WO 2015051850 A1 WO2015051850 A1 WO 2015051850A1 EP 2013071264 W EP2013071264 W EP 2013071264W WO 2015051850 A1 WO2015051850 A1 WO 2015051850A1
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galectin
bone
composition
agent
level
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PCT/EP2013/071264
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Johannes Grillari
Sylvia WEILNER
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Universität Für Bodenkultur Wien
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Priority to PCT/EP2014/071894 priority patent/WO2015052345A1/fr
Publication of WO2015051850A1 publication Critical patent/WO2015051850A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1732—Lectins

Definitions

  • 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.
  • osteoporosis 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 factor for osteoporosis.
  • 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.
  • 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.
  • MSCs mesenchymal stem cells
  • MSCs mesenchymal stem cells
  • MSCs mesenchymal stem cells
  • Galectin-3 (NG_017089.1 RefSeqGene, NR_003225.2, Isoforml :
  • 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 19, 433-440).
  • CRD carbohydrate-recognition domain
  • 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.
  • phosphorylation of Serine 96 was shown to inhibit degradation of ⁇ -Catenin, an important mediator of Wnt-signalling (Song, S., et al, 2009, Cancer Res 69, 1343-1349).
  • Galectin-3 can be found intracellular ⁇ , in the extracellular matrix and the circulation.
  • 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.
  • 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
  • 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.
  • Galectin-3 levels in human plasma may serve as a biomarker indicating how permissive the systemic environment is to osteogenic differentiation.
  • 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
  • agents with the ability to increase the level of Galectin-3 in mesenchymal stem cells selected from
  • Galectin-3 or fragments or variants or derivatives thereof or ii. nucleic acid molecules encoding Galectin-3 or fragments or
  • agents with the ability to decrease the level of Galectin-3 in mesenchymal stem cells selected from agents inhibiting Galectin-3 expression in mesenchymal stem
  • Galectin-3 may be any isoform of the protein.
  • a variant Galectin-3 polypeptide or a fragment or a nucleic acid molecule encoding such variant or fragment may be used.
  • chemically similar amino acids may be substituted for amino acids in the Galectin-3 protein sequence (to provide conservative amino acid substitutions).
  • 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.
  • the therapeutically active agent is a Galectin-3 derivative in which Galectin-3 is coupled to a chemical moiety that effects an increase of its half-life, activity or uptake in bone.
  • 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.
  • derivatization may be achieved by genetic modification that results in an N-terminal cyclic conformation (Cao et al., 2012,
  • Intralipid® an FDA-approved fat emulsion
  • Galectin-3 an FDA-approved fat emulsion
  • the therapeutically active agent is a peptide
  • Galectin-3 phosphorylation may act as a scavenger of ⁇ -Catenin, excess Galectin-3 may compete with ⁇ -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.
  • a Galectin-3 peptide has a length of about 8 - 30 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 ai., 2007 (Tissue Eng 13, 1 173-1 183) and Wolbank ef a/., 2009 (Tissue Eng Part A 15, 1843-1854).
  • 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).
  • ALP early osteogenic marker alkaline phosphatase
  • ON osteonectin
  • osteocalcin OC
  • Galectin-3 refers both to the naturally occurring protein and its therapeutically active
  • 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 .
  • 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.
  • Galectin-3 is the effective agent in a pharmaceutical
  • composition to be administered systemically/parenterally e.g. by subcutaneous bolus injection.
  • bone-targeting molecules 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.
  • 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).
  • 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, 2011 , Calcif. Tissue Int. 88 51 1-520).
  • 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).
  • 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).
  • the invention relates to Galectin-3, or a fragment or variant thereof, linked to a bone-targeting molecule.
  • Galectin-3 or a gene construct containing the Galectin-3 encoding DNA is contained in a delivery vehicle.
  • 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.
  • DOTAP dioleoyl trimethylammonium propane
  • 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 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
  • GHDD ghosal hematodiaphyseal dysplasia syndrome
  • osteoporosis osteogenesis
  • osteopenia Paget's disease, osteomyelitis, hypercalcemia, osteonecrosis, hyperparathyroidism, lytic bone metastases, periodontitis, and bone loss due to immobilization.
  • osteoporosis includes any form of osteoporosis.
  • osteoporosis includes primary osteoporosis, post-menopausal and age-related osteoporosis, endocrine osteoporosis (including hyperthyroidism, hyperparathyroidism, Gushing's syndrome, and
  • osteoporosis including osteogenesis imperfecta, homocystinuria, Menkes' syndrome, Riley-Day syndrome
  • 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, immunosuppressive 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
  • DXA or DEXA dual-energy X-ray absorptiometry
  • QCT quantitative computed tomography
  • QUS qualitative ultrasound
  • SPA single photon absorptiometry
  • DPA dual photon absorptiometry
  • DXR digital X-ray radiogrammetry
  • SEXA single energy X-ray absorptiometry
  • bone density which is considered to be an indicator also of bone mass.
  • 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.
  • 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.
  • 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.
  • 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 person's Galectin-3 level in plasma.
  • Galectin-3 level is used as the only test for diagnosing a disorder correlating an aberrant BMD.
  • 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.
  • 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.
  • 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.
  • PAM protein activated matrix
  • 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 wel! as methods for incorparting the therapeutic agent into such composites are provided by Lauzon et al., 2012 (Journal of Controlled Release 162, 502-520), and by Soundrapandian et al., 2009 (AAPS
  • biomaterial matrices are described in US 20130195863.
  • a Galectin-3 protein or peptide may be operably linked to a CPP ("cell penetrating peptide").
  • CPPs also known as protein transduction domains (PTDs)
  • PTDs protein transduction domains
  • a CPP-linked Galectin-3 may be obtained by recombinant production of the respective Galectin-3/CPP fusion protein/peptide.
  • the Galectin-3-containing composition contains components which are pharmaceutically acceptable. These may be in particular isotonic, sterile, saline solutions (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.
  • 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.
  • Vectors may be prepared from different type of viruses, including adenoviruses, adeno-associated viruses (AAV), herpes viruses (HSV), lentiviruses and retroviruses.
  • AAV adeno-associated viruses
  • HSV herpes viruses
  • lentiviruses lentiviruses
  • retroviruses retroviruses.
  • adenovirus vector is preferably used.
  • 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
  • a third generation adenovirus vector in which all viral coding sequences are deleted 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).
  • the Galectin-3 therapeutic may be administered in the form of a naked DNA or mRNA molecule.
  • 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.
  • 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.
  • BMP-2 bone morphogenic protein 2
  • 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
  • the promoter cloned upstream of the Galectin-3 encoding DNA sequence is the COL or the RUNX promoter, preferably the RUNX promoter.
  • 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.
  • viral or non-viral vectors As reviewed by Pelled et al.. 2010 (Tissue Engineering: Part B, Volume 16, 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.
  • the DNA molecule 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.
  • an adenoviral vector is used, as described for expressing bone morphogenetic protein (BMP) Egermann et ai, 2006 (Hum Gene Ther. May;17(5):507-17).
  • BMP bone morphogenetic protein
  • in vivo electroporation or sonoporation may be used to deliver the therapeutic locally.
  • 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.
  • 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).
  • localizing Galectin-3 at the site of interest e.g. the fracture site, e.g. by transgene expression
  • Galectin-3 DNA/RNA to a delivery system (e.g. by adsorption, entrapment or immobilization, or by covalent binding; Luginbuehl et ai, 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 a/., 1996 (Proc Natl Acad Sci USA 93, 5753).
  • the DNA may be condensed by chemical vectors such as
  • polyethyleneimine, liposomes or calcium-phosphate precipitates are examples of polyethyleneimine, liposomes or calcium-phosphate precipitates.
  • GAMs Useful matrices (GAMs, "gene-activated matrices") have been described above in the context with matrices for the delivery of the Galectin-3 protein.
  • 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.
  • 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.
  • the delivery vehicle e.g. a liposome
  • incorporation of the bone- targeting molecule is achieved by linking it to the surface of the delivery vehicle. The same applies for a CPP.
  • a Galectin-3 therapeutic of the invention 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 .
  • 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 .
  • the inhibitor of Galectin-3 expression may be selected from any RNAi (RNA interference) molecule including siRNAs, miRNAs, LNAs, phosphorothioate RNAs, antisense
  • RNAi RNA interference
  • oligonucleotides oligonucleotides, ribozymes and aptamers.
  • 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 prevents it.
  • 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.
  • an inhibitor of Galectin-3, linked to a bone-targeting molecule is the effective agent in a pharmaceutical composition to be administered
  • GAM matrix
  • bolus a matrix or bolus
  • 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 .
  • CPPs also known as protein transduction domains (PTDs)
  • PTDs protein transduction domains
  • 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%) is indicative for an aberrant bone mineral density.
  • the Galectin-3 protein level is determined by using an antibody binding specifically to Galectin-3.
  • neither the type of antibody nor the Galectin-3 epitope that it recognizes is critical.
  • the antibody recognizes the full length of the Galectin-3 molecule
  • Galectin-3 levels are determined using an ELISA assay.
  • ELISA refers to enzyme-linked immunosorbent assay (or EIA).
  • an ELISA method is a "direct ELISA," wherein the Galectin-3 antigen in a sample is detected.
  • 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.
  • an "indirect ELISA” is used.
  • 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.
  • a "sandwich ELISA” is used, where Galectin-3 (e.g.
  • a test sample 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • the Galectin-3 level may be determined directly in the plasma, i.e. without a preceding enrichment of MVs.
  • Endothelial cells were isolated from human umbilical veins as described
  • HUVECs were cultivated in gelatin-precoated flasks in EGM (Lonza) at 37°C in a humidified atmosphere with 5% C0 2 . 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 ⁇ -galactosidase (SA- ⁇ - 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-p-gal positive) cells were allowed to secrete into HUVEC medium, depending on the experiment, for 48 hours.
  • SA- ⁇ - gal senescence- associated ⁇ -galactosidase
  • ASCs Human adipose-derived stem cells
  • Subcutaneous adipose tissue was obtained during outpatient tumescence liposuction under local anestesia.
  • ASCs were isolated as described (Wolbank et al., 2007, Tissue Eng 13, 1173-1183; Wolbank et 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% C0 2 and 95% air humidity. Cells were passaged once or twice a week at a split ratio of 1 :2 according to the growth rate.
  • osteogenic differentiation All differentiation protocols were carried out in 24 well cell culture plates.
  • ASCs were seeded at a density of 2x10 3 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 ⁇ ascorbate-2-phosphat, 10 mM ⁇ -glycerolphosphate and 10 nM vitamine-D3) up to 4 weeks.
  • ASCs Human adipose-derived stem cells
  • ASCs were transfected using Neon® Transfection System (Life technologies). Cells were transfected according to the manufactures protocol. Briefly, 1x10 5 ASCs resuspended in 10 ⁇ buffer R, were mixed with 1 g of DNA or 10 pmol of siRNA and loaded into the Neon TM pipet tip. Subsequently cells were
  • ON- TARGETplus Non-targeting Pool (20 nmol) (D-001810-10-20, Dharmacon), ON- TARGETplus SMARTpool, Human LGALS3, (5 nmol) (L-010606-00-0005, Dharmacon).
  • HUVECs were transfected using Neon® Transfection System (Life technologies). Cells were transfected according to the manufactures protocol. Briefly; 5x10 5 HUVECs resuspended in 100 ⁇ buffer R were mixed with 5 pg 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
  • 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).
  • ALP early osteogenic marker alkaline phosphate
  • ON osteogenic marker osteonectin
  • OC late osteogenic marker osteocalcin
  • 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 NMJ3031 18
  • MVs were purified by filtration and differential centrifugation as described by
  • 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 ⁇ of monoclonal CD63 antibody (ab8219 Abeam) were mixed with 200 ⁇ of C1 solution. Washed beads were first mixed with prepared antibody solution and 250 ⁇ of C2 solution were added afterwards. Beads were incubated at 37°C on a roller over night.
  • MVs 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).
  • TEM transmission electron microscopy
  • Philips model CM 12 electron microscope Philips, Eindhoven, NL
  • MVs were incubated for at least 4 hours with hybridization buffer as described 98.
  • 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, ml-1 yeast RNA, 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.
  • 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.
  • 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.
  • ASCs were seeded one day before exposing them to plasma derived MVs for 72 hours. After 3 days osteogenic differentiation was induced as described (Woibank et a/., 2009,Tissue Eng Part A 15, 1843-1854).
  • 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.
  • Microvesicular Galectin-3 is elevated in healthy young donors
  • 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).
  • 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.
  • 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.
  • 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.
  • Endothelial cells are a possible source of Galectin-3 containing plasma derived microvesicles Since endothelial cells Sine 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.
  • HUVECs human umbilical vein endothelial cells
  • Endothelial microvesicles deliver genetic information to adipose-derived mesenchymal stem cells
  • 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.
  • 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.
  • MVs of senescent cells effect the differentiation capacity of ASCs differently when compared to MVs of early passage cells.
  • 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.
  • 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.
  • Vesicular Galectin-3 influences osteogenic differentiation capacity
  • 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.
  • Galectin-3 Overexpression of Galectin-3 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. 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

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Abstract

La présente invention concerne des compositions et des méthodes destinées au traitement, à la prophylaxie et au diagnostic de troubles associés à une densité minérale osseuse aberrante. Plus particulièrement, l'invention concerne l'augmentation des taux de Galectine-3 dans les cellules souches mésenchymateuses pour augmenter l'ostéogenèse. Les compositions comprenant de la galectine-3 ou des acides nucléiques codant pour la galectine-3 peuvent être administrées par voie systémique ou locale et sont utiles dans le traitement de maladies telles que l'ostéoporose et dans l'accélération de la cicatrisation osseuse. L'invention concerne également des méthodes permettant de diagnostiquer des maladies osseuses, dans lesquelles est mesuré le taux de Galectine-3.
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WO2017044940A1 (fr) * 2015-09-10 2017-03-16 Washington State University Nanovésicules à membrane cellulaire et leurs procédés d'utilisation
WO2018015761A1 (fr) * 2016-07-21 2018-01-25 University Of Leeds Matrices biocompatibles pour le transfert de molécules biologiques
US11427638B2 (en) 2019-01-30 2022-08-30 Truebinding, Inc. Anti-Gal3 antibodies and uses thereof

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