WO2020124229A1 - Stabilized osteocalcin and uses thereof - Google Patents

Stabilized osteocalcin and uses thereof Download PDF

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WO2020124229A1
WO2020124229A1 PCT/CA2019/051843 CA2019051843W WO2020124229A1 WO 2020124229 A1 WO2020124229 A1 WO 2020124229A1 CA 2019051843 W CA2019051843 W CA 2019051843W WO 2020124229 A1 WO2020124229 A1 WO 2020124229A1
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ocn
osteocalcin
rocn
gly
glycosylated
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PCT/CA2019/051843
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French (fr)
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Mathieu FERRON
Omar AL RIFAI
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Adaerata, Limited Partnership
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present disclosure relates to stabilized osteocalcin and uses thereof. More specifically, the present disclosure is concerned with osteocalcin (OCN) stabilized by O-glycosylation of at least one of its residue and uses of this OCN.
  • OCN osteocalcin
  • Sequence Listing G12810-701_ST25 is submitted herewith as an ASCII compliant text file named Sequence Listing G12810-701_ST25, that was created on December 12, 2019 and having a size of 53 kilobytes.
  • the content of the aforementioned file named Sequence Listing G12810-701_ST25 is hereby incorporated by reference in its entirety.
  • Osteocalcin is a bone-derived hormone regulating glucose metabolism
  • Osteocalcin is a short protein, 46 amino acid-long in the mouse and 49 amino acid-long in human produced and secreted specifically by osteoblasts. Inactivation of the two genes encoding osteocalcin ( Bglapl and Bglap2) has profound metabolic consequences in mice fed a normal chow diet (Lee et al., 2007). Most strikingly, osteocalcin deficient mice (Ocn ' ) are characterized by an increased adiposity, low circulating levels of insulin, reduced peripheral insulin sensitivity and decreased glucose tolerance. They also display liver steatosis and signs of inflammation in the liver and white adipose tissue.
  • osteocalcin causes a marked reduction in global energy expenditure, which likely contributes to the increased fat mass of these animals.
  • Intermittent injections of recombinant osteocalcin in lean or obese mice or rats resulted in phenotypes opposite to the ones observed in the Ocrr'- mice, i.e., they increased energy expenditure, reduced fat mass, improved insulin sensitivity and prevented liver steatosis (Ferron et al., 2012; Ferron et al., 2008; Huang et al., 2017; and Gupte et al., 2014).
  • mouse models in which the circulating level of the active form of osteocalcin is increased are characterized by increased insulin secretion, increased b-cell proliferation, improved insulin sensitivity, increased energy expenditure and reduced fat mass (Lee et al., 2007; Ferron et al., 2015).
  • osteocalcin The acute effect of osteocalcin on insulin secretion appears to be mediated by calcium (Ca 2+ ) signaling in b-cells (Hinoi et al., 2008).
  • a receptor to which osteocalcin binds in pancreatic b-cells a G protein-coupled receptor called GPRC6A that acts through G-Protein a-Subunit (Gsa) and C-AMP Response Element-binding protein (CREB), mediates osteocalcin regulation of b-cell proliferation, insulin expression and insulin secretion.
  • This receptor is also expressed in myofibers and regulates the functions of osteocalcin myofibers during exercise (Mera et al., 2016b) and the effect of osteocalcin on male fertility.
  • osteocalcin stimulates the expression of adiponectin, a hormone that regulates bone mass accrual in animals fed a normal diet, but this action appears to be independent of GPRC6A that is not expressed in adipocytes (Kajimura et al., 2013).
  • osteocalcin suppresses lipolysis, promotes directly glucose uptake and can suppress the secretion of pro-inflammatory cytokines in white adipocytes (Lee et al., 2007; Hill et al., 2014).
  • Osteocalcin also promotes mitochondria biogenesis in the muscle when injected in obese mice (Ferron et al., 2012) and stimulates the expression of genes involved in thermogenesis (Pgd crand Ucp1) in brown adipocytes in vivo and ex vivo (Ferron et al., 2008).
  • osteocalcin also stimulates insulin secretion in rat and in human islets ex vivo (Sabek et al., 2015; Gao et al., 2016a; Gao et al., 2016b; and Kover et al., 2015).
  • genetic studies further support an impact of osteocalcin polymorphisms in glucose and energy metabolism in humans (Korostishevsky et al., 2012; Das et al., 2010).
  • osteocalcin serum level is an independent risk factor for the development of type 2 diabetes (Liu et al., 2015).
  • osteocalcin differs from insulin that is an anabolic hormone.
  • Osteocalcin supports muscle function during exercise through an additional mechanism: it stimulates the production and release of interleukin-6 (IL-6), one of the first myokines ever identified (Pedersen et al., 2008; Steensberg et al., 2000).
  • IL-6 circulating levels increase after exercise in humans and rodents in a manner that is proportional to the length of exercise and the amount of muscles involved (Febbraio et al., 2004; Febbraio et al., 2002; and Nielsen et al., 2007).
  • IL-6 is expressed in cultured myotubes and myofibers, and skeletal muscle is the major source of circulating IL-6 during exercise (Keller et al., 2001 ; Steenbergen et al., 2002). During exercise, IL-6 acts in an autocrine, paracrine and endocrine manner to promote skeletal muscle nutrient utilization, glucose production in the liver and lipolysis in white adipose tissue (Pedersen et al., 2008).
  • Osteocalcin increases skeletal muscle function and adaptation to exercise
  • osteocalcin In addition to its role in energy metabolism, osteocalcin modulates the reproductive function of males.
  • the hormone acts on the Leydig cells of the testis to activate the production of cAMP, which results in a CREB-dependent transcription of at least four genes involved in testosterone synthesis: StAR, Cyp11, 3b-H80 and Cyp17. Therefore, osteocalcin stimulates the production of testosterone in the testis and is essential to male reproductive system maturation.
  • Ocn'- male mice have reduced circulating testosterone level and sperm count, smaller testes and seminal vesicles, and are subfertile (Oury et al., 201 1 ).
  • injection of recombinant osteocalcin can increase serum level of testosterone and sperm count in WT mice (Oury et al., 2013b).
  • osteocalcin In addition to its peripheral action, osteocalcin crosses the blood-brain barrier, binds to neurons of the brainstem, midbrain, and hippocampus, enhances the synthesis of monoamine neurotransmitters and inhibits GABA synthesis. Consequently, osteocalcin prevents anxiety and depression, and favors learning and memory (Oury et al., 2013a). Interestingly, intermittent injections of recombinant osteocalcin in 16-month-old mice can improve learning and memory, and reduced anxiety (Khrimian et al., 2017).
  • GPRC6A is the osteocalcin receptor in b-cells and in testis
  • osteocalcin mediates its functions through the binding of at least one specific receptor: GPRC6A.
  • This G protein-coupled receptor shares some sequence identity with the calcium sensing receptors (CASR), which is involved in calcium homeostasis through the regulation of parathyroid hormone (PTH) release.
  • CCR calcium sensing receptors
  • PTH parathyroid hormone
  • GPRC6A was proposed to be a cation-sensing receptor, and more recently, a receptor for amino acids, steroids and osteocalcin (Pi et al., 2012; Wei et al., 2014).
  • Gprcea 1 - mice phenocopied the osteocalcin-deficient animals with regard to their defects in insulin secretion and glucose tolerance (Pi et al., 2008).
  • inactivation of Gprc6a specifically in the pancreas resulted in reduced b-cell proliferation and decreased insulin secretion in response to glucose (Wei et al., 2014; Pi et al., 2008).
  • osteocalcin capacity to induce insulin secretion is abrogated in GprcQa '- islets (Wei et al., 2014; Pi et al., 2016).
  • GPRC6A also acts as an osteocalcin receptor in Leydig cells, where osteocalcin dependent signaling promotes testosterone synthesis (Oury et al., 2011 ; De Toni et al., 2014).
  • osteocalcin The endocrine function of osteocalcin is regulated by gamma-carboxylation
  • OCN To become active as a hormone, OCN undergoes a series of posttranslational modifications.
  • 3 glutamic acid (Glu) residues of OCN positioned at residues nos. 17, 21 , and 24 using the numbering of the circulating human OCM fragment (or nos. 68, 72 and 75 using the numbering the full encoded protein in human); 13, 17, and 20 using the numbering of the circulating mouse OCM fragment (or nos. 62, 66 and 69 using the numbering the full encoded protein in mouse) are modified into y-carboxyglutamic acid residues (Gla) through g-carboxylation, an enzymatic process reguiring vitamin K.
  • Glu glutamic acid
  • Gla residues increases OCN calcium-binding properties and results in its association with hydroxyapatite in the bone extracellular matrix (ECM).
  • ECM bone extracellular matrix
  • OCN then undergoes a second posttranslational modification during the resorption phase of bone remodeling.
  • the acidic environment generated by osteoclasts induces a loss of g-carboxylation on the first Glu residue (Glu17 in human; Glu13 in mouse) of OCN presents in bone ECM, thereby generating undercarboxylated Glu17-OCN (ucOCN) that is released in blood (Ferron et al., 2010a; Lacombe et al., 2013).
  • osteocalcin is first synthesized as a prohormone (pro-osteocalcin or pro-OCN). Yet until recently, the biological importance of pro-OCN maturation in regulating osteocalcin and the identity of the endopeptidase responsible for pro-OCN cleavage in osteoblasts were unknown. Based on biochemical and genetic arguments the proprotein convertase furin was identified by the inventors as the endopeptidase responsible for pro- OCN processing in osteoblasts (Al Rifai et al., 2017).
  • pro-OCN The proteolysis of pro-OCN is critical for the activation of this hormone, since inactivation of furin in osteoblasts in mice results in decreased circulating levels of active osteocalcin, impaired glucose tolerance and reduced energy expenditure. At the mechanistic level, it appears that the retention of the pro-peptide in osteocalcin reduces its ability to be decarboxylated during the process of bone resorption.
  • FIGs. 1A-B Osteocalcin is O-glycosylated in mouse osteoblasts.
  • FIG. 1A List of the most abundant forms of osteocalcin identified in mouse osteoblasts supernatant. The form for which a tandem mass spectrometry (MS/MS) spectrum is shown in FIG. 2 is in bold.
  • FIG. 1 B Schematic representation of the identified glycan adducts.
  • FIGs. 2A-B position of sugar on mature mouse OCN and nature of various peptides fragments (identified as Y and b) characterized in FIG. 2B.
  • the lines below each Y and b indicates the starting position of peptide and its direction and the number beside the y and b provides the size of the peptide in terms of amino acid number);
  • FIG. 2B Annotated HCD MS/MS spectrum of a modified form of osteocalcin (HexNAc-Hex-NANA + 3 Gla + S-S) (SEQ ID NO: 1 )) showing the m/z value of the different peptide fragments (y and b) of osteocalcin.
  • the m/z value of the mature unfragmented osteocalcin is 1 180.9501 (M +5 H) +5 and mass accuracy with the annotated Osteocalcin modified form is 4.6 ppm.
  • FIGs. 3A-B Mouse osteocalcin is O-glycosylated in vitro.
  • FIG. 3A Western blot analysis on the supernatant of control and core i 3-Gal-T-specific molecular chaperone (COSMO) (C1GALT1C1, core 1 3-Gal-T- specific molecular chaperone) KO HEK293 transfected with mouse OCN-V5.
  • FIG. 3B Western blot analysis on the supernatant of CHO and CHO-ldID cells transfected with mouse OCN-V5.
  • CHO-ldID cells were treated or not with 1 mM of N-AcetylGalactoseAmine (GalNAc) alone or in combination with 0.1 mM Galactose (Gal).
  • FIGs. 4A-B Mouse osteocalcin is O-glycosylated in vitro.
  • FIG. 4A Western blot analysis on the supernatant of osteoblasts transfected with mouse OCN-V5 and treated or not with 2mM of GalNAc-bn (a pharmacological inhibitor of N-acetylgalactosamine Transferases (GalNAc-Ts)).
  • FIG. 4B Western blot analysis of osteocalcin deglycosylation assay.
  • FIGs. 5A-C Mouse osteocalcin O-glycosylation is independent of its processing and y-carboxylation. OCN processing and g-carboxylation (Gla) in osteoblasts (FIG. 5A) and HEK293 (FIGs.
  • FIG. 6 Mouse osteocalcin O-glycosylation is independent of its processing in vivo. Western blot analysis of osteocalcin deglycosylation assay. Bone extract of Furin m and Furin 0sb -'- mice were treated or not with O-glycosidase and neuraminidase for 4 hours at 37°C and analyzed by western blot using OCN antibody.
  • FIGs. 7A-C Mouse osteocalcin is O-glycosylated on the serine 57 residue.
  • FIG. 7A Mature mouse osteocalcin amino acid sequence (SEQ ID NO: 1 ).
  • FIGs. 7B-C Western blot analysis on the supernatant of osteoblasts transfected with WT and different mutated forms of mouse OCN-V5.
  • FIG. 7B Western blot analysis on the supernatant of osteoblasts transfected with WT and different mutated forms of mouse OCN-V5.
  • SST/AAA mutant (Serine 54, Serine 57 and Threonine 64 were mutated to Alanine), STT/AAA (Serine 78, Threonine 85 and Threonine 94 were mutated to Alanine), 6ST/6A (Serine 54, Serine 57, Threonine 64, Serine 78, Threonine 85 and Threonine 94 were mutated to Alanine).
  • FIG. 70 SST/AAA mutant
  • S54A Serine 54 was mutated to Alanine
  • S57A Serine 57 was mutated to Alanine
  • T64A Threonine 64 was mutated to Alanine
  • SST/AAA mutant Serine 54, Serine 57 and Threonine 64 were mutated to Alanine
  • FIGs. 8A-D Osteocalcin O-glycosylation on the Serine 57 residue is independent of its processing and y-carboxylation.
  • Western blot analysis using anti V5 antibody (FIG. 8A and FIG. 80)) and ELISA analysis of % carboxylated OCN (FIG. 8B and FIG. 8D).
  • FIGs. 9A-B Different N-acetylgalactosamine Transferases (GalNAc-Ts) O-glycosylated OCN.
  • FIG. 9A Galnts expression in undifferentiated osteoblasts, results are represented as copy number of Galnt relative to Actb.
  • FIG. 9B Western blot analysis of OCN O-glycosylation in HEK293 deficient in COSMC or specific GALNTs.
  • OCN-V5 were transfected in HEK293, COSMC or GALNTs deficient HEK293. Cell supernatant was analyzed by western blot using anti V5 antibody.
  • FIGs. 10A-C Purification of O-glycosylated mouse OCN. Coomassie staining of purified O-glycosylated mouse OCN (O-gly OCN) compared to non-O-glycosylated mouse OCN (OCN).
  • FIG. 10B Purity of the purified mouse OCN . Annotated HOD MS/MS spectrum of purified non O-glycosylated mouse OCN (OCN).
  • FIG. 10C Production of pure O-glycosylated mouse OCN . Annotated HCD MS/MS spectrum of purified O-glycosylated mouse OCN (0- gly OCN). 2 different O-glycosylation adducts were detected and annotations thereof are shown .
  • FIG. 12 The decrease in OCN half-life is sensitive to temperature.
  • O-gly OCN O-glycosylated OCN
  • OCN non-O-glycosylated OCN
  • HI heat inactivated
  • FIGs. 13A-B In vivo half-life of O-glycosylated OCN (O-gly OCN) and non-O-glycosylated OCN (OCN) in fed condition.
  • Serum was collected at the indicated time points and OCN levels were then measured using total OCN ELISA assay. Results are given as mean ⁇ SEM. **, p ⁇ 0.01 ; ***, p ⁇ 0.001 using 2-way ANOVA for repeated measurements with Bonferroni multiple comparisons testing.
  • FIG. 14 OCN O-glycosylation increases its half-life in vivo in a dose dependent manner in fed condition.
  • O-gly OCN O-glycosylated OCN
  • OCN non-O-glycosylated OCN
  • OCN levels were measured at the indicated time points using total OCN ELISA assay. Results are given as mean ⁇ SEM. *, p ⁇ 0.05; using 2-way ANOVA for repeated measurements with Bonferroni multiple comparisons testing.
  • FIGs. 15A-B OCN O-glycosylation increase its half-life in vivo in fasting condition.
  • O-gly OCN O-glycosylated OCN
  • OCN non-O-glycosylated OCN
  • I. P OCN deficient male mice
  • I. P OCN
  • Serum was collected at the indicated time points and OCN levels were measured at the indicated time points using total OCN ELISA assay as an absolute concentration (ng/ml) (FIG.
  • FIGs. 16A-C Human OCN is not naturally O-glycosylated, but Y63S mutation (Tyrosine 63 in human pro-OCN was mutated to Serine) is sufficient to induce its glycosylation.
  • FIG. 16A Amino acid alignment of mouse (SEQ ID NO: 1 ) and human (SEQ ID NO: 3) mature OCN, highlighted residues represent the alignment of mouse OCN potential O- glycosylation sites compare to human OCN. None of them is conserved. The star indicates the site of O-glycosylation in mouse OCN.
  • FIG. 16B shows the site of O-glycosylation in mouse OCN.
  • FIG. 16C Purification of O-glycosylated human OCN from the Fclg construct (FIGs. 23 and 24D). Coomassie staining of purified O-glycosylated human OCN (O-gly hOCN) compared to non-O-glycosylated human OCN (hOCN).
  • FIGs. 17A-B FIG. 17A. Purity of the purified human OCN (hOCN). Annotated HOD MS/MS spectrum of purified non O-glycosylated human OCN (hOCN). FIG. 17B. Production of pure O-glycosylated human OCN (hOCN). Annotated HOD MS/MS spectrum of purified O-glycosylated human OCN (O-gly hOCN). One O-glycosylation adduct (HexNAc, Flex, 2NANA) was detected and annotations thereof are shown .
  • FIGs. 18A-G Nucleotide and amino acid sequences of mouse and human pre-pro-OCN.
  • FIGs. 18A-C nucleotide sequence (FIG. 18A) (SEQ ID NO: 4) and amino acid sequence (FIG. 18B) (SEQ ID NO: 5) of mouse pre-pro-OCN, identifying expressly in FIG. 18C the various domains of native mouse pre-pro-OCN (SEQ ID NO: 6).
  • FIGs. 18D-G nucleotide sequence (FIG. 18D) (SEQ ID NO: 6) and amino acid sequence (FIG. 18E) (SEQ ID NO: 7) of human pre- pro-OCN, identifying expressly in FIGs.
  • FIG. 18F-G the various domains of native human pre-pro-OCN (FIG. 18F) (SEQ ID NO: 7) and Y63S mutated human pre-pro-OCN which is artificially O-glycosylated (FIG. 18G) (SEQ ID NO: 8).
  • FIG. 20 The decrease in hOCN half-life is sensitive to temperature.
  • O-gly hOCN O-glycosylated hOCN
  • hOCN non-O-glycosylated hOCN
  • OCN deficient mice Ocn-/- heparin plasma
  • HI heat inactivated
  • hOCN levels were measured at the indicated time points using total hOCN ELISA assay. Results are given as mean ⁇ SEM. ** , p ⁇ 0.01 ; using one-way ANOVA with Bonferroni multiple comparisons testing.
  • FIG. 21 Construct’s map to produce and purify glycosylated mouse OCN fused to Fc of hlgG1. Partial map of the plasmid showing the key features allowing the expression and purification of glycosylated mouse OCN from mammalian cells. The enzymatic restriction sites used for the cloning are shown above the map.
  • CMV promoter for transcription initiation
  • KOZAK sequence for translation
  • SP Signal peptide to allow protein trafficking in the secretory pathway
  • Fc of hlgG1 Fc of human immunoglobulin (hlgG1 ) to allow affinity purification (e.g., using protein G or A column or beads)
  • Hinge region protein spacer between Fc domain and mouse OCN to provide flexibility to OCN and facilitate cleavage by thrombin; thrombin cleavage site to release mouse OCN following digestion using thrombin
  • Poly(A) signal transcription termination signal.
  • FIG. 22B Amino acid sequence of mouse OCN fused to Fc of hlgG1 (SEQ ID NO: 10).
  • FIG. 220 Schematic representation of amino acid sequence of mouse OCN fused to Fc of hlgG1 (SEQ ID NO: 10). Arrow: cleavage site by thrombin.
  • FIG. 23 Construct’s map to produce and purify glycosylated human OCN Y63S (Gly-rhOCN) fused to Fc of hlgG1. Partial map of the plasmid showing the key features allowing the expression and purification of glycosylated human OCN from mammalian cells. The enzymatic restriction sites used for the cloning are shown above the map. The position of the Y63S mutation in human OCN allowing its glycosylation is shown under the map.
  • CMV promoter for transcription initiation
  • KOZAK sequence for translation
  • SP Signal peptide to allow protein trafficking in the secretory pathway
  • Fc of hlgG1 Fc of human immunoglobulin to allow affinity purification (e.g., using protein G or A column or beads)
  • Hinge region protein spacer between Fc domain and human OCN to provide flexibility to OCN and facilitate cleavage by thrombin; thrombin cleavage site to release mouse OCN following digestion using thrombin
  • Poly(A) signal transcription termination signal.
  • FIGs. 24A-E Nucleotide sequence of human OCN fused to Fc of hlgG1 (SEQ ID NO: 1 1).
  • FIG. 24B Amino acid sequence of human OCN fused to Fc of hlgG1 (SEQ ID NO: 12).
  • FIG. 24C Nucleotide sequence of glycosylated human OCN Y63S fused to Fc of hlgG1 (SEQ ID NO: 13).
  • FIG. 24D Amino acid sequence of glycosylated human OCN Y63S fused to Fc of hlgG1 (SEQ ID NO: 14).
  • FIG. 24E Schematic representation of amino acid sequence of glycosylated human OCN Y63S fused to Fc of hlgG1. Arrow: cleavage site by thrombin.
  • FIG. 25 Mouse OCN and glycosylated human OCN constructs are well expressed in HEK293 cells. Expression analysis of pcDNA3-FchlgG1 ; pcDNA3-FchlgG1 -mOCN; pcDNA3-FchlgG1-mOCN (S57A); pcDNA3-FchlgG1 -hOCN; pcDNA3-FchlgG1 -hOCN (Y63S) and pcDNA3 construct transfected in HEK293. Western blot analysis on using HRP goat antihuman Fc antibody and human OCN antibody.
  • FIGs. 26A-B Generation of stable clones secreting high amounts of glycosylated mouse and human osteocalcin.
  • ELISA measurements of mouse and human OCN ELISA measurements of mouse and human OCN .
  • FIG. 26A mouse OCN level in the supernatant of clones expressing glycosylated mouse OCN fused to hlgG1 Fc domain.
  • FIG. 26B human OCN level in the supernatant of clones expressing glycosylated human OCN Y63S (Gly-rhOCN) fused to hlgG1 Fc domain. Results are normalized to total protein content in the cell lysate.
  • FIGs. 27A-B Table presenting mouse (SEQ ID NO: 1) and human (SEQ ID NO: 3) mature osteocalcin amino acid sequences and reported coding single nucleotide polymorphisms (SNPs) in mature human osteocalcin protein.
  • SNPs single nucleotide polymorphisms
  • the present disclosure describes a human osteocalcin modified to include an O-glycosylation.
  • the inventors engineered an artificially glycosylated human osteocalcin and demonstrated that this increased the stability of the active protein in human plasma. They also showed that ex vivo O-glycosylation does not interfere with osteocalcin y- carboxylation or processing by furin.
  • the disclosure also describes a method to express and purify a large amount of O-glycosylated mouse and human osteocalcin and provides a method of increasing the stability of human osteocalcin.
  • the present disclosure also provides a method of using the O-glycosylated osteocalcin in the treatment or prevention of an osteocalcin associated disease or condition, or of a symptom thereof.
  • Glycosylated recombinant osteocalcin comprising the sequence of formula (I):
  • Item 2 The gly-rOCN of item 1 , wherein the at least one of X9, X12 or X19 is O-glycosylated S or T.
  • Item 3 The gly-rOCN of item 1 , wherein X12 is O-glycosylated S or T.
  • Item 4 The gly-rOCN of any one of items 1 to 3, wherein
  • X2 is L
  • X 3 is T
  • X 5 is W
  • X 7 is G
  • Xe is A
  • X 9 is P
  • X11 is P
  • Xi3 is P
  • Xi4 is D
  • Xi5 is P
  • Xi9 is R
  • X25 is L
  • X26 is N
  • X33 is A
  • X 3 6 is I
  • X37 is G
  • X42 is Y
  • X43 is R
  • X44 is R
  • X45 is F; X47 is G; and
  • X' 2 is P
  • X' 4 is P
  • X's is R
  • X's is V
  • X' 7 is E
  • X's is P or G
  • X'g is A
  • X'ii isS or H
  • X'i3 is R or L
  • X'i 4 isG, Y or E;
  • X'i 5 isT, AorS;
  • X'i9 isG orS
  • Item 7 The gly-rOCN of item 4, wherein up to 5 of X2-X9, Xu, X13 -X15, X19, X25-X27, X30-X31, X33, X36-X37, X40, X42-X45, and X47-X48 as defined in item 4, is substituted by its corresponding X’ defined in item 4.
  • Item 8 The gly-rOCN of any one of items 1 to 3, wherein
  • X 2 is L or P
  • X 3 is T or C
  • X 4 is Q or P
  • X 5 is W or R
  • Cb is L or V
  • X 7 is G or E
  • Xs is A, P or G
  • X 9 is P or A
  • X 11 is P, S or H
  • Xi 3 is P, R or L
  • Xi 4 is D, G, Y or E;
  • Xi 5 is P, T, A or S;
  • Xi 9 is R, G or S;
  • X 26 is N or S
  • X27 is P or L
  • S; X43 is R, W or Q;
  • X 44 is R, C, H or P
  • X45 is F or Y
  • X47 is G, S or R;
  • X48 is P, S or L.
  • YLYQWLGAPVPX12PDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV wherein X i2 is O-glycosylated S or T.
  • Item 10 The gly-rOCN of any one of items 1 -9, wherein X12 is O-glycosylated S.
  • Item 1 The gly-rOCN of any one of items 1 -10, in its circulating form.
  • Item 12 The gly-rOCN of any one of items 1 -1 1 , in its active form.
  • Item 13 An isolated nucleic acid molecule encoding the rOCN defined in any one of items 1-12.
  • Item 14 An expression vector comprising the nucleic acid molecule defined in item 13.
  • Item 15 A host cell transformed or transfected with the vector defined in item 14.
  • Item 16 A method of producing the gly-rOCN defined in any one of items 1-12, comprising culturing the host cell defined in item 14 under conditions suitable to effect expression of the gly-rOCN and recovering the gly-rhOCN.
  • Item 17 A method of increasing the stability in blood of osteocalcin (OCN) comprising O-glycosylating at least one residue of the OCN using the method defined in item 16.
  • OCN osteocalcin
  • Item 18 A method of preventing or treating an osteocalcin associated disease or condition, or symptom thereof, comprising administering to a subject in need thereof a therapeutically effective amount of the gly-rOCN defined in any one of items 1 -12.
  • OCN osteocalcin
  • Such formula encompasses the human OCN (hOCN) as described in e.g., NP_954642.1 and any functional OCN.
  • any functional OCN comprising one or more of the polymorphisms described in FIGs. 27 A- B.
  • the term“circulating form” also sometimes called“mature OCN” herein see e.g., FIGs. 18F and 23 refers to, when the OCN is produced in cellulo, the OCN fragment remaining after the signal peptide and the propeptide have been removed (see e.g., FIG. 18F).
  • OCN active form refers to the OCN which has undergone posttranslational modifications so that it does not have a y-carboxyglutamic acid residue (Gla) at position 17 using the numbering of the human circulating form (or 68 using the numbering of the full encoded human OCN). This active form is also called uncarboxylated. OCN active form may be completely decarboxylated or have Gla residues at positions 21 and/or 24, using the numbering of the human circulating fragment (or 72 and 75 using the numbering of the full encoded human OCN).
  • Ga y-carboxyglutamic acid residue
  • the OCN active form is also meant to refer to the rOCN as modified by post-translational modification such as by glycolisation in addition to those expressly mentioned herein, acetylation, amidation, blockage, formylation, gamma-carboxyglutamic acid hydroxylation, methylation, phosphorylation, pyrrolidone carboxylic acid, and sulfatation.
  • post-translational modification such as by glycolisation in addition to those expressly mentioned herein, acetylation, amidation, blockage, formylation, gamma-carboxyglutamic acid hydroxylation, methylation, phosphorylation, pyrrolidone carboxylic acid, and sulfatation.
  • osteocalcin or“rOCN” is used herein to refer to a protein encoded by a genetically manipulated nucleic acid inserted into a host cell.
  • the term “recombinant osteocalcin” or“rOCN” refers to an OCN that comprises at least one modification as compared to a native (e.g., human) OCN .
  • a native OCN e.g., human
  • it refers to an OCN (e.g., hOCN) comprising a substitution of at least one of its residues by a serine or a threonine (e.g., P60S, Y63S/T, R70S/T).
  • a threonine e.g., P60S, Y63S/T, R70S/T.
  • it refers to a hOCN comprising a Y63S/T substitution.
  • the rOCN of the present disclosure consists of a biologically active fragment of a consensus sequence derived from the circulating form of known hOCNs (e.g., hOCN with known polymorphisms see e.g., FIGs. 27A and B) i.e. YXXXXXXXXVXXXXLEPXREVCEXXXDCXXLXDHXXFQXAXXXXYXXV (SEQ ID NO: 15) wherein each X is independently any amino acid or is any amino acid at the corresponding position of any of the hOCN described in FIGs. 27A-B.
  • the circulating form corresponds to amino acid residues 52-100 of hOCN.
  • the rOCN of the present disclosure encompasses fragments 52-99, 52-98, 52-97, 52-96, 52-95, and 52-94; 53-100, 53-99, 53-98, 53-97, 53-96, 53-95, and 53-94; 54-100, 54-99, 54-98, 54-97, 54-96, 54-95, and 54-94; 55-100, 55-99, 55-98, 55-97, 55-96, 55-95, and 55- 94; and 56-100, 56-99, 56-98, 56-97, 56-96, 56-95, and 56-94 of the sequence of hOCN .
  • each X is the amino acid or is any amino acid at the corresponding position of any of the hOCNs described in FIGs. 27A-B.
  • biological activity refers to an OCN able to perform known OCN biological activities in vivo.
  • OCN direct and indirect “biological activities” include binding to GPRC6A in pancreatic b-cells, increasing calcium (Ca 2+ ) signaling in b-cells, increasing b-cell proliferation, increasing energy expenditure, reducing fat mass, increasing insulin expression, increasing insulin secretion, increasing insulin sensitivity, decreasing liver steatosis, increasing adiponectin expression, decreasing lipolysis, increasing glucose uptake, decreasing secretion of pro-inflammatory cytokines in white adipocytes, increasing mitochondria biogenesis in muscle, increasing the expression of genes involving in thermogenesis in brown adipocytes, increasing uptake and catabolism of glucose and fatty acids (FAs) in muscle during exercise, increasing subject endurance during exercise, increasing aerobic endurance, increasing muscle mass gain, increasing muscle function during exercise; increasing production of cAMP in the Leydig cells of the testis, increasing CR
  • the order can differ from the above.
  • the purification domain and/or domain for increasing the stability of the OCN can be in C-terminal.
  • the construct may further optionally comprise a few additional amino acids, e.g., 1 , 2, 3, etc. between adjacent pairs of the above domains that are the result of the cloning strategy used to produce the construct which may introduce exogenous amino acids in these locations.
  • the signal peptides encompassed by the present disclosure include any peptide that enables the efficient secretion of the rOCN of the present disclosure.
  • useful signal peptides include the native hOCN signal peptide e.g., as predicted by SignIP 4.1 ( http://www.cbs.dtu.dk/services/SignalP ) and any other heterologous signal peptide that enables the efficient secretion of the rOCN of the present disclosure such as the hlgG1 signal peptide Mefglswvflvailkgvqc (SEQ ID NO: 16).
  • Other signal peptides can also be used such as the albumin signal peptide.
  • Other useful signal peptides can be identified on http://www.signalpeptide.de/ and Haryardi 2015.
  • any domain that can be affinity purified by e.g., which can be affinity purified with a protein A- or protein G-sepharoseTM columns is encompassed by the present disclosure.
  • the purification domains that can be used in the present disclosure include, without being so limited Fragment crystallizable region (Fc) fragments, GST, MBP, 3XFLAG peptide, 6XHIS, V5 and 8XHIS. They can be on the N-terminal or the C-terminal of the protein. In a specific embodiment, it is FC (e.g., IgFC).
  • Useful Fc fragments for the present disclosure include FC fragments of IgG that comprise the hinge (the case being, the hinge may serve as spacer), and the CPI2 and CH3 domains.
  • FC fragments can also be used to stabilize the protein in vivo.
  • the purification domain can contain between 6 and 500 amino acid residues or between 6 and 450, or between 6 and 400, or between 6 and 350.
  • Insertion of spacers between fusion protein domains can increase bioactivity by augmenting distance between domains alleviating potential repulsive forces between different segments (e.g., rOCN and purification/stabilization domain) of the construct resulting in increased rOCN folding and/or increased rOCN’s expression level and/or secretion. It is generally placed between the purification/stabilization domain and the rOCN to increase its flexibility. When the purification domain is to be cleaved out by a peptidase, the spacer also facilitates peptidase cleavage releasing the rOCN portion of the construct.
  • the spacer used in working embodiments described below comprises the FC lgG-1 hinge region AEPKSCDKTHTCPPCP (SEQ ID NO: 17).
  • the GS residues at the C terminal of this sequence in the construct is the result of the cloning strategy used. Additional useful spacers that can be use include i.e.
  • flexible linker structures rich in small hydrophilic amino acids that maintain distance between the two connected domains and improve their folding such as (EAAAK)n (SEQ ID NO: 18); (GGGGS)n (SEQ ID NO: 19); or (XPXPXP)n (SEQ ID NO: 20), wherein x is any amino acid; wherein n is any one of 1 to 5, more specifically 1 , 2, 3, 4 or 5.
  • the spacer can contain between 1 and 200 amino acid residues.
  • any peptide motif recognized by any endopeptidase is encompassed by the present disclosure. Without being so limited, it includes peptide motives recognized and cleaved by such as thrombin, Human rhinovirus (HRV) type 14 3C protease (Prescission), Tobacco Etch Virus (TEV) protease, Factor Xa, enterokinase.
  • HRV Human rhinovirus
  • TMV Tobacco Etch Virus
  • enterokinase enterokinase.
  • the endopeptidase cleavage site used in working embodiments described below is LVPf ⁇ GS (SEQ ID NO: 21 ). Without being so limited, the cleavage site can contain between 2 and 10 amino acid residues.
  • transcription termination signal any transcription termination signal may be used.
  • the transcription termination signal used in working embodiments described below is a poly(A) signal.
  • the termination signal can generally contain between 5 and 7 amino acid residues.
  • O-glycosylation refers to attachment of a N-acetyl-galactosamine to an oxygen atom in a serine or threonine residue by the by the enzyme UDP-N-acetyl-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase (EC number 2.4.1.41). It is followed by the attachment of other carbohydrates, such as GlcNac, 2 GlcNAc (branched), Gal and GlcNac, etc., and then sialic acid such as N-acetylneuraminic acid.
  • the glycosylated form of the rOCN of the present disclosure is referred to as gly-rOCN.
  • the present disclosure encompasses a method of producing the gly-rOCN of the present disclosure comprising culturing a host cell transformed or transfected with a vector comprising a nucleic acid molecule encoding the rOCN under conditions suitable to effect expression of the gly-rOCN and recovering the gly-rhOCN.
  • the present disclosure encompasses nucleic acids comprising nucleotide sequences encoding the above-mentioned rOCN.
  • the nucleic acid may be codon-optimized.
  • the nucleic acid can be a DNA or an RNA.
  • the nucleic acid sequence can be deduced by the skilled artisan on the basis of the disclosed amino acid sequences.
  • the nucleic acid encodes one of the amino acid sequences as presented in any one of FIGs. 18A-18G and 24AA-24E (orthologues and/or consensuses).
  • Vectors comprising the above-mentioned nucleic acids.
  • the vectors can be of any type suitable, e.g., for expression of said polypeptides or propagation of genes encoding said polypeptides in a particular organism.
  • the organism may be of eukaryotic origin (e.g., human cell).
  • the specific choice of vector depends on the host organism and is known to a person skilled in the art.
  • the vector comprises transcriptional regulatory sequences or a promoter operably-linked to a nucleic acid comprising a sequence encoding a rOCN of the disclosure.
  • a first nucleic acid sequence is“operably-linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
  • enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous.
  • Transcriptional regulatory sequences or “transcriptional regulatory elements” are generic terms that refer to DNA sequences, such as initiation and termination signals (terminators), enhancers, and promoters, splicing signals, polyadenylation signals, etc., which induce or control transcription of protein coding sequences with which they are operably-linked.
  • useful “promotors’ for used in the vectors of the present disclosure include the CMV promoter, EF1 a promoter, CAG promoter, PGK1 promoter, TRE (inducible) promoter, etc. Additional useful promoters may be found on https://blog.addgene.org/plasmids-101 -the-promoter-region.
  • the“host cell” comprises a vector comprising a nucleic acid molecule encoding the rOCN.
  • the host cell is a mammalian cell including but not limited to HEK293.
  • HEK293 cells have been used as a host for expressing the rOCN of the present disclosure in the Examples presented herein, a person of ordinary skill in the art will understand that a number of other hosts may be used to produce recombinant proteins according to methods that are routine in the art. Representative methods are disclosed in Maniatis, et al. Cold Springs Harbor Laboratory (1989).
  • the rOCN may be cleaved by a host’s enzyme (first by e.g., furin to remove the propeptide and then by a signal peptidase (i.e. the mammalian subunits are SPC12, SPC18, SPC21 , SPC22/23 and SPC25, named according to their molecular weight) so as to produce a secreted/circulating form of the rOCN.
  • a host e.g., furin to remove the propeptide and then by a signal peptidase (i.e. the mammalian subunits are SPC12, SPC18, SPC21 , SPC22/23 and SPC25, named according to their molecular weight) so as to produce a secreted/circulating form of the rOCN.
  • a host e.g., furin to remove the propeptide and then by a signal peptidase (i.e. the mammalian subunits are SPC12
  • condition suitable to effect expression of the polypeptide is meant to refer to any culture medium that will enable production of the gly-rOCN of the present disclosure. Without being so limited, it includes media prepared with a buffer, bicarbonate and/or HEPES, ions like chloride, phosphate, calcium, sodium, potassium, magnesium, iron, carbon sources like simple sugars, amino acids, potentially lipids, nucleotides, vitamins and growth factors like insulin; regular commercially available media like alpha-MEM, DMEM, Ham’s-F12 and IMDM supplemented with 2-4 mM L-glutamine and 10% Fetal bovine serum; regular commercially available animal protein free media like HycloneTM SFM4CHO, Sigma CHO DHFR-, Cambrex POWERTM CHO CD supplemented with 2-4 mM L-glutamine. These media are desirably prepared without thymidine, and hypoxanthine to maintain selective pressure allowing stable protein-product expression.
  • ions like chloride, phosphate, calcium,
  • the present disclosure also encompasses a method of increasing the stability in blood of OCN (e.g., human OCN) comprising O-glycosylating at least one residue of the OCN using the method of producing a gly-rOCN of the present disclosure.
  • OCN e.g., human OCN
  • the present disclosure also encompasses a method of preventing or treating an osteocalcin associated disease or condition, or symptom thereof, comprising administering to a subject in need thereof a therapeutically effective amount of the gly-rOCN (or nucleic acid molecule encoding the rOCN, vector comprising the nucleic acid molecule, host cell comprising the vector, or composition comprising any one of the gly-rOCN, nucleic acid molecule, vector or host cell) (hereinafter any one of the gly-rOCN, nucleic acid molecule encoding the rOCN, vector comprising the nucleic acid molecule, host cell comprising the vector and composition may be designated“agent”) of the present disclosure.
  • osteocalcin associated disease or condition refers to any disease or condition characterized at least in part by a defective osteocalcin level or function. Without being so limited, it includes diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), frailty associated with aging, cognitive disorder and male reproductive disorder. A symptom thereof includes any absence or decrease in one of the OCN“biological activities” as defined herein.
  • the term“subject” is meant to refer to any mammal including human, and any mammal having a non- naturally glycosylated OCN including pets such as cats, pigs, dogs, etc. In a particular embodiment, it refers to a human.
  • the term“subject in need thereof” in a method of administering a gly-rOCN of the present disclosure is meant to refer to a subject that would benefit from receiving a gly-rOCN of the present disclosure. In specific embodiments, it refers to a subject that already has at least one osteocalcin associated disease or condition, or symptom thereof or to a subject likely to develop at least one osteocalcin associated disease or condition, or symptom thereof.
  • the term“prevent/preventing/prevention” or“treat/treating/treatmenf” refers to eliciting the desired biological response, i.e., a prophylactic and therapeutic effect, respectively in a subject.
  • the therapeutic effect comprises one or more of a decrease/reduction in the severity, intensity and/or duration of the osteocalcin associated disease or condition, or symptom thereof following administration of the polypeptide, nucleic acid or vectors or host cells (“agent”) of the present disclosure when compared to its severity, intensity and/or duration in the subject prior to treatment or as compared to that/those in a non-treated control subject having the infection or any symptom thereof.
  • a prophylactic effect may comprise a delay in the onset of the osteocalcin associated disease or condition, or symptom thereof in an asymptomatic subject at risk of experiencing the osteocalcin associated disease or condition, or symptom thereof at a future time; or a decrease/reduction in the severity, intensity and/or duration of an osteocalcin associated disease or condition, or symptom thereof occurring following administration of the agent of the present disclosure, when compared to the timing of their onset or their severity, intensity and/or duration in a non-treated control subject (i.e.
  • asymptomatic subject at risk of experiencing the osteocalcin associated disease or condition, or symptom thereof at risk of experiencing the osteocalcin associated disease or condition, or symptom thereof); and/or a decrease/reduction in the progression of any pre-existing osteocalcin associated disease or condition, or symptom thereof in a subject following administration of the agent of the present disclosure when compared to the progression of osteocalcin associated disease or condition, or symptom thereof in a non-treated control subject having such pre-existing osteocalcin associated disease or condition, or symptom thereof.
  • the agent of the present disclosure in a therapeutic treatment, is administered after the onset of the osteocalcin associated disease or condition, or symptom thereof.
  • a prophylactic treatment the agent of the present disclosure is administered before the onset of the osteocalcin associated disease or condition, or symptom thereof or after the onset thereof but before the progression thereof.
  • the term“decrease” or“reduction” refers to a reduction (e.g., in a symptom) of at least 10% as compared to a control subject (a subject not treated with an agent of the present disclosure), in an embodiment of at least 20% lower, in a further embodiment of at least 30% lower, in a further embodiment of at least 40% lower, in a further embodiment of at least 50% lower, in a further embodiment of at least 60% lower, in a further embodiment of at least 70% lower, in a further embodiment of at least 80% lower, in a further embodiment of at least 90% lower, in a further embodiment of 100% (complete inhibition).
  • the term“increase” or“increasing” refers to an increase (e.g., in an OCN biological activity or OCN stability) of at least 10% as compared to a control, in an embodiment of at least 20% higher, in a further embodiment of at least 30% higher, in a further embodiment of at least 40% higher, in a further embodiment of at least 50% higher, in a further embodiment of at least 60% higher, in a further embodiment of at least 70% higher, in a further embodiment of at least 80% higher, in a further embodiment of at least 90% higher, in a further embodiment of 100% higher, in a further embodiment of 200% higher, etc.
  • The“control” for use as reference in the method disclosed herein of preventing or treating an osteocalcin associated disease or condition, or any symptom thereof may be e.g., a control subject that has an osteocalcin associated disease or condition, or any symptom thereof, and that is not treated with an agent present disclosure.
  • the “control” for use as reference in the method disclosed herein of stabilizing an OCN may be e.g., an OCN that is not glycosylated.
  • Gly-rOCN of the present disclosure can be administered by routes such as parenterally (e.g., intravenously intramuscularly, subcutaneously, intradermally), intranasally or orally (Mizokami et al., 2013; Mizokami et al., 2014).
  • the route of administration can depend on a variety of factors, such as the environment and therapeutic goals.
  • pharmaceutical compositions of the disclosure can be in the form of a liquid, solution, suspension, pill, capsule, tablet, gelcap, powder, gel, ointment, cream, nebulae, spray, mist, atomized vapor, aerosol, or phytosome.
  • preparations can be in the forms of liquids, can include pharmaceutically acceptable liquid carriers such as sterile aqueous or non-aqueous solvents, suspensions or emulsions.
  • pharmaceutically acceptable liquid carriers such as sterile aqueous or non-aqueous solvents, suspensions or emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters.
  • Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils.
  • Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose, and the like.
  • tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents.
  • the tablets can be coated by methods known in the art.
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspension, or they can be presented as a dry product for constitution with saline or other suitable liquid vehicle before use.
  • Dietary supplements of the disclosure also can contain pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles, preservatives, buffer salts, flavoring, coloring, and sweetening agents as appropriate.
  • Preparations for oral administration also can be suitably formulated to give controlled release of the active ingredients.
  • preparations can be in the form of sprays, drops, gels, ointments, creams etc. and can be administered in the form of nasal atomizers, nebulizers, etc.
  • any amount of a pharmaceutical composition can be administered to a subject.
  • the dosages will depend on many factors including the mode of administration and the age of the subject.
  • the amount of gly-rOCN of the disclosure contained within a single dose will be an amount that effectively prevent, or treat an osteocalcin associated disease or condition, or symptom thereof without inducing significant toxicity.
  • therapeutically effective amount is meant to refer to an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects.
  • gly-rOCN in accordance with the present disclosure can be administered to subjects in doses ranging from 0.001 to 500 mg/kg/day and, in a more specific embodiment, about 0.1 to about 100 mg/kg/day, and, in a more specific embodiment, about 0.2 to about 20 mg/kg/day.
  • the allometric scaling method of Mahmood et al. can be used to extrapolate the dose from mice to human.
  • the dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient.
  • the therapeutically effective amount of the gly-rOCN may also be measured directly.
  • the effective amount may be given daily or weekly or fractions thereof.
  • a pharmaceutical composition of the disclosure can be administered in an amount from about 0.001 mg up to about 500 mg per kg of body weight per day (e.g., 0.05, 0.01 , 0.1 , 0.2, 0.3, 0.5, 0.7, 0.8, 1 mg, 2 mg, 3 mg, 4mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 50 mg, 100 mg, or 250 mg). Dosages may be provided in either a single or multiple dosage regimens.
  • the effective amount is a dose that ranges from about 0.1 to about 100 mg/kg/day, from about 0.2 mg to about 20 mg of the gly-rOCN per day, about 1 mg to about 10 mg of the gly-rOCN per day, from about .07 mg to about 210 mg of the gly-rOCN per week, 1.4 mg to about 140 mg of the gly-rOCN per week, about 0.3 mg to about 300 mg of the gly-rOCN every three days, about 0.4 mg to about 40 mg of the gly-rOCN every other day, and about 2 mg to about 20 mg of the gly-rOCN every other day.
  • the optimal daily dose will be determined by methods known in the art and will be influenced by factors such as the age of the patient as indicated above and other clinically relevant factors.
  • patients may be taking medications for other diseases or conditions. The other medications may be continued during the time that a gly-rOCN is given to the patient, but it is particularly advisable in such cases to begin with low doses to determine if adverse side effects are experienced.
  • Preparations containing a gly-rOCN may be provided to patients in combination with pharmaceutically acceptable carrier.
  • pharmaceutically acceptable such as pharmaceutically acceptable carrier, excipient, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular inhibitor is administered.
  • aqueous or non-aqueous solvents, suspensions or emulsions can be used with the disclosure.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters.
  • Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils.
  • Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose, and the like.
  • the pharmaceutical compositions of the present disclosure can be delivered in a controlled release system.
  • polymeric materials including polylactic acid, polyorthoesters, cross-linked amphipathic block copolymers and hydrogels, polyhydroxy butyric acid and polydihydropyrans can be used (see also Smolen and Ball, Controlled Drug Bioavailability, Drug product design and performance, 1984, John Wiley & Sons; Ranade and Hollinger, Drug Delivery Systems, pharmacology and toxicology series, 2003, 2 nd edition, CRRC Press), in another embodiment, a pump may be used (Saudek ef a/., 1989, N. Engl. J. Med. 321 : 574).
  • the gly-rOCN of the present disclosure could be in the form of a lyophilized powder using appropriate excipient solutions (e.g., sucrose) as diluents.
  • excipient solutions e.g., sucrose
  • nucleotide molecules or proteins according to the present disclosure can be introduced into individuals in a number of ways.
  • osteoblasts can be isolated from the afflicted individual, transformed with a nucleotide construct (e.g., vector) according to the instant disclosure and reintroduced to the afflicted individual in a number of ways, including an intravenous injection.
  • the nucleotide construct can be administered directly to the afflicted individual, for example, by injection.
  • the nucleotide construct can also be delivered through a vehicle such as a liposome, which can be designed to be targeted to a specific cell type and engineered to be administered through different routes.
  • the gly-rOCN of the present disclosure could also be advantageously delivered through gene therapy.
  • Useful gene therapy methods include those described in W006060641A2, US7179903 and W00136620A2 to Genzyme using for instance an adenovirus vector for the therapeutic protein and targeting hepatocytes as protein producing cells.
  • a “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • Gene delivery are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction.
  • exogenous polynucleotide sometimes referred to as a "transgene”
  • Such methods include a variety of well-known techniques such as vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • the introduced polynucleotide may be stably or transiently maintained in the host cell.
  • Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
  • a "viral vector” is defined as a recombinantly produced virus or viral; particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors such as those described in W006002203A2, alphavirus vectors and the like.
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy.
  • a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene.
  • Ads adenoviruses
  • Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071. Ads are easy to grow and do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos.
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, Wl). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation.
  • the gly-rOCN of the present disclosure may also be used in combination with at least one other active ingredient to treat or prevent an osteocalcin associated disease or condition, or symptom thereof.
  • kits for treating or preventing an osteocalcin associated disease or condition, or symptom thereof comprising a nucleic acid, a protein or a ligand in accordance with the present disclosure.
  • it may comprise a bone targeted composition of the present disclosure or a vector encoding same, and instructions to administer said composition or vector to a subject to treat or prevent an osteocalcin associated disease or condition, or symptom thereof.
  • kits may further comprise at least one other active agent able to prevent or treat an osteocalcin associated disease or condition, or symptom thereof.
  • a compartmentalized kit in accordance with the present disclosure includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • the term "about” has its ordinary meaning. In embodiments, it may mean plus or minus 10% of the numerical value qualified.
  • the Furin m and Furin 0sb -/- mice were generated by breeding Funn m with OCA/-Cre-transgenic mice that express Ore recombinase under the control of human OCN promoter as described previously (Al Rifai et al., 2017).
  • Ocn-I- mice were generated using homologous recombination to replace Ocn1 ( Bglapl ) and Ocn2 ( Bglap2 ) genes in the mouse Ocn cluster with a neomycin resistance cassette (Ducy et al., 1996).
  • Mouse pro-OCN cDNA was cloned into a plRES2-EGFP-V5 plasmid in EcoRI and Agel cloning sites.
  • SST/AAA pro- OCN, STT/AAA pro-OCN and 6ST/6A pro-OCN mutant were purchased originally from GeneArts.
  • pcDNA3 human pre- pro-OCN cDNA was originally purchased from GenScript. Each construct was used as PCR template for amplification and to introduce EcoRI and Agel cloning sites and cloned in plRES2-EGFP-V5 plasmid. Point mutations in mouse pro- OCN (S54A; S57A, T64A) and Y63S in human pro-OCN were generated by site directed mutagenesis using specific primers (Table I).
  • Galntl6-For ACCGAGACTAGCAGTT CCCT (SEQ ID NO: 80) M QPCR primer
  • the cDNA coding of the Fc and hinge region of human immunoglobulin flanked with Hindlll-BamHI restriction sites was amplified using standard PCR and pTT5-Fd_CTL vector as template (Zhang et al., 2009).
  • the PCR product was cloned in pcDNA3.1 -myc-His B in Hindll-BmaHI cloning site, generating the pcDNA3.1 -Fc-hinge-myc-His vector.
  • cDNA coding for thrombin-hOCN was generated using plRES2-EGFP-hOCN (Y63S)-V5 as template, to which a thrombin cleavage site was added at the N-terminus and Bgll l-EcoRI restriction site were introduced by standard PCR amplifications.
  • Thrombin-hOCN (Y63S) product was cloned in the pcDNA3.1 -Fc-hinge-myc-His vector.
  • the generated vector pcDNA3.1 -Fc-hinge-thrombin-hOCN (Y63S) is an expression vector of human OCN fusion protein composed of the Fc and hinge region of human lgG1 , thrombin cleavage site and human OCN (Y63S), engineered to produce hlgG1 Fc fused to human glycosylated OCN which can be affinity purified using protein A- or protein G-sepharoseTM columns.
  • a thrombin cleavage site was designed to allow the purification of OCN following thrombin digestion on the column.
  • pcDNA3.1 -Fc-hinge-thrombin-mOCN expressing wild type mature mouse OCN fused to Fc was generated following the same procedure but using different primers.
  • osteoblasts were treated with the g-carboxylation inhibitor warfarin (50 pM; Santa Cruz Biotechnology), or with the N-acetylgalactosamine transferase inhibitor GalNAc-bn (2 mM; Sigma) and the proprotein convertase inhibitor Dec-RVKR-CMK (SEQ ID NO: 2) (50 pM; Tocris) combined with 22 pM vitamin K1 (Sandoz).
  • warfarin 50 pM; Santa Cruz Biotechnology
  • GalNAc-bn 2 mM; Sigma
  • proprotein convertase inhibitor Dec-RVKR-CMK SEQ ID NO: 2
  • CHO cells Chinese hamster ovary (CHO) cells, originally purchased from ATCC, and Chinese hamster ovary IdID cells (CHO- IdlD; originating from the M. Krieger laboratory (Kingsley et al., 1986)) were cultured in DMEM-F12 containing PS and 5% FBS for CHO cells or 3% FBS for CHO-ldID cells and transfected using LipofectamineTM 2000 reagent (Life technology) following standard protocol. Secretion was performed in DMEM-F12 media supplemented with 50IU/ml penicillin and 50pg/ml Streptomycin and 22mM vitamin Ki.
  • CHO-ldID culture, transfection and secretion media was supplemented with 0.1 mM galactose and/or 1 mM A/-acetylgalactosamine (GlaNAc) to rescue the O-glycosylation defect.
  • GalNAc A/-acetylgalactosamine
  • HEK 293 cells were treated with the g-carboxylation inhibitor warfarin (50 pM; Santa Cruz Biotechnology), or with the N-acetylgalactosamine transferase inhibitor GalNAc- bn (2 mM; Sigma) and the proprotein convertase inhibitor Dec-RVKR-CMK (SEQ ID NO: 2) (50 pM; Tocris) combined with 22 pM vitamin K1 (Sandoz).
  • Antibody used in examples herein anti-V5 (mouse, clone V5-10, V8012; Sigma-Aldrich), anti— b-actin (mouse, clone AC-15, A5441 ; Sigma-Aldrich), anti-GFP (mouse, clones 7.1 and 13.1 , 1 1814460001 ; Sigma), anti-Gla OCN goat antibody which recognizes amino acids 11 -26 of carboxylated mature osteocalcin and anti-CTERM OCN goat antibody which recognizes amino acids 26-46 of mature mouse OCN (Ferron et al., 2010b).
  • Flushed mouse femur and tibia from Furin m and Furin 0sb -/- were homogenized in lysis buffer containing (20 mM Tris- HCI, pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1 % Triton, 1 mM PMSF, and 1 x protease inhibitors cocktail). Tissue homogenates were then centrifuged for 10 minutes at 4000 rpm to remove tissue debris. In vitro de- glycosylation assay was performed on 10pg of bone homogenate.
  • proteins were denatured in denaturing buffer at 95°C for 5 min and incubated with O-glycosidase which removes O-linked GalNAc and neuraminidase which removes N-Acetylneuraminic acid (NANA), for 4 hours at 37°C following the NEB kit protocol (NEB; E0540S). Samples were resolved on 15% Tris tricine gel and using anti-CTERM OCN goat antibody.
  • Flushed femur and tibia from wild type mice were homogenized in lysis buffer containing (20 mM Tris-HCI, pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1 % Triton, 1 mM PMSF, and 1 x protease inhibitors cocktail). 10Opg of protein homogenate was diluted in 1.6 ml of 100 mM phosphate buffer pH 7.4 and incubated overnight at 4°C with anti-OCN antibody. After overnight incubation, samples were centrifuged at 10000 rpm for 10 minutes, supernatant was incubated with protein-G agarose beads pre-washed with 1X PBS.
  • Samples were diluted in 25% ACN 0.3%TFA and loaded onto a 50x4.6 mm PLRP-S 300A column (Agilent Technologies) connected to an AccelaTM pump (Thermo Scientific) and an RTC autosampler (Pal systems).
  • the buffers used for chromatography were 0.1 % formic acid (buffer A) and 100% acetonitrile/0.1 % formic acid (buffer B). Proteins and peptides were eluted with a two-slope gradient at a flowrate of 120 pL/min. Solvent B first increased from 12 to 50% in 4.5 min and then from 50 to 70% in 1.5 min.
  • the HPLC system was coupled to a Q ExactiveTM mass spectrometer (Thermo Scientific) through an electrospray Ion Source.
  • the spray and S-lens voltages were set to 3.6 kV and 60 V, respectively.
  • Capillary temperature was set to 225 °C.
  • Full scan MS survey spectra (m/z 600-2000) in profile mode were acquired in the OrbitrapTM with a resolution of 70,000 with a target value at 3e6.
  • the 4 most intense protein/peptide ions were fragmented in the HCD collision cell and analyzed in the OrbitrapTM with a target value at 5e5 and a normalized collision energy at 33.
  • Data processing protocol The identification of the different forms of osteocalcin was performed by manual de novo sequencing.
  • HEK 293 Human embryonic kidney cells HEK 293 were originally purchased from ATCC. To generate stable clone expressing glycosylated human and mouse OCN, HEK 293 were transfected with pcDNA3.1-Fc-hinge-Thrombin-hOCN (Y63S) and pcDNA3.1 -Fc-hinge-Thrombin-OCN respectively using LipofectamineTM 2000 reagent. Following 48 hours of transfection, cells were trypsinized and resuspended in sorting buffer containing (1X sterile PBS, 2% FBS and 1 mM EDTA).
  • TM 102F12 clones expressing mouse OCN fusion protein and 22H5 clone expressing human OCN fusion protein were cultured in triple 175cm 2 plates. After reaching 100% confluency, cells were kept to secretes in secretion media (EMEM media supplemented with 1 % FBS and 10 pM warfarin) for 72 hours. Secretion media was collected, filtered with 0.45 pm filter, and media was buffer exchanged with 10X Binding buffer (0.2 M phosphate buffer, pH 7). Protein supernatant was then loaded into protein A affinity column (HiTrap protein A high performance, GE29-0485-76) using automated pump (GE AKTA Prime Plus Liquid Chromatography System).
  • secretion media EMEM media supplemented with 1 % FBS and 10 pM warfarin
  • Colum were then washed with 10 ml 1X biding buffer (0.02 M phosphate buffer, pH 7) and 5 ml of filtered 1X PBS.
  • OCN fusion protein was digested with thrombin (27-0846-01 , GE healthcare) and eluted with 1X PBS, thrombin was pulled down using benzamidine sepharose beads (17-5123-10, GE healthcare). Fc region was then eluted from column using acetate buffer pH 3 and buffer exchanged with 1 M Tris pH 9.
  • Mouse and human OCN purity were assessed using Coomassie staining and MS analysis compared.
  • Mouse OCN was quantified using the ELISA assay as described previously (Ferron et al., 2010b). Human OCN measurements was performed using human homemade ELISA described below.
  • Human OCN measurements Human OCN measurements were performed using human OCN ELISA developed in the inventors’ laboratory. In brief, the ELISA plate was coated with human Glu OCN antibody (mouse, 4B6 clone recognize 3X Glu hOCN) overnight at room temperature (RT). The ELISA plate was then washed twice with wash buffer (1X PBS, 0.05% tween) and blocked with blocking buffer (3% FA free BSA in 1X PBS) for 4 hours at RT. Assay buffer (3% FA free BSA in 1X PBS), standards and samples were loaded and incubated overnight at 4°C.
  • wash buffer (1X PBS, 0.05% tween
  • Assay buffer 3% FA free BSA in 1X PBS
  • the ELISA plate was washed 6 times with wash buffer, incubated with human OCN antibody coupled to HRP (mouse, 4C5 clone recognize C-terminal of hOCN) for 1 hour with shaking at RT. After another 6 washes with the wash buffer, the plate was tapped firmly and TMB substrate was added. After color development (15-30 minutes of incubation in dark), HCI was added to stop the reaction and absorbance was measured at 450nm using ELISA plate reader (“homemade ELISA”).
  • mice OCN ex vivo half-life assay was performed on plasma from four independent Ocn-I- mice collected in lithium heparin tube, glycosylated OCN and non-glycosylated OCN were incubated in plasma at 37°C and OCN level was measured at indicated time points using a total mouse OCN ELISA assay as described previously (Ferron et al., 2010b). Human OCN half-life assay was performed ex vivo using Ocn-I- mice plasma and human OCN was measured using homemade ELISA described below.
  • plasma was heat inactivated for 30 minutes at 56°C, to inactivate proteases, or treated with 10 mM EDTA, protease inhibitor cocktail EDTA free (PI) (7X, Roche), Phenylmethylsulfonyl Fluoride (PMSF) (1 mM, Sigma), 4-benzenesulfonyl fluoride hydrochloride (AEBSF) (2.5 mM, Sigma), Pepstatin A (Pep A) (10 pM, Sigma), 50 pM RVKR, and Benzamidine sepharose beads (BAM).
  • 10 mM EDTA protease inhibitor cocktail EDTA free
  • PMSF Phenylmethylsulfonyl Fluoride
  • AEBSF 4-benzenesulfonyl fluoride hydrochloride
  • Ocn-I- male mice were injected intraperitoneally with 12ng/g, 40ng/g or 80 ng/g of O- glycosylated OCN or non-O-glycosylated OCN. Serum OCN level was analyzed at indicated time points using total mouse OCN ELISA.
  • mouse or human proteins were prepared in 3.5% BSA prepared in standard saline solution. The in vivo half-life study was performed on fed or fasted Ocn-I- male mice and it is indicated in the figure legend.
  • OCN was immunoprecipitated from the supernatant of primary osteoblasts and from mouse bone protein extracts using polyclonal goat antibodies recognizing its C-terminus region (Ferron et al., 2010b), and characterized without tryptic digestion by HLPC Reverse- phase followed by mass spectrometry (MS) and tandem mass spectrometry (MS/MS).
  • MS mass spectrometry
  • MS/MS tandem mass spectrometry
  • this glycan is composed of one N-acetylgalactosamine (GalNAc), one galactose (Gal) and one or two N-Acetylneuraminic acid (NANA) (FIG. 1 B).
  • N N-acetylgalactosamine
  • NANA N-Acetylneuraminic acid
  • MS/MS spectrum of one of the glycosylated form of osteocalcin suggests that the glycan is not linked to the only asparagine (N) residue of osteocalcin, but instead is covalently linked to the second serine (S8 in mature mouse osteocalcin or S57 in prepro-osteocalcin) (deduced from the mass of various peptide fragments (y38 peptide and b6 peptide) in FIGs. 2A versus mass of unfragmented mature mouse OCN). Given the type of sugars composing the glycan and the site of linkage, these results strongly suggest that osteocalcin is 0- glycosylated in
  • the inventors next confirmed that mouse osteocalcin is subjected to O-glycosylation using cell-based assays coupled to SDS-PAGE analyses.
  • the osteocalcin molecular weight is reduced in HEK 293 cells lacking core i 3-Gal-T-specific molecular chaperone (COSMC), a protein essential to the addition of Gal on the O-linked GalNAc (FIG. 3A).
  • COSMC 3-Gal-T-specific molecular chaperone
  • FIG. 3B the osteocalcin apparent molecular weight is also reduced compared to the parental CHO cell line (FIG. 3B).
  • GalNAc-Ts N-acetylgalactosamine transferases
  • Vitamin K-dependent gamma-carboxylation and processing of the pro-osteocalcin by the proprotein convertase furin are two posttranslational modifications regulating osteocalcin endocrine function (Ferron et al., 2015; Al Rifai et al., 2017). Whether O-glycosylation can interfere with the gamma-carboxylation or the processing of osteocalcin or vice versa was unknown. The inventors therefore tested whether pharmacologically blocking gamma-carboxylation or furin, using warfarin or Dec-RVKR-CMK (RVKR (SEQ ID NO: 2)) respectively, impacts osteocalcin O-glycosylation.
  • the inventors next aimed to determine which amino acid(s) within osteocalcin is glycosylated.
  • Mature mouse osteocalcin contains 3 serine residues and 3 threonine residues (FIG. 7A), the two types of amino acids on which 0- glycosylation can occur. Accordingly, mutation of all 6 serine and threonine residues into alanine abrogates osteocalcin glycosylation in primary osteoblasts (FIG. 7B). Further mutagenesis analyses revealed that the O-g lycosy I ati o n site resides within the N-terminal part of the protein on Ser54, Ser57 or Thr64 (see SST/AAA band on FIG. 7B where the O-gly OCN protein is missing vs.
  • the inventors assessed the impact of the Ser57Ala O-glycosylation-free mutant on osteocalcin carboxylation and processing by furin.
  • the Ser57Ala mutant is still efficiently processed by furin, since a molecular shift is observed following treatment with RVKR (SEQ ID NO: 2). It is also carboxylated to a comparable level compared to the WT protein, independently of the processing by furin (FIG. 8B).
  • blocking its carboxylation using warfarin did not affect the processing of Ser57Ala osteocalcin mutant (FIGs. 8C-D).
  • the first enzymatic step in the O-glycosylation of protein consists of the transfer of a N-acetyl-D-galactosamine (GalNAc) to a serine or threonine residue.
  • This reaction is catalyzed by a family of glycosyltransferases residing in the Golgi, the Polypeptide N-acetylgalactosamine transferases (GalNAc-Ts ).
  • GalNAc-Ts Polypeptide N-acetylgalactosamine transferases
  • the inventors first characterized the relative mRNA expression level of the 19 members of the Galnt gene family in primary fully differentiated osteoblasts.
  • Galntd and GaM5 are expressed in HEK293, but not in CHO (Kato et al., 2006), and the inventors’ expression analysis shows that they are also expressed in primary mouse osteoblasts, making those two enzymes interesting candidates. Nevertheless, in HEK293 cells in which the Galnt3 and/or Galnt6 genes have been inactivated osteocalcin is still normally glycosylated (FIG. 9B). The impact of the deletion of Galntl, Galnt2 and Galnt3 was also tested since Galntl and Galnt2 are highly expressed in osteoblasts. The deletion of these 3 enzymes partially prevented osteocalcin glycosylation (FIG. 9B, last lane), suggesting that they may redundantly initiate the O-glycosylation of this protein. In summary, these results suggest that multiple GalNAc-Ts are able to initiate the O-glycosylation osteocalcin.
  • EXAMPLE 6 Glycosylated mouse osteocalcin is more stable ex vivo in plasma
  • the inventors purified the glycosylated and non-glycosylated mouse osteocalcin, produced in HEK293 cells and bacteria respectively. The purity of both forms was assessed using Coomassie staining and proteomics analysis (FIG. 10A-C). MS/MS spectrum showed a single abundant peak at 5168.51 for non-glycosylated OCN (FIG. 10B), This peak is shifted to 6202.9 and 7150.22 for the glycosylated OCN (FIG. 10C) The stability of purified protein was tested in Ocn-I- mouse plasma incubated at 37°C. As shown in FIG.
  • non-glycosylated mouse osteocalcin OCN
  • OCN non-glycosylated mouse osteocalcin
  • EXAMPLE 7 Glycosylated mouse osteocalcin’s half-life ex vivo is sensitive to temperature
  • the instability of non-O-glycosylated mouse OCN is dependent on the temperature (37 °C vs 4 °C) and on a proteolitic activity in the plasma which is inactivated when the plasma is heat inactivated (H I) prior to the experiment.
  • EXAMPLE 8 OCN O-glycosylation increase its half-life in vivo in fed conditions
  • FIGs. 13A-B presents the results obtained with doses of 12ng/g and 40ng/g, respectively and FIG. 14 presents results obtaining with doses of 40ng/g or 80ng/g.
  • glycosylated protein Gly OCN
  • OCN non-glycosylated version
  • the inventors next examined the half-life of O-glycosylated and non-glycosylated mouse ucOCN in vivo by injecting an equal dose (40ng/g) of each protein in fed Ocn-I- mice.
  • the maximum serum level of ucOCN reached after 30 minutes of injection was 1.5 times higher with the glycosylated protein compared to the non-glycosylated form.
  • glycosylated ucOCN could be detected in the serum up to 3 hours following the injection and was further increased with 80ng/g of injection, while non-glycosylated ucOCN was already undetectable after 2 hours and slightly increased with the higher dose. (FIG. 14).
  • the inventors next examined the stability of glycosylated and non-glycosylated mouse osteocalcin in vivo by injecting an equal dose of each protein in fasting Ocn-I- mice and by measuring the level of osteocalcin in the serum using an ELISA assay.
  • the maximum serum concentrations of ucOCN was reduced at 30 min compared to the level reached in fed animals, regardless of glycosylation status.
  • the level of glycosylated ucOCN remained higher compare to that of the non-glycosylated form for the following 90 minutes. (FIGs. 15A-B).
  • the circulating levels of osteocalcin in adult humans are 9-42 ng/ml (Mayo Clinic), while they are much higher (200- 500 ng/ml) in adult mice (Ferron et al., 2015; Ferron et al., 2010b).
  • the inventors therefore tested whether O- glycosylation could increase the stability and half-life of human osteocalcin.
  • the inventors purified the glycosylated (Y63S) and non-glycosylated human osteocalcin, produced in HEK293 cells and bacteria, respectively.
  • the purity of both forms was assessed using Coomassie staining and proteomics analysis (FIGs. 16C; 17A and B).
  • MS/MS spectrum showed a single abundant peak at 5850.78 for the non-glycosylated hOCN (FIG 17A), This peak is shifted to 6809.10 for the glycosylated hOCN.
  • EXAMPLE 12 Engineering DNA constructs and cell lines to produce and purified recombinant glycosylated mouse and human osteocalcin proteins
  • O-glycosylated osteocalcin can only be produced in mammalian cells, which possess the enzymatic machinery required for the O-glycosylation of proteins.
  • the inventors therefore engineered recombinant DNA constructs to allow the expression and the purification of large amounts of mouse and human O-glycosylated osteocalcin from human cells.
  • FIG. 21 The map of the DNA construct allowing the production and purification of mouse O-glycosylated osteocalcin is presented in FIG. 21 , its nucleotide sequence is shown in FIG. 22A and the protein sequence to be expressed from this construct is shown in FIGs. 22B-C.
  • FIG. 23 The map of the DNA construct allowing the production and purification of human O-glycosylated osteocalcin is presented in FIG. 23, its nucleotide sequence is shown in FIG. 24C and the protein sequence to be expressed from this construct is shown in FIGs. 24D-E.
  • These constructs allow the expression under the control of the CMV promoter of a recombinant osteocalcin protein in fusion with the Fc region of human immunoglobulin G1 (hlgG1 Fc).
  • the presence of an upstream signal peptide allows the secretion of the fusion protein in the supernatant.
  • the hlgG1 Fc domain can be used to purify the recombinant protein from the supernatant using protein G- or protein A- SepharoseTM columns.
  • the hlgG1 hinge domain followed by a thrombin cleavage site was included between the Fc region and the mature osteocalcin sequence to allow cleavage by thrombin a peptidase which cleaves peptides containing the sequence“LeuValProArg ⁇ GlySer” (SEQ ID NO: 21 ).
  • This feature allows the removal of the Fc domain to release mature glycosylated osteocalcin at the end of the purification process.
  • the FC could be retained to increase the in vivo stability of the rOCN.
  • the construct to produce glycosylated human osteocalcin also contains the Y63S mutation to create the artificial glycosylation site (FIGs. 23 and
  • the inventors next generated and screened using ELISA assays large number of clones of HEK 293 cells stably transfected with each of the constructs.
  • the inventors could identify several individual clones secreting mouse osteocalcin in the range of 5 to 40 mg/L (FIG. 26A) or human osteocalcin protein in the range of 1-3 mg/L (FIG. 26B).
  • Constructs with a thrombin cleavage site were also generated and used to purify human and mouse glycosylated OCN (see e.g., FIGs. 10A-C and 16C, 17A-B).
  • the clones with the highest level of secretion are selected and adapted to suspension culture in serum free media to further increase the amount of fusion protein secreted.
  • Ferron, M., J. Wei, T. Yoshizawa, P. Ducy, and G. Karsenty An ELISA-based method to quantify osteocalcin carboxylation in mice. Biochem Biophys Res Commun, 2010b. 397(4): p. 691-6. Ferron, M., M.D. McKee, R.L. Levine, P. Ducy, and G. Karsenty, Intermittent injections of osteocalcin improve glucose metabolism and prevent type 2 diabetes in mice. Bone, 2012. 50(2): p. 568-75.

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Abstract

The present disclosure provides a glycosylated recombinant osteocalcin (gly-rOCN), comprising the sequence of formula (I): YX2X3X4X5X6X7X8X9VX11X12X13X14X15LEPX19REVCEX25X26X27DCX30X31LX33DHX36X37FQX40AX42X43X44X45YX47X48V, wherein X2-X9, X11 -X15, X19, X25-X27, X30-X31, X33, X36-X37, X40, X42-X45, and X47-X48 are each independently any amino acid; at least one of X9, X12 and X19 is O-glycosylated. This gly-rOCN has an increased stability in blood as compared to a non-O-glycosylated OCN.

Description

STABILIZED OSTEOCALCIN AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. provisional application Serial No. 62/781 ,957, filed on December 19, 2018. The document above is incorporated herein in its entirety by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
N.A.
FIELD OF THE DISCLOSURE
The present disclosure relates to stabilized osteocalcin and uses thereof. More specifically, the present disclosure is concerned with osteocalcin (OCN) stabilized by O-glycosylation of at least one of its residue and uses of this OCN.
REFERENCE TO SEQUENCE LISTING
Pursuant to 37 C.F.R. 1.821 (c), a sequence listing is submitted herewith as an ASCII compliant text file named Sequence Listing G12810-701_ST25, that was created on December 12, 2019 and having a size of 53 kilobytes. The content of the aforementioned file named Sequence Listing G12810-701_ST25 is hereby incorporated by reference in its entirety.
BACKGROUND OF THE DISCLOSURE
Osteocalcin is a bone-derived hormone regulating glucose metabolism
Osteocalcin is a short protein, 46 amino acid-long in the mouse and 49 amino acid-long in human produced and secreted specifically by osteoblasts. Inactivation of the two genes encoding osteocalcin ( Bglapl and Bglap2) has profound metabolic consequences in mice fed a normal chow diet (Lee et al., 2007). Most strikingly, osteocalcin deficient mice (Ocn ' ) are characterized by an increased adiposity, low circulating levels of insulin, reduced peripheral insulin sensitivity and decreased glucose tolerance. They also display liver steatosis and signs of inflammation in the liver and white adipose tissue. In addition, the absence of osteocalcin causes a marked reduction in global energy expenditure, which likely contributes to the increased fat mass of these animals. Intermittent injections of recombinant osteocalcin in lean or obese mice or rats resulted in phenotypes opposite to the ones observed in the Ocrr'- mice, i.e., they increased energy expenditure, reduced fat mass, improved insulin sensitivity and prevented liver steatosis (Ferron et al., 2012; Ferron et al., 2008; Huang et al., 2017; and Gupte et al., 2014). Similarly, mouse models in which the circulating level of the active form of osteocalcin is increased, e.g., Esp'- or osteoblast-specific knockout of Ggcx, are characterized by increased insulin secretion, increased b-cell proliferation, improved insulin sensitivity, increased energy expenditure and reduced fat mass (Lee et al., 2007; Ferron et al., 2015). These results indicated what has become a hallmark of osteocalcin biology: it is necessary and sufficient to favor the physiological processes it regulates (Oury et al., 2013a; Mera et al., 2016b; Khrimian et al., 2017).
The acute effect of osteocalcin on insulin secretion appears to be mediated by calcium (Ca2+) signaling in b-cells (Hinoi et al., 2008). A receptor to which osteocalcin binds in pancreatic b-cells, a G protein-coupled receptor called GPRC6A that acts through G-Protein a-Subunit (Gsa) and C-AMP Response Element-binding protein (CREB), mediates osteocalcin regulation of b-cell proliferation, insulin expression and insulin secretion. This receptor is also expressed in myofibers and regulates the functions of osteocalcin myofibers during exercise (Mera et al., 2016b) and the effect of osteocalcin on male fertility. Remarkably, in white adipocytes osteocalcin stimulates the expression of adiponectin, a hormone that regulates bone mass accrual in animals fed a normal diet, but this action appears to be independent of GPRC6A that is not expressed in adipocytes (Kajimura et al., 2013). In addition, osteocalcin suppresses lipolysis, promotes directly glucose uptake and can suppress the secretion of pro-inflammatory cytokines in white adipocytes (Lee et al., 2007; Hill et al., 2014). Osteocalcin also promotes mitochondria biogenesis in the muscle when injected in obese mice (Ferron et al., 2012) and stimulates the expression of genes involved in thermogenesis (Pgd crand Ucp1) in brown adipocytes in vivo and ex vivo (Ferron et al., 2008). Altogether, these observations suggest that the protective effect of this hormone on obesity and insulin resistance could be, at least partially, caused by its capacity to enhance energy expenditure in muscle and brown adipose tissue.
Several cell-based assays provided evidence that osteocalcin also stimulates insulin secretion in rat and in human islets ex vivo (Sabek et al., 2015; Gao et al., 2016a; Gao et al., 2016b; and Kover et al., 2015). Likewise, genetic studies further support an impact of osteocalcin polymorphisms in glucose and energy metabolism in humans (Korostishevsky et al., 2012; Das et al., 2010). Finally, two meta-analysis demonstrated respectively that circulating levels of total osteocalcin are higher in subjects with normal glucose tolerance compared to patients with type 2 diabetes (Kunutsor et al., 2015) and that osteocalcin serum level is an independent risk factor for the development of type 2 diabetes (Liu et al., 2015).
A role for osteocalcin in adaptation to exercise
Considering the significant role of skeletal muscle in the maintenance of whole-body glucose homeostasis, it became important to determine whether osteocalcin influences any aspect of energy metabolism in skeletal muscle. Interestingly, circulating levels of bioactive osteocalcin triple in mice and also increase in human after a single bout of endurance exercise, suggesting that this hormone may be involved in the control of metabolism adaptation during exercise (Mera et al., 2016b). Analysis of 3-month-old Ocn'- mice and mice lacking GPRC6A only in skeletal muscle (Gprc6aMck -) revealed that when forced to run on a treadmill at a constant speed and until exhausted, Ocn '- and Gprc6aMck - mice run 20% to 30% less than control littermates (Mera et al., 2016b). Further investigation of this phenotype reveals that osteocalcin regulates the uptake and catabolism of glucose and fatty acids (FAs) in muscle during exercise. The uptake and utilization of these nutrients by contracting myofibers is absolutely essential for the adaptation to exercise. Consequently, the regulation of nutrient catabolism in skeletal muscle by osteocalcin certainly explains, at least in part, the decreased performance during exercise observed in Ocn '- and Gprc6aMck/· mice. Hence, in that respect osteocalcin differs from insulin that is an anabolic hormone.
Osteocalcin supports muscle function during exercise through an additional mechanism: it stimulates the production and release of interleukin-6 (IL-6), one of the first myokines ever identified (Pedersen et al., 2008; Steensberg et al., 2000). IL-6 circulating levels increase after exercise in humans and rodents in a manner that is proportional to the length of exercise and the amount of muscles involved (Febbraio et al., 2004; Febbraio et al., 2002; and Nielsen et al., 2007). Additionally, IL-6 is expressed in cultured myotubes and myofibers, and skeletal muscle is the major source of circulating IL-6 during exercise (Keller et al., 2001 ; Steenbergen et al., 2002). During exercise, IL-6 acts in an autocrine, paracrine and endocrine manner to promote skeletal muscle nutrient utilization, glucose production in the liver and lipolysis in white adipose tissue (Pedersen et al., 2008).
Osteocalcin increases skeletal muscle function and adaptation to exercise
Circulating levels of bioactive osteocalcin decline early in adult life in mice, monkeys and humans of both genders. This reduction in circulating bioactive osteocalcin occurs at the same time than the ability to perform exercise declines, at least in mice (Mera et al., 2016b). In prior work, the inventors showed that administration of exogenous osteocalcin to wild-type (WT) mice increase their endurance during exercise. A single injection of exogenous osteocalcin immediately before exercise or a chronic delivery of this hormone for one month not only improved the exercise capacity of young mice but also restored aerobic endurance in older mice to level similar to the ones seen in young adult mice (Mera et al., 2016b). Moreover, chronic delivery of osteocalcin also favors a gain in muscle mass in older mice (Mera et al., 2016a). These experiments demonstrating that osteocalcin signaling in myofibers is necessary and sufficient to increase muscle function during exercise highlighting the therapeutic potential of osteocalcin to reverse the age- induced decline in exercise capacity and muscle mass observed in humans.
Osteocalcin stimulates testosterone production and male fertility
In addition to its role in energy metabolism, osteocalcin modulates the reproductive function of males. Here, the hormone acts on the Leydig cells of the testis to activate the production of cAMP, which results in a CREB-dependent transcription of at least four genes involved in testosterone synthesis: StAR, Cyp11, 3b-H80 and Cyp17. Therefore, osteocalcin stimulates the production of testosterone in the testis and is essential to male reproductive system maturation. As a consequence, Ocn'- male mice have reduced circulating testosterone level and sperm count, smaller testes and seminal vesicles, and are subfertile (Oury et al., 201 1 ). In contrast, injection of recombinant osteocalcin can increase serum level of testosterone and sperm count in WT mice (Oury et al., 2013b).
Osteocalcin influences brain functions
In addition to its peripheral action, osteocalcin crosses the blood-brain barrier, binds to neurons of the brainstem, midbrain, and hippocampus, enhances the synthesis of monoamine neurotransmitters and inhibits GABA synthesis. Consequently, osteocalcin prevents anxiety and depression, and favors learning and memory (Oury et al., 2013a). Interestingly, intermittent injections of recombinant osteocalcin in 16-month-old mice can improve learning and memory, and reduced anxiety (Khrimian et al., 2017).
GPRC6A is the osteocalcin receptor in b-cells and in testis
As alluded above and like most known peptide hormones, osteocalcin mediates its functions through the binding of at least one specific receptor: GPRC6A. This G protein-coupled receptor shares some sequence identity with the calcium sensing receptors (CASR), which is involved in calcium homeostasis through the regulation of parathyroid hormone (PTH) release. Based on ex vivo experiments, GPRC6A was proposed to be a cation-sensing receptor, and more recently, a receptor for amino acids, steroids and osteocalcin (Pi et al., 2012; Wei et al., 2014). Gprcea 1- mice phenocopied the osteocalcin-deficient animals with regard to their defects in insulin secretion and glucose tolerance (Pi et al., 2008). Moreover, inactivation of Gprc6a specifically in the pancreas resulted in reduced b-cell proliferation and decreased insulin secretion in response to glucose (Wei et al., 2014; Pi et al., 2008). In addition, osteocalcin capacity to induce insulin secretion is abrogated in GprcQa '- islets (Wei et al., 2014; Pi et al., 2016). Further genetic experiments in mice demonstrated that GPRC6A also acts as an osteocalcin receptor in Leydig cells, where osteocalcin dependent signaling promotes testosterone synthesis (Oury et al., 2011 ; De Toni et al., 2014).
Many lines of evidence suggest that the GPRC6A function as an osteocalcin receptor is conserved in humans. First, human osteocalcin can bind and activate human GPRC6A receptor like what was reported for the mouse proteins (De Toni et al., 2014). Second and more directly, a mutation in the human GPRC6A gene that disrupts GPRC6A trafficking to the plasma membrane is associated with insulin resistance and testicular failure in humans, two phenotypes caused by osteocalcin deficiency in mice (Oury et al., 2013b). Third, polymorphisms in the human GPRC6A gene were shown to be also associated with insulin resistance and testicular failure (De Toni et al., 2016; Di Nisio et al., 2017).
The endocrine function of osteocalcin is regulated by gamma-carboxylation
To become active as a hormone, OCN undergoes a series of posttranslational modifications. First, prior to its secretion, 3 glutamic acid (Glu) residues of OCN (positioned at residues nos. 17, 21 , and 24 using the numbering of the circulating human OCM fragment (or nos. 68, 72 and 75 using the numbering the full encoded protein in human); 13, 17, and 20 using the numbering of the circulating mouse OCM fragment (or nos. 62, 66 and 69 using the numbering the full encoded protein in mouse) are modified into y-carboxyglutamic acid residues (Gla) through g-carboxylation, an enzymatic process reguiring vitamin K. The presence of Gla residues increases OCN calcium-binding properties and results in its association with hydroxyapatite in the bone extracellular matrix (ECM). OCN then undergoes a second posttranslational modification during the resorption phase of bone remodeling. The acidic environment generated by osteoclasts induces a loss of g-carboxylation on the first Glu residue (Glu17 in human; Glu13 in mouse) of OCN presents in bone ECM, thereby generating undercarboxylated Glu17-OCN (ucOCN) that is released in blood (Ferron et al., 2010a; Lacombe et al., 2013). Both the g-carboxylated and ucOCN forms are detected in the general circulation, but a number of studies performed on genetically engineered mouse models or using cell-based assays provided evidences that OCN endocrine functions are negatively regulated by g-carboxylation and that ucOCN represent the active form of this hormone (Ferron et al., 2008; Zhou et al., 2013). For instance, inactivation of Ggcx, the gene encoding the g-carboxylase, in osteoblasts results in increased circulating levels of undercarboxylated osteocalcin and improved glucose tolerance in mice (Ferron et al., 2015). A cross-sectional study in post-menopausal obese women also demonstrated that the level of osteocalcin carboxylation on its Glu17 residue positively correlates with insulin resistance and low-grade inflammation (Bonneau et al., 2017), suggesting that g-carboxylation of osteocalcin negatively regulates the function of this hormone in humans as it does in rodents.
Regulation of osteocalcin by the proprotein convertase furin Like many other peptide hormones, osteocalcin is first synthesized as a prohormone (pro-osteocalcin or pro-OCN). Yet until recently, the biological importance of pro-OCN maturation in regulating osteocalcin and the identity of the endopeptidase responsible for pro-OCN cleavage in osteoblasts were unknown. Based on biochemical and genetic arguments the proprotein convertase furin was identified by the inventors as the endopeptidase responsible for pro- OCN processing in osteoblasts (Al Rifai et al., 2017). The proteolysis of pro-OCN is critical for the activation of this hormone, since inactivation of furin in osteoblasts in mice results in decreased circulating levels of active osteocalcin, impaired glucose tolerance and reduced energy expenditure. At the mechanistic level, it appears that the retention of the pro-peptide in osteocalcin reduces its ability to be decarboxylated during the process of bone resorption.
There is a need for improving the stability of osteocalcin in circulation to achieve more efficient OCN-based therapies.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
FIGs. 1A-B. Osteocalcin is O-glycosylated in mouse osteoblasts. FIG. 1A. List of the most abundant forms of osteocalcin identified in mouse osteoblasts supernatant. The form for which a tandem mass spectrometry (MS/MS) spectrum is shown in FIG. 2 is in bold. FIG. 1 B. Schematic representation of the identified glycan adducts.
FIGs. 2A-B. FIG. 2A: position of sugar on mature mouse OCN and nature of various peptides fragments (identified as Y and b) characterized in FIG. 2B. The lines below each Y and b indicates the starting position of peptide and its direction and the number beside the y and b provides the size of the peptide in terms of amino acid number); FIG. 2B: Annotated HCD MS/MS spectrum of a modified form of osteocalcin (HexNAc-Hex-NANA + 3 Gla + S-S) (SEQ ID NO: 1 )) showing the m/z value of the different peptide fragments (y and b) of osteocalcin. The m/z value of the mature unfragmented osteocalcin is 1 180.9501 (M+5H)+5 and mass accuracy with the annotated Osteocalcin modified form is 4.6 ppm.
FIGs. 3A-B. Mouse osteocalcin is O-glycosylated in vitro. FIG. 3A. Western blot analysis on the supernatant of control and core i 3-Gal-T-specific molecular chaperone (COSMO) (C1GALT1C1, core 1 3-Gal-T- specific molecular chaperone) KO HEK293 transfected with mouse OCN-V5. FIG. 3B. Western blot analysis on the supernatant of CHO and CHO-ldID cells transfected with mouse OCN-V5. CHO-ldID cells were treated or not with 1 mM of N-AcetylGalactoseAmine (GalNAc) alone or in combination with 0.1 mM Galactose (Gal).
FIGs. 4A-B. Mouse osteocalcin is O-glycosylated in vitro. FIG. 4A. Western blot analysis on the supernatant of osteoblasts transfected with mouse OCN-V5 and treated or not with 2mM of GalNAc-bn (a pharmacological inhibitor of N-acetylgalactosamine Transferases (GalNAc-Ts)). FIG. 4B. Western blot analysis of osteocalcin deglycosylation assay. Bone extract of C57B6J mice were treated or not with O-glycosidase, which removes O-linked GalNAc and neuramidase, which removes N-Acetylneuraminic acid (NANA), for 4 hours at 37°C and analyzed by western blot using OCN antibody. FIGs. 5A-C. Mouse osteocalcin O-glycosylation is independent of its processing and y-carboxylation. OCN processing and g-carboxylation (Gla) in osteoblasts (FIG. 5A) and HEK293 (FIGs. 5B-C) transfected with mouse OCN-V5 and treated or not with 2mM of the pharmacological inhibitor of GalNAc-Ts GalNAc-bn, 50pM of the g-carboxylation inhibitor Warfarin and 50pM of the proprotein convertase inhibitor Dec-RVKR-CMK (SEQ ID NO: 2). ELISA analysis of % carboxylated (i.e. % Gla) OCN (FIG. 5A (upper panel) and FIG. 5B), and Western blot analysis using anti V5 antibody and/or anti-Gla OCN antibody (FIG. 5A (lower panel) and FIG. 50).
FIG. 6. Mouse osteocalcin O-glycosylation is independent of its processing in vivo. Western blot analysis of osteocalcin deglycosylation assay. Bone extract of Furinm and Furin0sb-'- mice were treated or not with O-glycosidase and neuraminidase for 4 hours at 37°C and analyzed by western blot using OCN antibody.
FIGs. 7A-C. Mouse osteocalcin is O-glycosylated on the serine 57 residue. FIG. 7A. Mature mouse osteocalcin amino acid sequence (SEQ ID NO: 1 ). FIGs. 7B-C. Western blot analysis on the supernatant of osteoblasts transfected with WT and different mutated forms of mouse OCN-V5. FIG. 7B. SST/AAA mutant (Serine 54, Serine 57 and Threonine 64 were mutated to Alanine), STT/AAA (Serine 78, Threonine 85 and Threonine 94 were mutated to Alanine), 6ST/6A (Serine 54, Serine 57, Threonine 64, Serine 78, Threonine 85 and Threonine 94 were mutated to Alanine). FIG. 70. S54A (Serine 54 was mutated to Alanine), S57A (Serine 57 was mutated to Alanine), T64A (Threonine 64 was mutated to Alanine), and SST/AAA mutant (Serine 54, Serine 57 and Threonine 64 were mutated to Alanine).
FIGs. 8A-D. Osteocalcin O-glycosylation on the Serine 57 residue is independent of its processing and y-carboxylation. OCN processing and y-carboxylation in osteoblasts transfected with mouse OCN-V5 or S57A (Serine 57 was mutated to Alanine) mouse OCN-V5 and treated or not with 50pM Dec-RVKR-CMK (SEQ ID NO: 2) (FIGs. 8A-B) or 50pM Warfarin (FIGs. 8C-D). Western blot analysis using anti V5 antibody (FIG. 8A and FIG. 80)) and ELISA analysis of % carboxylated OCN (FIG. 8B and FIG. 8D).
FIGs. 9A-B. Different N-acetylgalactosamine Transferases (GalNAc-Ts) O-glycosylated OCN. FIG. 9A. Galnts expression in undifferentiated osteoblasts, results are represented as copy number of Galnt relative to Actb. FIG. 9B. Western blot analysis of OCN O-glycosylation in HEK293 deficient in COSMC or specific GALNTs. OCN-V5 were transfected in HEK293, COSMC or GALNTs deficient HEK293. Cell supernatant was analyzed by western blot using anti V5 antibody.
FIGs. 10A-C. Purification of O-glycosylated mouse OCN. Coomassie staining of purified O-glycosylated mouse OCN (O-gly OCN) compared to non-O-glycosylated mouse OCN (OCN). FIG. 10B. Purity of the purified mouse OCN . Annotated HOD MS/MS spectrum of purified non O-glycosylated mouse OCN (OCN). FIG. 10C. Production of pure O-glycosylated mouse OCN . Annotated HCD MS/MS spectrum of purified O-glycosylated mouse OCN (0- gly OCN). 2 different O-glycosylation adducts were detected and annotations thereof are shown .
FIGs. 1 1. Ex vivo half-life of O-glycosylated mouse OCN (O-gly OCN) and non-O-glycosylated mouse OCN (OCN) in OCN deficient mice (Ocn-/-) heparin plasma (n=3-6 plasma). O-gly OCN and OCN were incubated separately in normal plasma at 37°C over 5 hours. OCN levels were measured at the indicated time points using total OCN ELISA assay. Results are given as mean ±SEM. *, p<0.05; ***, pO.001 using 2-way ANOVA for repeated measurements with Bonferroni multiple comparisons testing.
FIG. 12. The decrease in OCN half-life is sensitive to temperature. Ex vivo half-life of O-glycosylated OCN (O-gly OCN) and non-O-glycosylated OCN (OCN) in OCN deficient mice (Ocn-/-) heparin plasma (n=3 plasma). O-gly OCN or OCN was incubated for 2 hours in normal plasma at 37°C or 4°C, and in heat inactivated (HI) plasma (56°C for 30 min), to inactivate protease in the serum. OCN levels were measured at the indicated time points using total OCN ELISA assay. Results are given as mean ±SEM. ***, p<0.001 using 2-way ANOVA for non repeated measurements with Bonferroni multiple comparisons testing.
FIGs. 13A-B. In vivo half-life of O-glycosylated OCN (O-gly OCN) and non-O-glycosylated OCN (OCN) in fed condition. O-gly OCN (n=3 mice) or OCN (n=5 mice) were injected intraperitoneally in OCN deficient male mice (Ocn-/-) at a dose of 12 ng/g (FIG. 13A), and O-gly OCN (n=9 mice) or OCN (n=9 mice) were injected intraperitoneally in OCN deficient male mice (Ocn-/-) at a dose of 40 ng/g (FIG. 13B). Serum was collected at the indicated time points and OCN levels were then measured using total OCN ELISA assay. Results are given as mean ±SEM. **, p<0.01 ; ***, p<0.001 using 2-way ANOVA for repeated measurements with Bonferroni multiple comparisons testing.
FIG. 14: OCN O-glycosylation increases its half-life in vivo in a dose dependent manner in fed condition. In vivo half- life of O-glycosylated OCN (O-gly OCN) and non-O-glycosylated OCN (OCN). O-gly OCN (n=4 mice) or OCN (n=4 mice) were injected intraperitoneally (I. P) in OCN deficient male mice (Ocn-/-) at a dose of 40ng/g or 80ng/g and serum was collected 120 minutes post-injection. OCN levels were measured at the indicated time points using total OCN ELISA assay. Results are given as mean ±SEM. *, p< 0.05; using 2-way ANOVA for repeated measurements with Bonferroni multiple comparisons testing.
FIGs. 15A-B: OCN O-glycosylation increase its half-life in vivo in fasting condition. In vivo half-life of O-glycosylated OCN (O-gly OCN) and non-O-glycosylated OCN (OCN) in fasting condition. OCN deficient male mice (Ocn-/-) were fasted for 16 hours, O-gly OCN (n=5 mice) or OCN (n=5 mice) was injected intraperitoneally (I. P) at a dose of 40ng/g. Serum was collected at the indicated time points and OCN levels were measured at the indicated time points using total OCN ELISA assay as an absolute concentration (ng/ml) (FIG. 15A) and as a percentage of remaining OCN at the indicated time points based on the concentration at T30 (FIG. 15B). Results are given as mean ±SEM. *, p< 0.05; **, p<0.01 using 2-way ANOVA for repeated measurements with Bonferroni multiple comparisons testing.
FIGs. 16A-C Human OCN is not naturally O-glycosylated, but Y63S mutation (Tyrosine 63 in human pro-OCN was mutated to Serine) is sufficient to induce its glycosylation. FIG. 16A. Amino acid alignment of mouse (SEQ ID NO: 1 ) and human (SEQ ID NO: 3) mature OCN, highlighted residues represent the alignment of mouse OCN potential O- glycosylation sites compare to human OCN. None of them is conserved. The star indicates the site of O-glycosylation in mouse OCN. FIG. 16B. Western blot analysis of human OCN, human pro-OCN, and human OCN and human pro- OCN gain of O-glycosylation mutant Y63S from supernatant of cells transfected with human OCN-V5. WT and the gain of O-glycosylation mutant Y63S were transfected in primary mouse osteoblasts and treated or not with Dec-RVKR- CMK (SEQ ID NO: 2). Cell supernatant was analyzed by western blot. FIG. 16C. Purification of O-glycosylated human OCN from the Fclg construct (FIGs. 23 and 24D). Coomassie staining of purified O-glycosylated human OCN (O-gly hOCN) compared to non-O-glycosylated human OCN (hOCN).
FIGs. 17A-B. FIG. 17A. Purity of the purified human OCN (hOCN). Annotated HOD MS/MS spectrum of purified non O-glycosylated human OCN (hOCN). FIG. 17B. Production of pure O-glycosylated human OCN (hOCN). Annotated HOD MS/MS spectrum of purified O-glycosylated human OCN (O-gly hOCN). One O-glycosylation adduct (HexNAc, Flex, 2NANA) was detected and annotations thereof are shown .
FIGs. 18A-G. Nucleotide and amino acid sequences of mouse and human pre-pro-OCN. FIGs. 18A-C: nucleotide sequence (FIG. 18A) (SEQ ID NO: 4) and amino acid sequence (FIG. 18B) (SEQ ID NO: 5) of mouse pre-pro-OCN, identifying expressly in FIG. 18C the various domains of native mouse pre-pro-OCN (SEQ ID NO: 6). FIGs. 18D-G. nucleotide sequence (FIG. 18D) (SEQ ID NO: 6) and amino acid sequence (FIG. 18E) (SEQ ID NO: 7) of human pre- pro-OCN, identifying expressly in FIGs. 18F-G the various domains of native human pre-pro-OCN (FIG. 18F) (SEQ ID NO: 7) and Y63S mutated human pre-pro-OCN which is artificially O-glycosylated (FIG. 18G) (SEQ ID NO: 8).
FIG. 19. Human OCN O-glycosylation increases its half-life ex vivo. Ex vivo stability of O-glycosylated human OCN (0- gly-OCN) and non-O-glycosylated human OCN (hOCN) in OCN deficient mice (Ocn-/-) heparin plasma (n=4 plasma for each of O-gly-OCN and hOCN)) . O-gly hOCN and hOCN were incubated separately in normal plasma plasma at 37°C over 5 hours. hOCN levels were measured at the indicated time points using human OCN ELISA assay. Results are given as mean ±SEM. ***, p<0.001 using 2-way ANOVA for repeated measurements with Bonferroni multiple comparisons testing.
FIG. 20: The decrease in hOCN half-life is sensitive to temperature. Ex vivo half-life of O-glycosylated hOCN (O-gly hOCN) and non-O-glycosylated hOCN (hOCN) in OCN deficient mice (Ocn-/-) heparin plasma (n=3 plasma). O-gly hOCN or hOCN was incubated for 2 hours in normal plasma at 37°C or 4°C, and/or in heat inactivated (HI) plasma at 37°C. hOCN levels were measured at the indicated time points using total hOCN ELISA assay. Results are given as mean ±SEM. **, p<0.01 ; using one-way ANOVA with Bonferroni multiple comparisons testing.
FIG. 21. Construct’s map to produce and purify glycosylated mouse OCN fused to Fc of hlgG1. Partial map of the plasmid showing the key features allowing the expression and purification of glycosylated mouse OCN from mammalian cells. The enzymatic restriction sites used for the cloning are shown above the map. The list of the key features included in the construct: CMV promoter: for transcription initiation; KOZAK sequence: for translation; SP: Signal peptide to allow protein trafficking in the secretory pathway; Fc of hlgG1 : Fc of human immunoglobulin (hlgG1 ) to allow affinity purification (e.g., using protein G or A column or beads); Hinge region: protein spacer between Fc domain and mouse OCN to provide flexibility to OCN and facilitate cleavage by thrombin; thrombin cleavage site to release mouse OCN following digestion using thrombin; and Poly(A) signal: transcription termination signal. FIGs. 22A-C. FIG. 22k. Nucleotide sequence of mouse OCN fused to Fc of hlgG1 (SEQ ID NO: 9). FIG. 22B. Amino acid sequence of mouse OCN fused to Fc of hlgG1 (SEQ ID NO: 10). FIG. 220: Schematic representation of amino acid sequence of mouse OCN fused to Fc of hlgG1 (SEQ ID NO: 10). Arrow: cleavage site by thrombin.
FIG. 23. Construct’s map to produce and purify glycosylated human OCN Y63S (Gly-rhOCN) fused to Fc of hlgG1. Partial map of the plasmid showing the key features allowing the expression and purification of glycosylated human OCN from mammalian cells. The enzymatic restriction sites used for the cloning are shown above the map. The position of the Y63S mutation in human OCN allowing its glycosylation is shown under the map. The list of the key features included in the construct: CMV promoter: for transcription initiation; KOZAK sequence: for translation; SP: Signal peptide to allow protein trafficking in the secretory pathway; Fc of hlgG1 : Fc of human immunoglobulin to allow affinity purification (e.g., using protein G or A column or beads); Hinge region: protein spacer between Fc domain and human OCN to provide flexibility to OCN and facilitate cleavage by thrombin; thrombin cleavage site to release mouse OCN following digestion using thrombin; and Poly(A) signal: transcription termination signal.
FIGs. 24A-E. FIG. 24A: Nucleotide sequence of human OCN fused to Fc of hlgG1 (SEQ ID NO: 1 1). FIG. 24B. Amino acid sequence of human OCN fused to Fc of hlgG1 (SEQ ID NO: 12). FIG. 24C: Nucleotide sequence of glycosylated human OCN Y63S fused to Fc of hlgG1 (SEQ ID NO: 13). FIG. 24D. Amino acid sequence of glycosylated human OCN Y63S fused to Fc of hlgG1 (SEQ ID NO: 14). FIG. 24E Schematic representation of amino acid sequence of glycosylated human OCN Y63S fused to Fc of hlgG1. Arrow: cleavage site by thrombin.
FIG. 25. Mouse OCN and glycosylated human OCN constructs are well expressed in HEK293 cells. Expression analysis of pcDNA3-FchlgG1 ; pcDNA3-FchlgG1 -mOCN; pcDNA3-FchlgG1-mOCN (S57A); pcDNA3-FchlgG1 -hOCN; pcDNA3-FchlgG1 -hOCN (Y63S) and pcDNA3 construct transfected in HEK293. Western blot analysis on using HRP goat antihuman Fc antibody and human OCN antibody.
FIGs. 26A-B. Generation of stable clones secreting high amounts of glycosylated mouse and human osteocalcin. ELISA measurements of mouse and human OCN . (FIG. 26A) mouse OCN level in the supernatant of clones expressing glycosylated mouse OCN fused to hlgG1 Fc domain. (FIG. 26B) human OCN level in the supernatant of clones expressing glycosylated human OCN Y63S (Gly-rhOCN) fused to hlgG1 Fc domain. Results are normalized to total protein content in the cell lysate.
FIGs. 27A-B. Table presenting mouse (SEQ ID NO: 1) and human (SEQ ID NO: 3) mature osteocalcin amino acid sequences and reported coding single nucleotide polymorphisms (SNPs) in mature human osteocalcin protein. The artificial glycosylation site tested in examples described herein (Y63S) is shown in red.
SUMMARY OF THE DISCLOSURE
The present disclosure describes a human osteocalcin modified to include an O-glycosylation. The inventors engineered an artificially glycosylated human osteocalcin and demonstrated that this increased the stability of the active protein in human plasma. They also showed that ex vivo O-glycosylation does not interfere with osteocalcin y- carboxylation or processing by furin. The disclosure also describes a method to express and purify a large amount of O-glycosylated mouse and human osteocalcin and provides a method of increasing the stability of human osteocalcin. The present disclosure also provides a method of using the O-glycosylated osteocalcin in the treatment or prevention of an osteocalcin associated disease or condition, or of a symptom thereof.
More specifically, in accordance with the present disclosure, there are provided the following items:
Item 1. Glycosylated recombinant osteocalcin (gly-rOCN), comprising the sequence of formula (I):
YX2X3X4X5X6X7X8X9VX11X12X13X14X15LEPX19REVCEX25X26X27DCX30X31 LX33DHX36X37FQX40AX42X43X44X45YX47X48V, wherein X2-X9, Xu -X15, X19, X25-X27, X30-X31 , X33, X36-X37, X40, X42-X45, and X47-X48 are each independently any amino acid; and at least one of X9, X12 and X19 is O-glycosylated.
Item 2. The gly-rOCN of item 1 , wherein the at least one of X9, X12 or X19 is O-glycosylated S or T.
Item 3. The gly-rOCN of item 1 , wherein X12 is O-glycosylated S or T.
Item 4. The gly-rOCN of any one of items 1 to 3, wherein
X2 is L;
X3 is T;
X4 is Q;
X5 is W;
Cb is L;
X7 is G;
Xe is A;
X9 is P;
X11 is P;
Xi3 is P;
Xi4 is D;
Xi5 is P;
Xi9 is R;
X25 is L;
X26 is N;
X27 is P;
X30 is D;
X31 is E;
X33 is A;
X36 is I;
X37 is G;
X4o is E;
X42 is Y;
X43 is R;
X44 is R;
X45 is F; X47 is G; and
X48 is P,
with the proviso that up to 28 of X2-X9, Xu, X13 -X15, Xi 9, X25-X27, X30-X31, X33, X36-X37, X40, X42-X45, and X47-X48 are substituted by their corresponding X’ as follows:
X'2 is P;
X’3isC;
X'4 is P;
X's is R;
X's is V;
X'7 is E;
X's is P or G;
X'g is A;
X'ii isS or H;
X'i3 is R or L;
X'i4 isG, Y or E;
X'i5 isT, AorS;
X'i9 isG orS;
Figure imgf000012_0001
as defined in item 4, are substituted by their corresponding X' defined in item 4. The gly-rOCN of item 4, wherein up to 19, 18, 17, 16, 15, 14, 13, 12 or 11 of X2-X9, Xu ,Xi3 -Xis,
Figure imgf000013_0001
X30-X31,
Figure imgf000013_0002
as defined in item 4, are substituted by their corresponding X’ defined in item 4.
Item 6. The gly-rOCN of item 4, wherein up to 10 of
Figure imgf000013_0003
as defined in item 4, are substituted by their corresponding X’ defined in item 4.
The gly-rOCN of item 4, wherein up to 9, 8, 7 or 6 of X2-X9, Xu, X13 -X15, X19, X25-X27, X30-X31, X33, X36-X37, X40, X42- X45, and X47-X48 as defined in item 4, is substituted by its corresponding X’ defined in item 4.
Item 7. The gly-rOCN of item 4, wherein up to 5 of X2-X9, Xu, X13 -X15, X19, X25-X27, X30-X31, X33, X36-X37, X40, X42-X45, and X47-X48 as defined in item 4, is substituted by its corresponding X’ defined in item 4.
The gly-rOCN of item 4, wherein up to 4, 3, 2 or only 1 of X2-X9, Xu, X13 -X15, X19, X25-X27, X30-X31, X33, X36-X37, X40, X42-X45, and X47-X48 as defined in item 4, is substituted by its corresponding X’ defined in item 4.
Item 8. The gly-rOCN of any one of items 1 to 3, wherein
Figure imgf000013_0004
Figure imgf000013_0005
are as follows:
X2 is L or P;
X3 is T or C;
X4 is Q or P;
X5 is W or R;
Cb is L or V;
X7 is G or E;
Xs is A, P or G;
X9 is P or A;
X11 is P, S or H;
Xi3 is P, R or L;
Xi4 is D, G, Y or E;
Xi5 is P, T, A or S;
Xi9 is R, G or S;
X26 is N or S;
X27 is P or L;
S;
Figure imgf000013_0006
X43 is R, W or Q;
X44 is R, C, H or P;
X45 is F or Y;
X47 is G, S or R; and
X48 is P, S or L.
Item 9. The gly-rOCN of item 1 , wherein the sequence of formula (I) is
YLYQWLGAPVPX12PDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV, wherein Xi2 is O-glycosylated S or T. Item 10. The gly-rOCN of any one of items 1 -9, wherein X12 is O-glycosylated S.
Item 1 1. The gly-rOCN of any one of items 1 -10, in its circulating form.
Item 12. The gly-rOCN of any one of items 1 -1 1 , in its active form.
Item 13. An isolated nucleic acid molecule encoding the rOCN defined in any one of items 1-12.
Item 14. An expression vector comprising the nucleic acid molecule defined in item 13.
Item 15. A host cell transformed or transfected with the vector defined in item 14.
Item 16. A method of producing the gly-rOCN defined in any one of items 1-12, comprising culturing the host cell defined in item 14 under conditions suitable to effect expression of the gly-rOCN and recovering the gly-rhOCN.
Item 17. A method of increasing the stability in blood of osteocalcin (OCN) comprising O-glycosylating at least one residue of the OCN using the method defined in item 16.
Item 18. A method of preventing or treating an osteocalcin associated disease or condition, or symptom thereof, comprising administering to a subject in need thereof a therapeutically effective amount of the gly-rOCN defined in any one of items 1 -12.
DEFINITIONS
As used herein, the term“osteocalcin” or“OCN” refers to a biologically active OCN comprising an amino acid sequence satisfying the formula (I)
YX2X3X4X5X6X7X8X9VX11X12X13X14X15LEPX19REVCEX25X26X27DCX30X31LX33DHX36X37FQX40AX42X43X44X45YX47X48V (SEQ ID NO: 15).
Such formula encompasses the human OCN (hOCN) as described in e.g., NP_954642.1 and any functional OCN. For example, it encompasses any functional OCN comprising one or more of the polymorphisms described in FIGs. 27 A- B. The term“circulating form” (also sometimes called“mature OCN” herein see e.g., FIGs. 18F and 23) refers to, when the OCN is produced in cellulo, the OCN fragment remaining after the signal peptide and the propeptide have been removed (see e.g., FIG. 18F).
The term“OCN active form” refers to the OCN which has undergone posttranslational modifications so that it does not have a y-carboxyglutamic acid residue (Gla) at position 17 using the numbering of the human circulating form (or 68 using the numbering of the full encoded human OCN). This active form is also called uncarboxylated. OCN active form may be completely decarboxylated or have Gla residues at positions 21 and/or 24, using the numbering of the human circulating fragment (or 72 and 75 using the numbering of the full encoded human OCN). The OCN active form is also meant to refer to the rOCN as modified by post-translational modification such as by glycolisation in addition to those expressly mentioned herein, acetylation, amidation, blockage, formylation, gamma-carboxyglutamic acid hydroxylation, methylation, phosphorylation, pyrrolidone carboxylic acid, and sulfatation.
The term“recombinant osteocalcin” or“rOCN” is used herein to refer to a protein encoded by a genetically manipulated nucleic acid inserted into a host cell.
More particularly, the term “recombinant osteocalcin” or“rOCN” refers to an OCN that comprises at least one modification as compared to a native (e.g., human) OCN . Without being so limited it refers to an OCN (e.g., hOCN) comprising a substitution of at least one of its residues by a serine or a threonine (e.g., P60S, Y63S/T, R70S/T). In a specific embodiment, it refers to a hOCN comprising a Y63S/T substitution.
In specific embodiments, the rOCN of the present disclosure consists of a biologically active fragment of a consensus sequence derived from the circulating form of known hOCNs (e.g., hOCN with known polymorphisms see e.g., FIGs. 27A and B) i.e. YXXXXXXXXVXXXXXLEPXREVCEXXXDCXXLXDHXXFQXAXXXXYXXV (SEQ ID NO: 15) wherein each X is independently any amino acid or is any amino acid at the corresponding position of any of the hOCN described in FIGs. 27A-B. Using the numbering of the residues of the full encoded sequence of amino acids of hOCN, the circulating form corresponds to amino acid residues 52-100 of hOCN. The rOCN of the present disclosure encompasses fragments 52-99, 52-98, 52-97, 52-96, 52-95, and 52-94; 53-100, 53-99, 53-98, 53-97, 53-96, 53-95, and 53-94; 54-100, 54-99, 54-98, 54-97, 54-96, 54-95, and 54-94; 55-100, 55-99, 55-98, 55-97, 55-96, 55-95, and 55- 94; and 56-100, 56-99, 56-98, 56-97, 56-96, 56-95, and 56-94 of the sequence of hOCN . Alternatively, using the numbering of the residues of circulating hOCN form (SEQ ID NO: 3 ) (i.e.1-49), the fragments are 1 -48, 1 -47, 1-46, 1 - 45, 1 -44, and 1 -43; 2-49, 2-48, 2-47, 2-46, 2-45, 2-44, and 2-43; 3-49, 3-48, 3-47, 3-46, 3-45, 3-44, and 3-43; 4-49, 4- 48, 4-47, 4-46, 4-45, 4-44, and 4-43; and 5-49, 5-48, 5-47, 5-46, 5-45, 5-44, and 5-43, respectively. In these consensus fragments, each X is the amino acid or is any amino acid at the corresponding position of any of the hOCNs described in FIGs. 27A-B.
As used herein, the term“biologically active OCN” refers to an OCN able to perform known OCN biological activities in vivo. Without being so limited, OCN (direct and indirect) “biological activities” include binding to GPRC6A in pancreatic b-cells, increasing calcium (Ca2+) signaling in b-cells, increasing b-cell proliferation, increasing energy expenditure, reducing fat mass, increasing insulin expression, increasing insulin secretion, increasing insulin sensitivity, decreasing liver steatosis, increasing adiponectin expression, decreasing lipolysis, increasing glucose uptake, decreasing secretion of pro-inflammatory cytokines in white adipocytes, increasing mitochondria biogenesis in muscle, increasing the expression of genes involving in thermogenesis in brown adipocytes, increasing uptake and catabolism of glucose and fatty acids (FAs) in muscle during exercise, increasing subject endurance during exercise, increasing aerobic endurance, increasing muscle mass gain, increasing muscle function during exercise; increasing production of cAMP in the Leydig cells of the testis, increasing CREB-dependent transcription of StAR, Cypl l, 3/3-HSD and Cyp17 genes involving in testosterone synthesis, increasing testosterone synthesis, increasing level of testosterone in serum, increasing sperm count; increasing OCN binding to neurons of the brainstem, midbrain, and hippocampus, increasing synthesis of monoamine neurotransmitters, inhibits GABA synthesis, decreasing anxiety and depression, and increasing learning and memory; increasing association of OCN with hydroxyapatite in the bone extracellular matrix (ECM).
In specific embodiments, the rOCN is expressed in a fusion protein comprising (i) a signal peptide (SP) to allow the OCN trafficking in the secretory pathway; (i) a domain to facilitate the purification of the rOCN, and/or if this domain remains on the construct for use in administration, to increase the in vivo stability of the OCN in circulation; (iii) a spacer between the purification/stabilization domain and the OCN to increase the flexibility of the OCN and/or, if the purification domain is to be removed by a peptidase (e.g., endopeptidase such as thrombin, enterokinase, etc.) and a cleavage site is included accordingly, the spacer can also be used to facilitate peptidase cleavage releasing the rOCN portion of the construct; (iv) optionally, an enzymatic cleavage site; (v) a transcription termination signal; or (vi) a combination of at least two of (i) to (v). The order can differ from the above. For example, the purification domain and/or domain for increasing the stability of the OCN can be in C-terminal. The construct may further optionally comprise a few additional amino acids, e.g., 1 , 2, 3, etc. between adjacent pairs of the above domains that are the result of the cloning strategy used to produce the construct which may introduce exogenous amino acids in these locations.
Signal peptide
The signal peptides encompassed by the present disclosure include any peptide that enables the efficient secretion of the rOCN of the present disclosure. Without being so limited such useful signal peptides include the native hOCN signal peptide e.g., as predicted by SignIP 4.1 ( http://www.cbs.dtu.dk/services/SignalP ) and any other heterologous signal peptide that enables the efficient secretion of the rOCN of the present disclosure such as the hlgG1 signal peptide Mefglswvflvailkgvqc (SEQ ID NO: 16). Other signal peptides can also be used such as the albumin signal peptide. Other useful signal peptides can be identified on http://www.signalpeptide.de/ and Haryardi 2015.
Purification domain
Any domain that can be affinity purified by e.g., which can be affinity purified with a protein A- or protein G-sepharose™ columns is encompassed by the present disclosure. The purification domains that can be used in the present disclosure include, without being so limited Fragment crystallizable region (Fc) fragments, GST, MBP, 3XFLAG peptide, 6XHIS, V5 and 8XHIS. They can be on the N-terminal or the C-terminal of the protein. In a specific embodiment, it is FC (e.g., IgFC). Useful Fc fragments for the present disclosure include FC fragments of IgG that comprise the hinge (the case being, the hinge may serve as spacer), and the CPI2 and CH3 domains. lgG-1 , lgG-2, lgG-3, lgG-3 and IgG- 4 for instance can be used. Without being so limited, FC fragments can also be used to stabilize the protein in vivo. Without being so limited, the purification domain can contain between 6 and 500 amino acid residues or between 6 and 450, or between 6 and 400, or between 6 and 350.
Spacer
Insertion of spacers between fusion protein domains can increase bioactivity by augmenting distance between domains alleviating potential repulsive forces between different segments (e.g., rOCN and purification/stabilization domain) of the construct resulting in increased rOCN folding and/or increased rOCN’s expression level and/or secretion. It is generally placed between the purification/stabilization domain and the rOCN to increase its flexibility. When the purification domain is to be cleaved out by a peptidase, the spacer also facilitates peptidase cleavage releasing the rOCN portion of the construct. Without being so limited, the spacer used in working embodiments described below comprises the FC lgG-1 hinge region AEPKSCDKTHTCPPCP (SEQ ID NO: 17). The GS residues at the C terminal of this sequence in the construct (see FIGs. 22B, 24B and 24D) is the result of the cloning strategy used. Additional useful spacers that can be use include i.e. flexible linker structures, rich in small hydrophilic amino acids that maintain distance between the two connected domains and improve their folding such as (EAAAK)n (SEQ ID NO: 18); (GGGGS)n (SEQ ID NO: 19); or (XPXPXP)n (SEQ ID NO: 20), wherein x is any amino acid; wherein n is any one of 1 to 5, more specifically 1 , 2, 3, 4 or 5. Without being so limited, the spacer can contain between 1 and 200 amino acid residues.
Endopeptidase cleavage site
Any peptide motif recognized by any endopeptidase is encompassed by the present disclosure. Without being so limited, it includes peptide motives recognized and cleaved by such as thrombin, Human rhinovirus (HRV) type 14 3C protease (Prescission), Tobacco Etch Virus (TEV) protease, Factor Xa, enterokinase. Without being so limited, the endopeptidase cleavage site used in working embodiments described below is LVPf^GS (SEQ ID NO: 21 ). Without being so limited, the cleavage site can contain between 2 and 10 amino acid residues.
Transcription termination signal
Any transcription termination signal may be used. Without being so limited, the transcription termination signal used in working embodiments described below is a poly(A) signal. Without being so limited, the termination signal can generally contain between 5 and 7 amino acid residues.
As used herein the term“O-glycosylation” refers to attachment of a N-acetyl-galactosamine to an oxygen atom in a serine or threonine residue by the by the enzyme UDP-N-acetyl-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase (EC number 2.4.1.41). It is followed by the attachment of other carbohydrates, such as GlcNac, 2 GlcNAc (branched), Gal and GlcNac, etc., and then sialic acid such as N-acetylneuraminic acid. The glycosylated form of the rOCN of the present disclosure is referred to as gly-rOCN.
Method of producing rOCN (e.g, qlv-rOCN)
gly-rOCN
The present disclosure encompasses a method of producing the gly-rOCN of the present disclosure comprising culturing a host cell transformed or transfected with a vector comprising a nucleic acid molecule encoding the rOCN under conditions suitable to effect expression of the gly-rOCN and recovering the gly-rhOCN.
The present disclosure encompasses nucleic acids comprising nucleotide sequences encoding the above-mentioned rOCN. The nucleic acid may be codon-optimized. The nucleic acid can be a DNA or an RNA. The nucleic acid sequence can be deduced by the skilled artisan on the basis of the disclosed amino acid sequences. In a specific embodiment, the nucleic acid encodes one of the amino acid sequences as presented in any one of FIGs. 18A-18G and 24AA-24E (orthologues and/or consensuses).
Vectors The present disclosure also encompasses vectors (plasmids) comprising the above-mentioned nucleic acids. The vectors can be of any type suitable, e.g., for expression of said polypeptides or propagation of genes encoding said polypeptides in a particular organism. The organism may be of eukaryotic origin (e.g., human cell). The specific choice of vector depends on the host organism and is known to a person skilled in the art. In an embodiment, the vector comprises transcriptional regulatory sequences or a promoter operably-linked to a nucleic acid comprising a sequence encoding a rOCN of the disclosure. A first nucleic acid sequence is“operably-linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since for example enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. "Transcriptional regulatory sequences" or “transcriptional regulatory elements" are generic terms that refer to DNA sequences, such as initiation and termination signals (terminators), enhancers, and promoters, splicing signals, polyadenylation signals, etc., which induce or control transcription of protein coding sequences with which they are operably-linked. Without being so limited, useful “promotors’ for used in the vectors of the present disclosure include the CMV promoter, EF1 a promoter, CAG promoter, PGK1 promoter, TRE (inducible) promoter, etc. Additional useful promoters may be found on https://blog.addgene.org/plasmids-101 -the-promoter-region.
As used herein the“host cell” comprises a vector comprising a nucleic acid molecule encoding the rOCN. In a specific embodiment, the host cell is a mammalian cell including but not limited to HEK293. Although HEK293 cells have been used as a host for expressing the rOCN of the present disclosure in the Examples presented herein, a person of ordinary skill in the art will understand that a number of other hosts may be used to produce recombinant proteins according to methods that are routine in the art. Representative methods are disclosed in Maniatis, et al. Cold Springs Harbor Laboratory (1989). The rOCN may be cleaved by a host’s enzyme (first by e.g., furin to remove the propeptide and then by a signal peptidase (i.e. the mammalian subunits are SPC12, SPC18, SPC21 , SPC22/23 and SPC25, named according to their molecular weight) so as to produce a secreted/circulating form of the rOCN. Any cell type with a functional O-glycosylation system can be used. Without being so limited Hamster kidney cells (HEK), Baby hamster Kidney cells, Chinese Hamster Ovary (CHO) cells, PerC6, L cell, C127 cells, 3T3 cells, BHK cells, COS-7 cells can also be used.
As used herein the terminology“conditions suitable to effect expression of the polypeptide” is meant to refer to any culture medium that will enable production of the gly-rOCN of the present disclosure. Without being so limited, it includes media prepared with a buffer, bicarbonate and/or HEPES, ions like chloride, phosphate, calcium, sodium, potassium, magnesium, iron, carbon sources like simple sugars, amino acids, potentially lipids, nucleotides, vitamins and growth factors like insulin; regular commercially available media like alpha-MEM, DMEM, Ham’s-F12 and IMDM supplemented with 2-4 mM L-glutamine and 10% Fetal bovine serum; regular commercially available animal protein free media like Hyclone™ SFM4CHO, Sigma CHO DHFR-, Cambrex POWER™ CHO CD supplemented with 2-4 mM L-glutamine. These media are desirably prepared without thymidine, and hypoxanthine to maintain selective pressure allowing stable protein-product expression. In examples presented herein, G-418 was as a positive selection (the plasmid contains a Neomycin resistance cassette).
Method of stabilizing
The present disclosure also encompasses a method of increasing the stability in blood of OCN (e.g., human OCN) comprising O-glycosylating at least one residue of the OCN using the method of producing a gly-rOCN of the present disclosure.
Method of preventing or treating
The present disclosure also encompasses a method of preventing or treating an osteocalcin associated disease or condition, or symptom thereof, comprising administering to a subject in need thereof a therapeutically effective amount of the gly-rOCN (or nucleic acid molecule encoding the rOCN, vector comprising the nucleic acid molecule, host cell comprising the vector, or composition comprising any one of the gly-rOCN, nucleic acid molecule, vector or host cell) (hereinafter any one of the gly-rOCN, nucleic acid molecule encoding the rOCN, vector comprising the nucleic acid molecule, host cell comprising the vector and composition may be designated“agent”) of the present disclosure.
As used herein the term“osteocalcin associated disease or condition, or symptom thereof refers to any disease or condition characterized at least in part by a defective osteocalcin level or function. Without being so limited, it includes diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), frailty associated with aging, cognitive disorder and male reproductive disorder. A symptom thereof includes any absence or decrease in one of the OCN“biological activities” as defined herein.
As used herein the term“subject” is meant to refer to any mammal including human, and any mammal having a non- naturally glycosylated OCN including pets such as cats, pigs, dogs, etc. In a particular embodiment, it refers to a human.
As used herein, the term“subject in need thereof” in a method of administering a gly-rOCN of the present disclosure is meant to refer to a subject that would benefit from receiving a gly-rOCN of the present disclosure. In specific embodiments, it refers to a subject that already has at least one osteocalcin associated disease or condition, or symptom thereof or to a subject likely to develop at least one osteocalcin associated disease or condition, or symptom thereof.
As used herein, the term“prevent/preventing/prevention” or“treat/treating/treatmenf, refers to eliciting the desired biological response, i.e., a prophylactic and therapeutic effect, respectively in a subject. In accordance with the present disclosure, the therapeutic effect comprises one or more of a decrease/reduction in the severity, intensity and/or duration of the osteocalcin associated disease or condition, or symptom thereof following administration of the polypeptide, nucleic acid or vectors or host cells (“agent”) of the present disclosure when compared to its severity, intensity and/or duration in the subject prior to treatment or as compared to that/those in a non-treated control subject having the infection or any symptom thereof. In accordance with the disclosure, a prophylactic effect may comprise a delay in the onset of the osteocalcin associated disease or condition, or symptom thereof in an asymptomatic subject at risk of experiencing the osteocalcin associated disease or condition, or symptom thereof at a future time; or a decrease/reduction in the severity, intensity and/or duration of an osteocalcin associated disease or condition, or symptom thereof occurring following administration of the agent of the present disclosure, when compared to the timing of their onset or their severity, intensity and/or duration in a non-treated control subject (i.e. asymptomatic subject at risk of experiencing the osteocalcin associated disease or condition, or symptom thereof); and/or a decrease/reduction in the progression of any pre-existing osteocalcin associated disease or condition, or symptom thereof in a subject following administration of the agent of the present disclosure when compared to the progression of osteocalcin associated disease or condition, or symptom thereof in a non-treated control subject having such pre-existing osteocalcin associated disease or condition, or symptom thereof. As used herein, in a therapeutic treatment, the agent of the present disclosure is administered after the onset of the osteocalcin associated disease or condition, or symptom thereof. As used herein, in a prophylactic treatment, the agent of the present disclosure is administered before the onset of the osteocalcin associated disease or condition, or symptom thereof or after the onset thereof but before the progression thereof.
As used herein, the term“decrease” or“reduction” (e.g., of a symptom of an osteocalcin associated disease or condition, or any symptom thereof) refers to a reduction (e.g., in a symptom) of at least 10% as compared to a control subject (a subject not treated with an agent of the present disclosure), in an embodiment of at least 20% lower, in a further embodiment of at least 30% lower, in a further embodiment of at least 40% lower, in a further embodiment of at least 50% lower, in a further embodiment of at least 60% lower, in a further embodiment of at least 70% lower, in a further embodiment of at least 80% lower, in a further embodiment of at least 90% lower, in a further embodiment of 100% (complete inhibition).
Similarly, as used herein, the term“increase” or“increasing” (e.g., of an OCN biological activity in a method of prevention or treatment of the present disclosure; or of OCN (e.g., human OCN) stability in a method of stabilizing OCN of the present disclosure) refers to an increase (e.g., in an OCN biological activity or OCN stability) of at least 10% as compared to a control, in an embodiment of at least 20% higher, in a further embodiment of at least 30% higher, in a further embodiment of at least 40% higher, in a further embodiment of at least 50% higher, in a further embodiment of at least 60% higher, in a further embodiment of at least 70% higher, in a further embodiment of at least 80% higher, in a further embodiment of at least 90% higher, in a further embodiment of 100% higher, in a further embodiment of 200% higher, etc. The“control” for use as reference in the method disclosed herein of preventing or treating an osteocalcin associated disease or condition, or any symptom thereof may be e.g., a control subject that has an osteocalcin associated disease or condition, or any symptom thereof, and that is not treated with an agent present disclosure. The “control” for use as reference in the method disclosed herein of stabilizing an OCN may be e.g., an OCN that is not glycosylated.
Route of administration
Gly-rOCN of the present disclosure can be administered by routes such as parenterally (e.g., intravenously intramuscularly, subcutaneously, intradermally), intranasally or orally (Mizokami et al., 2013; Mizokami et al., 2014). The route of administration can depend on a variety of factors, such as the environment and therapeutic goals. By way of example, pharmaceutical compositions of the disclosure can be in the form of a liquid, solution, suspension, pill, capsule, tablet, gelcap, powder, gel, ointment, cream, nebulae, spray, mist, atomized vapor, aerosol, or phytosome. For parenteral administrations, preparations can be in the forms of liquids, can include pharmaceutically acceptable liquid carriers such as sterile aqueous or non-aqueous solvents, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose, and the like. For oral administration, tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets can be coated by methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspension, or they can be presented as a dry product for constitution with saline or other suitable liquid vehicle before use. Dietary supplements of the disclosure also can contain pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles, preservatives, buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration also can be suitably formulated to give controlled release of the active ingredients. For intranasal administrations, preparations can be in the form of sprays, drops, gels, ointments, creams etc. and can be administered in the form of nasal atomizers, nebulizers, etc.
Dosage
Any amount of a pharmaceutical composition can be administered to a subject. The dosages will depend on many factors including the mode of administration and the age of the subject. Typically, the amount of gly-rOCN of the disclosure contained within a single dose will be an amount that effectively prevent, or treat an osteocalcin associated disease or condition, or symptom thereof without inducing significant toxicity. As used herein the term“therapeutically effective amount” is meant to refer to an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects. Typically, gly-rOCN in accordance with the present disclosure can be administered to subjects in doses ranging from 0.001 to 500 mg/kg/day and, in a more specific embodiment, about 0.1 to about 100 mg/kg/day, and, in a more specific embodiment, about 0.2 to about 20 mg/kg/day. The allometric scaling method of Mahmood et al. (Mahmood et al. 2003) can be used to extrapolate the dose from mice to human. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient.
The therapeutically effective amount of the gly-rOCN may also be measured directly. The effective amount may be given daily or weekly or fractions thereof. Typically, a pharmaceutical composition of the disclosure can be administered in an amount from about 0.001 mg up to about 500 mg per kg of body weight per day (e.g., 0.05, 0.01 , 0.1 , 0.2, 0.3, 0.5, 0.7, 0.8, 1 mg, 2 mg, 3 mg, 4mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 50 mg, 100 mg, or 250 mg). Dosages may be provided in either a single or multiple dosage regimens. For example, in some embodiments the effective amount is a dose that ranges from about 0.1 to about 100 mg/kg/day, from about 0.2 mg to about 20 mg of the gly-rOCN per day, about 1 mg to about 10 mg of the gly-rOCN per day, from about .07 mg to about 210 mg of the gly-rOCN per week, 1.4 mg to about 140 mg of the gly-rOCN per week, about 0.3 mg to about 300 mg of the gly-rOCN every three days, about 0.4 mg to about 40 mg of the gly-rOCN every other day, and about 2 mg to about 20 mg of the gly-rOCN every other day.
These are simply guidelines since the actual dose must be carefully selected and titrated by the attending physician based upon clinical factors unique to each patient or by a nutritionist. The optimal daily dose will be determined by methods known in the art and will be influenced by factors such as the age of the patient as indicated above and other clinically relevant factors. In addition, patients may be taking medications for other diseases or conditions. The other medications may be continued during the time that a gly-rOCN is given to the patient, but it is particularly advisable in such cases to begin with low doses to determine if adverse side effects are experienced.
Carriers/vehicles
Preparations containing a gly-rOCN may be provided to patients in combination with pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable” such as pharmaceutically acceptable carrier, excipient, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular inhibitor is administered.
Pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions or emulsions can be used with the disclosure. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose, and the like.
In yet another embodiment, the pharmaceutical compositions of the present disclosure can be delivered in a controlled release system. In one embodiment polymeric materials including polylactic acid, polyorthoesters, cross-linked amphipathic block copolymers and hydrogels, polyhydroxy butyric acid and polydihydropyrans can be used (see also Smolen and Ball, Controlled Drug Bioavailability, Drug product design and performance, 1984, John Wiley & Sons; Ranade and Hollinger, Drug Delivery Systems, pharmacology and toxicology series, 2003, 2nd edition, CRRC Press), in another embodiment, a pump may be used (Saudek ef a/., 1989, N. Engl. J. Med. 321 : 574).
The gly-rOCN of the present disclosure could be in the form of a lyophilized powder using appropriate excipient solutions (e.g., sucrose) as diluents.
Further, the nucleotide molecules or proteins according to the present disclosure can be introduced into individuals in a number of ways. For example, osteoblasts can be isolated from the afflicted individual, transformed with a nucleotide construct (e.g., vector) according to the instant disclosure and reintroduced to the afflicted individual in a number of ways, including an intravenous injection. Alternatively, the nucleotide construct can be administered directly to the afflicted individual, for example, by injection. The nucleotide construct can also be delivered through a vehicle such as a liposome, which can be designed to be targeted to a specific cell type and engineered to be administered through different routes.
The gly-rOCN of the present disclosure could also be advantageously delivered through gene therapy. Useful gene therapy methods include those described in W006060641A2, US7179903 and W00136620A2 to Genzyme using for instance an adenovirus vector for the therapeutic protein and targeting hepatocytes as protein producing cells.
A "gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic hosts, and may be used for gene therapy as well as for simple protein expression. "Gene delivery," "gene transfer," and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
A "viral vector" is defined as a recombinantly produced virus or viral; particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors such as those described in W006002203A2, alphavirus vectors and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy.
In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (MV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene. Adenoviruses (Ads) are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071. Ads are easy to grow and do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos. WO 95/00655 and WO 95/1 1984. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, Wl). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation.
The gly-rOCN of the present disclosure may also be used in combination with at least one other active ingredient to treat or prevent an osteocalcin associated disease or condition, or symptom thereof.
Kits
The present disclosure also relates to a kit for treating or preventing an osteocalcin associated disease or condition, or symptom thereof comprising a nucleic acid, a protein or a ligand in accordance with the present disclosure. For instance, it may comprise a bone targeted composition of the present disclosure or a vector encoding same, and instructions to administer said composition or vector to a subject to treat or prevent an osteocalcin associated disease or condition, or symptom thereof. Such kits may further comprise at least one other active agent able to prevent or treat an osteocalcin associated disease or condition, or symptom thereof. In addition, a compartmentalized kit in accordance with the present disclosure includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed.
No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.
Herein, the term "about" has its ordinary meaning. In embodiments, it may mean plus or minus 10% of the numerical value qualified.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present disclosure is illustrated in further details by the following non-limiting examples.
EXAMPLE 1 : Material and Methods
Animal models
The Furinm and Furin0sb-/- mice were generated by breeding Funnm with OCA/-Cre-transgenic mice that express Ore recombinase under the control of human OCN promoter as described previously (Al Rifai et al., 2017). Ocn-I- mice were generated using homologous recombination to replace Ocn1 ( Bglapl ) and Ocn2 ( Bglap2 ) genes in the mouse Ocn cluster with a neomycin resistance cassette (Ducy et al., 1996). Both strains used in this study were backcrossed on a C57BL/6J genetic background more than 10 times and maintained under 12-hour dark/12-hour light cycles in a specific pathogen-free animal facility (SPF) at IRCM. Male mice were used in all experiments, and they were fed on normal chow diet.
DNA constructs
Mouse pro-OCN cDNA was cloned into a plRES2-EGFP-V5 plasmid in EcoRI and Agel cloning sites. SST/AAA pro- OCN, STT/AAA pro-OCN and 6ST/6A pro-OCN mutant were purchased originally from GeneArts. pcDNA3 human pre- pro-OCN cDNA was originally purchased from GenScript. Each construct was used as PCR template for amplification and to introduce EcoRI and Agel cloning sites and cloned in plRES2-EGFP-V5 plasmid. Point mutations in mouse pro- OCN (S54A; S57A, T64A) and Y63S in human pro-OCN were generated by site directed mutagenesis using specific primers (Table I).
Figure imgf000026_0002
mOCN-For-EcoRI AATTGAATTCgCcaccatgaggaccctctctc (SEQ ID NO: 22) M Cloning of mOCN in plRES2-EGFP-V5
mOCN-Rev-Stop- AATTACCGGTctaaatagtgataccgtagatg (SEQ ID NO: 23) M
Agel Cloning of mOCN in plRES2-EGFP-V5, No V5 tagged protein mOCN-Rev-Agel AATTACCGGTaatagtgataccgtagatgcg (SEQ ID NO: 24) M Cloning of mOCN in plRES2-EGFP-V5
mOCNSTT-Age1 -
M
Rev AATTACCGGTaatagcgataccgtagatgcg (SEQ ID NO: 25) Cloning of S78A,T85A,T94A mOCN in plRES2-EGFP-V5
mOCN-T64A-For CTG GAG CCC GCC CGG GAG CAG (SEQ ID NO: 26) M Mutagenesis of Threonine 64 to Alanine in mOCN
mOCN-T64A-Rev ctg etc ccg ggc ggg etc cag (SEQ ID NO: 27) M Mutagenesis of Threonine 64 to Alanine in mOCN
mOCN-S54A-For 5’-taccttggagccGcCgtccccagccca-3’ (SEQ ID NO: 28) M Mutagenesis of Serine 54 to Alanine in mOCN
mOCN-S54A-Rev 5’-tgggctggggacggcggctccaaggta-3’ (SEQ ID NO: 29) M Mutagenesis of Serine 54 to Alanine in mOCN
mOCN-S57A-For 5’-gcctcagtccccGCcccagatcccctg-3’ (SEQ ID NO: 30) M Mutagenesis of Serine 57 to Alanine in mOCN
Figure imgf000026_0001
mOCN-S57A-Rev 5’-caggggatctggggcggggactgaggc-3’ (SEQ ID NO: 31 ) M Mutagenesis of Serine 57 to Alanine in mOCN
mOCNSST/AAA- Mutation of Alanine 64 to Threonine in mOCN S54A, S57A, T64A. To
M
A64T-For 5’-cccctggagcccAcTcgggagcagtgt-3’ (SEQ ID NO: 32) generate S54A, S57A mOCN
mOCNSST/AAA- Mutation of Alanine 64 to Threonine in mOCN S54A, S57A, T64A. To
5’-acactgctcccgagtgggctccagggg-3’ (SEQ ID NO: 33) M
A64T-Rev generate S54A, S57A mOCN
hOCN-EcoRI-For Aattgaattcgccaccatgagagccctcacactcct (SEQ ID NO: 34) FI Cloning human osteocalcin in plRES2-EGFP-V5
hOCN-Agel-Rev aatt accggt gaccgggccgtagaagcg (SEQ ID NO: 35) FI Cloning FI osteocalcin in plRES2-EGFP-V5
hOCN-Y63S-For gccccagtccccAGcccggatcccctg (SEQ ID NO: 36) FI Mutagenesis of Tyrosine 63 to Serine in hOCN
hOCN-Y63S-Rev Caggggatccgggctggggactggggc (SEQ ID NO: 37) FI Mutagenesis of Tyrosine 63 to Serine in hOCN
Hindlll-FchlgGI - AATT AAGCTT GCCACCAT GGAGTTT GGGCT G (SEQ ID Amplification of FC fragment† hinge region in pTT5FC-CTL plasmid,
For NO: 38) H and cloning in pcDNA3 in Hindl ll-BamHI
BamHI-FchlgG1 - AATT GGAT COT GGGCACGGT GGGCAT GT G (SEQ ID NO: Amplification of FC fragment† hinge region in pTT5FC-CTL plasmid,
Rev 39) H and cloning in pcDNA3 in Hindl ll-BamHI
BamHI-Thrombin- AATT ggatccCT GGTT CCGCGTGGAT CTtaccttggagcctcagtcc
mOCN-For (SEQ ID NO: 40) M Cloning of Thrombin mOCN in pcDNA3 FchlgGI using BamHI-EcoRI
EcoRI-mOCN-Rev AATTGAATTCctaaatagtgataccgtagatg (SEQ ID NO: 41) M Cloning of Thrombin mOCN in pcDNA3 FchlgGI using BamHI-EcoRI
Bgl 11-Thrombin- AATT agatctCTGGTTCCGCGT GGATCTtacctgtatcaatggctgg
hOCN-For (SEQ ID NO: 42) H Cloning of Thrombin hOCN in pcDNA3 Fchlgd using Bglll-EcoRI
EcoRI-hOCN-Rev AATTGAATTCctagaccgggccgtagaagcgc (SEQ ID NO: 43) FI Cloning of Thrombin hOCN in pcDNA3 Fchlgd using Bglll-EcoRI GalnT1 -For GCAGCATGTGAACAGCAATCA (SEQ ID NO: 44) M QPCR primer
GalnT1 -Rev GCT GAGGTAGCCCAGT CAAT C (SEQ ID NO: 45) M QPCR primer
GalnT2-For GGCAACTCCAAACTGCGACA (SEQ ID NO: 46) M QPCR primer
GalnT2-Rev T CAACAAACT GGGCCGGTG (SEQ ID NO: 47) M QPCR primer
GalnT3-For ACTT AGT GCCAT GTGACGCA (SEQ ID NO: 48) M QPCR primer
GalnT3-Rev GGGTTTCTGCAGCGGTTCTA (SEQ ID NO: 49) M QPCR primer
GalnT4-For CAAAACT GCCCCAAAGACGG (SEQ ID NO: 50) M QPCR primer
GalnT4-Rev CGCT CT GCT GCTAGCCTATT (SEQ ID NO: 51 ) M QPCR primer
GalnT5-For CCCT GAAACT GGCT GCTTGT (SEQ ID NO: 52) M QPCR primer
GalnT5-Rev AT GGAGAGAAATT CAGTCAGCAA (SEQ ID NO: 53) M QPCR primer
GalnT6-For CCAGCTCTGGCTGTTTGTCTA (SEQ ID NO: 54) M QPCR primer
GalnT6-Rev TTGGGCCAAGTAGCAT GT GA (SEQ ID NO: 55) M QPCR primer
GalnT7-For GCACAGGTTTACGCACATCA (SEQ ID NO: 56) M QPCR primer
Figure imgf000027_0001
GalnT7-Rev TTCCAGGCGGTTTT CAGTCC (SEQ ID NO: 57) M QPCR primer
GalnT9-For CAACTTT GGGCT GCGGTTAG (SEQ ID NO: 58) M QPCR primer
GalnT9-Rev CCCACATT GCTCTTGGGTCT (SEQ ID NO: 59) M QPCR primer
GalnT10-For GGAGTACCGCCACCTCTCAG (SEQ ID NO: 60) M QPCR primer
GalnTI 0-Rev AGGT CCCAGGCAATTTT GGT (SEQ ID NO: 61 ) M QPCR primer
GalnT 1 1-For GGCT GT ACCAAGT GT CCGTT (SEQ ID NO: 62) M QPCR primer
GalnTI 1-Rev GCAGGCAT GAC AAAACCAGG (SEQ ID NO: 63) M QPCR primer
GalnTI 2-For ACAACGGCTTTGCACCAT AC (SEQ ID NO: 64) M QPCR primer
GalnTI 2-Rev ACACT CTTGT GACACCCAGC (SEQ ID NO: 65) M QPCR primer
GalnTI 3-For CTGGCAAT GT GGAGGTT CTT (SEQ ID NO: 66) M QPCR primer
GalnTI 3-Rev AATT CAT CCAT CCACACTT CT GC (SEQ ID NO: 67) M QPCR primer
GalnTI 4-For T CTTT CCGAGT GT GGAT GTGT (SEQ ID NO: 68) M QPCR primer
GalnTI 4-Rev CCCAT CGGGGAAAACAT AAGGA (SEQ ID NO: 69) M QPCR primer
GalnTI 5-For CTGCGGTGGCTCTGTTGAAA (SEQ ID NO: 70) M QPCR primer
GalnTI 5-Rev CTGGGAT GTGCCT GT AGAAGG (SEQ ID NO: 71) M QPCR primer
GalnT16-For T GGT GACCAGCAAAT GT CAGA (SEQ ID NO: 72) M QPCR primer
GalnTI 6-Rev T CCGGT CGAAAT GT GAGGAG (SEQ ID NO: 73) M QPCR primer
GalnT18-For CAGAAGTGCT CGGGACAACA (SEQ ID NO: 74) M QPCR primer
GalnTI 8-Rev TTGGCT CT CCCT CT CAGACT (SEQ ID NO: 75) M QPCR primer
Galntl5-For AGTGAGCGCGTGGAATTAAG (SEQ ID NO: 76) M QPCR primer
Galntl5-Rev AGATTT GTCCT GT GGT GCGA (SEQ ID NO: 77) M QPCR primer
Wbscr17-For CTT AGGT GCT CT GGGGACCA (SEQ ID NO: 78) M QPCR primer
Wbscrl 7-Rev T GT ACAAGCT GCTCTT GACCT (SEQ ID NO: 79) M QPCR primer
Galntl6-For ACCGAGACTAGCAGTT CCCT (SEQ ID NO: 80) M QPCR primer
Galntl6-Rev GTCAT GCGCT CT GTTT CCAC (SEQ ID NO: 81 ) M QPCR primer
M=mouse
H=human
Figure imgf000028_0001
The cDNA coding of the Fc and hinge region of human immunoglobulin flanked with Hindlll-BamHI restriction sites was amplified using standard PCR and pTT5-Fd_CTL vector as template (Zhang et al., 2009). The PCR product was cloned in pcDNA3.1 -myc-His B in Hindll-BmaHI cloning site, generating the pcDNA3.1 -Fc-hinge-myc-His vector. cDNA coding for thrombin-hOCN (Y63S) was generated using plRES2-EGFP-hOCN (Y63S)-V5 as template, to which a thrombin cleavage site was added at the N-terminus and Bgll l-EcoRI restriction site were introduced by standard PCR amplifications. Thrombin-hOCN (Y63S) product was cloned in the pcDNA3.1 -Fc-hinge-myc-His vector. The generated vector pcDNA3.1 -Fc-hinge-thrombin-hOCN (Y63S) is an expression vector of human OCN fusion protein composed of the Fc and hinge region of human lgG1 , thrombin cleavage site and human OCN (Y63S), engineered to produce hlgG1 Fc fused to human glycosylated OCN which can be affinity purified using protein A- or protein G-sepharose™ columns. A thrombin cleavage site was designed to allow the purification of OCN following thrombin digestion on the column. pcDNA3.1 -Fc-hinge-thrombin-mOCN expressing wild type mature mouse OCN fused to Fc was generated following the same procedure but using different primers.
Cell culture and transfection
Primary osteoblasts were prepared as described previously (Ferron et al., 2015). In brief, calvariae from 3 days old mice were collected and washed with 1 c PBS, digested in aMEM containing 0.1 mg/ml collagenase type 2 (Worthington Biochemical Corporation) and 0.00075% trypsin. The first two digestions which last for 10 minutes were discarded. The next two 30 minutes digestions were collected, centrifuged and cultured in aMEM supplemented with 10% FBS, Pen Strep and L-Glutamine. Osteoblasts differentiation was induced by supplementing aMEM culture media with 5 mM b-glycerophosphate and 100 pg/ml L-ascorbic acid and replaced every 2 days for 21 days.
Primary osteoblasts were transfected using jetPRIME™ Reagent (Polypus transfection). After an overnight incubation, media were changed to secretion media (FBS-free aMEM plus 2mM L-glutamine, PS). After 24 hours of secretion, media were collected, and cells were lysed in protein lysis buffer (20 mM T ris-HC, pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1 % Triton, 1 mM PMSF, and 1 x protease inhibitor cocktail) and analyzed by Western blotting. In some experiments, osteoblasts were treated with the g-carboxylation inhibitor warfarin (50 pM; Santa Cruz Biotechnology), or with the N-acetylgalactosamine transferase inhibitor GalNAc-bn (2 mM; Sigma) and the proprotein convertase inhibitor Dec-RVKR-CMK (SEQ ID NO: 2) (50 pM; Tocris) combined with 22 pM vitamin K1 (Sandoz).
Chinese hamster ovary (CHO) cells, originally purchased from ATCC, and Chinese hamster ovary IdID cells (CHO- IdlD; originating from the M. Krieger laboratory (Kingsley et al., 1986)) were cultured in DMEM-F12 containing PS and 5% FBS for CHO cells or 3% FBS for CHO-ldID cells and transfected using Lipofectamine™ 2000 reagent (Life technology) following standard protocol. Secretion was performed in DMEM-F12 media supplemented with 50IU/ml penicillin and 50pg/ml Streptomycin and 22mM vitamin Ki. In the experiments, CHO-ldID culture, transfection and secretion media was supplemented with 0.1 mM galactose and/or 1 mM A/-acetylgalactosamine (GlaNAc) to rescue the O-glycosylation defect.
Experiment on COSMC knockout HEK293 cells and GALNTs deficient HEK293 cells were done in collaboration with Dr. Henrik Clausen (Goth et at, 2017). Human embryonic kidney cells HEK 293 were originally purchased from ATCC. Cells were transfected using Lipofectamine™ 2000 reagent and secretion was performed over 24 hours in EMEM supplemented with PS and 22mM VK. In some experiments, HEK 293 cells were treated with the g-carboxylation inhibitor warfarin (50 pM; Santa Cruz Biotechnology), or with the N-acetylgalactosamine transferase inhibitor GalNAc- bn (2 mM; Sigma) and the proprotein convertase inhibitor Dec-RVKR-CMK (SEQ ID NO: 2) (50 pM; Tocris) combined with 22 pM vitamin K1 (Sandoz).
For western blot analysis, proteins were resolved on 15% Tris tricine gel and blotted overnight with indicated antibody. Antibody used in examples herein: anti-V5 (mouse, clone V5-10, V8012; Sigma-Aldrich), anti— b-actin (mouse, clone AC-15, A5441 ; Sigma-Aldrich), anti-GFP (mouse, clones 7.1 and 13.1 , 1 1814460001 ; Sigma), anti-Gla OCN goat antibody which recognizes amino acids 11 -26 of carboxylated mature osteocalcin and anti-CTERM OCN goat antibody which recognizes amino acids 26-46 of mature mouse OCN (Ferron et al., 2010b).
In vitro de-qlvcosylation assay
Flushed mouse femur and tibia from Furinm and Furin0sb-/- were homogenized in lysis buffer containing (20 mM Tris- HCI, pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1 % Triton, 1 mM PMSF, and 1 x protease inhibitors cocktail). Tissue homogenates were then centrifuged for 10 minutes at 4000 rpm to remove tissue debris. In vitro de- glycosylation assay was performed on 10pg of bone homogenate. Briefly, proteins were denatured in denaturing buffer at 95°C for 5 min and incubated with O-glycosidase which removes O-linked GalNAc and neuraminidase which removes N-Acetylneuraminic acid (NANA), for 4 hours at 37°C following the NEB kit protocol (NEB; E0540S). Samples were resolved on 15% Tris tricine gel and using anti-CTERM OCN goat antibody.
Top-down Liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis of OCN in osteoblasts supernatant and on bone extract
Flushed femur and tibia from wild type mice were homogenized in lysis buffer containing (20 mM Tris-HCI, pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1 % Triton, 1 mM PMSF, and 1 x protease inhibitors cocktail). 10Opg of protein homogenate was diluted in 1.6 ml of 100 mM phosphate buffer pH 7.4 and incubated overnight at 4°C with anti-OCN antibody. After overnight incubation, samples were centrifuged at 10000 rpm for 10 minutes, supernatant was incubated with protein-G agarose beads pre-washed with 1X PBS. After for 4 hours of agitation at 4°C, beads were spin down, washed twice with 1X PBS and three times with 50 mM Ammonium Bicarbonate pH 8. OCN was then eluted with 100 pi of 0.5 M NH4OH, snap frozen in liquid nitrogen and evaporated under vacuum using Speedvac™ concentrator (Thermo scientific, savant SPD131 DDA).
Samples were diluted in 25% ACN 0.3%TFA and loaded onto a 50x4.6 mm PLRP-S 300A column (Agilent Technologies) connected to an Accela™ pump (Thermo Scientific) and an RTC autosampler (Pal systems). The buffers used for chromatography were 0.1 % formic acid (buffer A) and 100% acetonitrile/0.1 % formic acid (buffer B). Proteins and peptides were eluted with a two-slope gradient at a flowrate of 120 pL/min. Solvent B first increased from 12 to 50% in 4.5 min and then from 50 to 70% in 1.5 min. The HPLC system was coupled to a Q Exactive™ mass spectrometer (Thermo Scientific) through an electrospray Ion Source. The spray and S-lens voltages were set to 3.6 kV and 60 V, respectively. Capillary temperature was set to 225 °C. Full scan MS survey spectra (m/z 600-2000) in profile mode were acquired in the Orbitrap™ with a resolution of 70,000 with a target value at 3e6. The 4 most intense protein/peptide ions were fragmented in the HCD collision cell and analyzed in the Orbitrap™ with a target value at 5e5 and a normalized collision energy at 33. Data processing protocol: The identification of the different forms of osteocalcin was performed by manual de novo sequencing.
Galnts expression
RNA was extracted from undifferentiated and differentiated calvariae osteoblasts using TRIzol™ reagent (Invitrogen, Thermo Fisher Scientific) following standard protocol. RNA products were treated with DNAse 1 , and reversely transcribed using Poly dT oligo, random primers and MMLV transcriptase (Invitrogen). QPCR was performed on the genomic and cDNA products using specific primers and ViiA-7 QPCR machine. Results were represented as copy number normalized to Actb expression level.
Production of mouse and human OCN fused to the Fc region of human immunoglobulin
Human embryonic kidney cells HEK 293 were originally purchased from ATCC. To generate stable clone expressing glycosylated human and mouse OCN, HEK 293 were transfected with pcDNA3.1-Fc-hinge-Thrombin-hOCN (Y63S) and pcDNA3.1 -Fc-hinge-Thrombin-OCN respectively using Lipofectamine™ 2000 reagent. Following 48 hours of transfection, cells were trypsinized and resuspended in sorting buffer containing (1X sterile PBS, 2% FBS and 1 mM EDTA). Cells were sorted 5-10 cells/well in 96 well plates containing the selection media (EMEM, 10% FBS supplemented with G418 sulfate (500 pg/ml; multicell)). Following two weeks of selection, clones started to appear and the expression of mouse and human OCN was assessed using the homemade ELISA assay described previously (Ferron et al., 2010b) or in the next section, respectively. Clones that express high level of OCN were amplified and frozen in liquid Nitrogen.
Purification of mouse and human OCN fused to the Fc region of human immunoglobulin
TM 102F12 clones expressing mouse OCN fusion protein and 22H5 clone expressing human OCN fusion protein were cultured in triple 175cm2 plates. After reaching 100% confluency, cells were kept to secretes in secretion media (EMEM media supplemented with 1 % FBS and 10 pM warfarin) for 72 hours. Secretion media was collected, filtered with 0.45 pm filter, and media was buffer exchanged with 10X Binding buffer (0.2 M phosphate buffer, pH 7). Protein supernatant was then loaded into protein A affinity column (HiTrap protein A high performance, GE29-0485-76) using automated pump (GE AKTA Prime Plus Liquid Chromatography System). Colum were then washed with 10 ml 1X biding buffer (0.02 M phosphate buffer, pH 7) and 5 ml of filtered 1X PBS. To release OCN from the column, OCN fusion protein was digested with thrombin (27-0846-01 , GE healthcare) and eluted with 1X PBS, thrombin was pulled down using benzamidine sepharose beads (17-5123-10, GE healthcare). Fc region was then eluted from column using acetate buffer pH 3 and buffer exchanged with 1 M Tris pH 9. Mouse and human OCN purity were assessed using Coomassie staining and MS analysis compared. Mouse OCN was quantified using the ELISA assay as described previously (Ferron et al., 2010b). Human OCN measurements was performed using human homemade ELISA described below.
Human OCN measurements Human OCN measurements were performed using human OCN ELISA developed in the inventors’ laboratory. In brief, the ELISA plate was coated with human Glu OCN antibody (mouse, 4B6 clone recognize 3X Glu hOCN) overnight at room temperature (RT). The ELISA plate was then washed twice with wash buffer (1X PBS, 0.05% tween) and blocked with blocking buffer (3% FA free BSA in 1X PBS) for 4 hours at RT. Assay buffer (3% FA free BSA in 1X PBS), standards and samples were loaded and incubated overnight at 4°C. Following the incubation, the ELISA plate was washed 6 times with wash buffer, incubated with human OCN antibody coupled to HRP (mouse, 4C5 clone recognize C-terminal of hOCN) for 1 hour with shaking at RT. After another 6 washes with the wash buffer, the plate was tapped firmly and TMB substrate was added. After color development (15-30 minutes of incubation in dark), HCI was added to stop the reaction and absorbance was measured at 450nm using ELISA plate reader (“homemade ELISA”).
Ex vivo and in vivo half-life assay of O-qlvcosylated and non-O-qlvcosylated mouse and human OCN protein
For mouse OCN ex vivo half-life assay was performed on plasma from four independent Ocn-I- mice collected in lithium heparin tube, glycosylated OCN and non-glycosylated OCN were incubated in plasma at 37°C and OCN level was measured at indicated time points using a total mouse OCN ELISA assay as described previously (Ferron et al., 2010b). Human OCN half-life assay was performed ex vivo using Ocn-I- mice plasma and human OCN was measured using homemade ELISA described below. In some experiment, plasma was heat inactivated for 30 minutes at 56°C, to inactivate proteases, or treated with 10 mM EDTA, protease inhibitor cocktail EDTA free (PI) (7X, Roche), Phenylmethylsulfonyl Fluoride (PMSF) (1 mM, Sigma), 4-benzenesulfonyl fluoride hydrochloride (AEBSF) (2.5 mM, Sigma), Pepstatin A (Pep A) (10 pM, Sigma), 50 pM RVKR, and Benzamidine sepharose beads (BAM).
For the in vivo half-life assays, Ocn-I- male mice were injected intraperitoneally with 12ng/g, 40ng/g or 80 ng/g of O- glycosylated OCN or non-O-glycosylated OCN. Serum OCN level was analyzed at indicated time points using total mouse OCN ELISA. In all the ex vivo and in vivo study, mouse or human proteins were prepared in 3.5% BSA prepared in standard saline solution. The in vivo half-life study was performed on fed or fasted Ocn-I- male mice and it is indicated in the figure legend.
EXAMPLE 2: Mouse osteocalcin is O-glycosylated
To better characterize the posttranslational modifications present on mouse osteocalcin, OCN was immunoprecipitated from the supernatant of primary osteoblasts and from mouse bone protein extracts using polyclonal goat antibodies recognizing its C-terminus region (Ferron et al., 2010b), and characterized without tryptic digestion by HLPC Reverse- phase followed by mass spectrometry (MS) and tandem mass spectrometry (MS/MS). This revealed that most of the forms of osteocalcin present in the sample have a monoisotopic mass exceeding the expected 5243.45 Da, ranging from 5783.68 to 6190.74 Da (FIG. 1A). In depth analysis suggested that this difference could be explained by the presence of a glycan on osteocalcin (FIG. 1A). According to the various monoisotopic mass observed, the inventors could predict that this glycan is composed of one N-acetylgalactosamine (GalNAc), one galactose (Gal) and one or two N-Acetylneuraminic acid (NANA) (FIG. 1 B). MS/MS spectrum of one of the glycosylated form of osteocalcin suggests that the glycan is not linked to the only asparagine (N) residue of osteocalcin, but instead is covalently linked to the second serine (S8 in mature mouse osteocalcin or S57 in prepro-osteocalcin) (deduced from the mass of various peptide fragments (y38 peptide and b6 peptide) in FIGs. 2A versus mass of unfragmented mature mouse OCN). Given the type of sugars composing the glycan and the site of linkage, these results strongly suggest that osteocalcin is 0- glycosylated in osteoblast.
The inventors next confirmed that mouse osteocalcin is subjected to O-glycosylation using cell-based assays coupled to SDS-PAGE analyses. First, the osteocalcin molecular weight is reduced in HEK 293 cells lacking core i 3-Gal-T- specific molecular chaperone (COSMC), a protein essential to the addition of Gal on the O-linked GalNAc (FIG. 3A). Second, when expressed in CHO-ldID cells which have defective UDP-Gal/UDP-GalNAc 4-epimerase and are hence deficient in O-glycosylation, osteocalcin apparent molecular weight is also reduced compared to the parental CHO cell line (FIG. 3B). Importantly, addition of Gal and GalNAc in the culture media, which rescues the O-glycosylation defect of CHO-ldID, restored the molecular shift in the secreted osteocalcin, further supporting that it is subject to 0- glycosylation. Third, treatment of primary osteoblasts with GalNAc-bn, an inhibitor of the enzyme responsible for initiating O-glycosylation, namely N-acetylgalactosamine transferases (GalNAc-Ts), decreases the apparent molecular weight of the osteocalcin secreted in the media (FIG. 4A). Finally, treatment of bone extracts with neuraminidase and O-glycosidase, which remove respectively NANA and O-linked GalNAc, also decreases the apparent molecular weight of osteocalcin by SDS-PAGE (FIG. 4B). Together these results confirm that mouse osteocalcin is O-glycosylated in primary osteoblasts and in vivo.
EXAMPLE 3: Mouse osteocalcin O-glycosylation is independent of its carboxylation and of its processing by furin
Vitamin K-dependent gamma-carboxylation and processing of the pro-osteocalcin by the proprotein convertase furin are two posttranslational modifications regulating osteocalcin endocrine function (Ferron et al., 2015; Al Rifai et al., 2017). Whether O-glycosylation can interfere with the gamma-carboxylation or the processing of osteocalcin or vice versa was unknown. The inventors therefore tested whether pharmacologically blocking gamma-carboxylation or furin, using warfarin or Dec-RVKR-CMK (RVKR (SEQ ID NO: 2)) respectively, impacts osteocalcin O-glycosylation. Conversely, they also verified whether inhibiting O-glycosylation using a pharmacological inhibitor of N- acetylgalactosamine Transferases, namely the compound GalNAc-bn affects OCN’s processing or its gamma- carboxylation. The results of these analyses suggest that these three modifications do not influence each other significantly (FIGs. 5A-C). The inventors also tested whether furin processing may influence osteocalcin 0- glycosylation in vivo. As shown in FIG. 6, both mature osteocalcin present in control bones and pro-osteocalcin present in furin-deficient bones is deglycosylated by neuramidase and O-glycosidase, indicating that pro-osteocalcin is normally glycosylated. Altogether, these results support the notion that osteocalcin O-glycosylation is not influenced by the OCN’s carboxylation status or by the removal of its propeptide by furin. Moreover, blocking O-glycosylation does not prevent the processing of pro-osteocalcin by furin.
EXAMPLE 4: Osteocalcin is O-glycosylated on a single serine residue
The inventors next aimed to determine which amino acid(s) within osteocalcin is glycosylated. Mature mouse osteocalcin contains 3 serine residues and 3 threonine residues (FIG. 7A), the two types of amino acids on which 0- glycosylation can occur. Accordingly, mutation of all 6 serine and threonine residues into alanine abrogates osteocalcin glycosylation in primary osteoblasts (FIG. 7B). Further mutagenesis analyses revealed that the O-g lycosy I ati o n site resides within the N-terminal part of the protein on Ser54, Ser57 or Thr64 (see SST/AAA band on FIG. 7B where the O-gly OCN protein is missing vs. the STT/AAA band on FIG. 7B where the O-gly OCN protein is present). Single amino acid mutagenesis allowed the identification of Ser57 as the site of O-glycosylation of osteocalcin in osteoblasts (FIG. 7C), a result consistent with the LC-MS/MS analysis of osteocalcin isolated from bone which also suggested that Ser57 is the site of glycosylation (FIGs. 2A-B).
The inventors then assessed the impact of the Ser57Ala O-glycosylation-free mutant on osteocalcin carboxylation and processing by furin. As shown in FIG. 8A, the Ser57Ala mutant is still efficiently processed by furin, since a molecular shift is observed following treatment with RVKR (SEQ ID NO: 2). It is also carboxylated to a comparable level compared to the WT protein, independently of the processing by furin (FIG. 8B). In addition, blocking its carboxylation using warfarin did not affect the processing of Ser57Ala osteocalcin mutant (FIGs. 8C-D). These results further support the conclusion that osteocalcin O-glycosylation does not influence the other known post-translational modifications present in this protein.
EXAMPLE 5: Several polypeptide N-acetylgalactosamine transferases redundantly initiate osteocalcin O-glycosylation
The first enzymatic step in the O-glycosylation of protein consists of the transfer of a N-acetyl-D-galactosamine (GalNAc) to a serine or threonine residue. This reaction is catalyzed by a family of glycosyltransferases residing in the Golgi, the Polypeptide N-acetylgalactosamine transferases (GalNAc-Ts ). In an effort to identify one or several GalNAc- Ts involved in osteocalcin O-glycosylation the inventors first characterized the relative mRNA expression level of the 19 members of the Galnt gene family in primary fully differentiated osteoblasts. This analysis revealed that several Galnts are expressed at detectable levels with the most strongly expressed one being Galntl and Galnt2 (FIG. 9A). The generated data indicates that osteocalcin is glycosylated on Ser57 in HEK293 cells and in primary osteoblasts (FIGs. 7A-C, and data not shown). Flowever, in CHO cells, mutation of the Ser57 is not sufficient to abrogate the glycosylation (data not shown). These observations suggest that the Ga/nf(s) responsible of osteocalcin carboxylation on Ser57 in osteoblasts may be expressed in HEK, but not in CHO. It is known that Galntd and GaM5 are expressed in HEK293, but not in CHO (Kato et al., 2006), and the inventors’ expression analysis shows that they are also expressed in primary mouse osteoblasts, making those two enzymes interesting candidates. Nevertheless, in HEK293 cells in which the Galnt3 and/or Galnt6 genes have been inactivated osteocalcin is still normally glycosylated (FIG. 9B). The impact of the deletion of Galntl, Galnt2 and Galnt3 was also tested since Galntl and Galnt2 are highly expressed in osteoblasts. The deletion of these 3 enzymes partially prevented osteocalcin glycosylation (FIG. 9B, last lane), suggesting that they may redundantly initiate the O-glycosylation of this protein. In summary, these results suggest that multiple GalNAc-Ts are able to initiate the O-glycosylation osteocalcin.
The results presented above suggest that O-glycosylation is not regulating the processing of pro-osteocalcin by furin or the secretion (protein is detected in medium both when it is glycosylated and when it is not) of mature osteocalcin by osteoblasts.
EXAMPLE 6: Glycosylated mouse osteocalcin is more stable ex vivo in plasma The inventors purified the glycosylated and non-glycosylated mouse osteocalcin, produced in HEK293 cells and bacteria respectively. The purity of both forms was assessed using Coomassie staining and proteomics analysis (FIG. 10A-C). MS/MS spectrum showed a single abundant peak at 5168.51 for non-glycosylated OCN (FIG. 10B), This peak is shifted to 6202.9 and 7150.22 for the glycosylated OCN (FIG. 10C) The stability of purified protein was tested in Ocn-I- mouse plasma incubated at 37°C. As shown in FIG. 1 1 , non-glycosylated mouse osteocalcin (OCN) is rapidly degraded in the condition of this assay and shows a half-life of about 30 minutes. In contrast, the glycosylated protein (O-gly OCN) is much more stable, and its level does not decline, even after 5 hours of incubation (FIG. 1 1 ).
EXAMPLE 7: Glycosylated mouse osteocalcin’s half-life ex vivo is sensitive to temperature
The inventors tested the thermic stability of glycosylated and non-glycosylated mouse osteocalcin, produced in HEK293 cells and bacteria, respectively (FIG. 12) in OCN deficient mice (Ocn-/-) mouse plasma incubated at 37°C and 4°C under normal and heat inactivated (HI) plasma conditions, to inactivate proteases in the plasma.
As shown in FIG. 12, the instability of non-O-glycosylated mouse OCN is dependent on the temperature (37 °C vs 4 °C) and on a proteolitic activity in the plasma which is inactivated when the plasma is heat inactivated (H I) prior to the experiment.
EXAMPLE 8: OCN O-glycosylation increase its half-life in vivo in fed conditions
The inventors next examined the stability of glycosylated and non-glycosylated mouse osteocalcin in vivo by injecting an equal dose of each protein in fed Ocn-I- mice and by measuring the level of osteocalcin in the serum using an ELISA assay. FIGs. 13A-B presents the results obtained with doses of 12ng/g and 40ng/g, respectively and FIG. 14 presents results obtaining with doses of 40ng/g or 80ng/g.
The maximum serum level of osteocalcin reached after 30 minutes following the injection was 3 times higher with the glycosylated protein (Gly OCN) compared to the non-glycosylated version (OCN) (FIGs. 13A-B). Moreover, glycosylated osteocalcin can be detected at significant concentration in the serum up to 3 hours following the injection, while the non-glycosylated protein is already undetectable after 2 hours.
The inventors next examined the half-life of O-glycosylated and non-glycosylated mouse ucOCN in vivo by injecting an equal dose (40ng/g) of each protein in fed Ocn-I- mice. In fed mice, the maximum serum level of ucOCN reached after 30 minutes of injection was 1.5 times higher with the glycosylated protein compared to the non-glycosylated form. Moreover, glycosylated ucOCN could be detected in the serum up to 3 hours following the injection and was further increased with 80ng/g of injection, while non-glycosylated ucOCN was already undetectable after 2 hours and slightly increased with the higher dose. (FIG. 14).
These results establish that O-glycosylation stabilizes mouse osteocalcin protein in vitro and in vivo.
EXAMPLE 9: OCN O-glycosylation increase its half-life in vivo in fasting condition
The inventors next examined the stability of glycosylated and non-glycosylated mouse osteocalcin in vivo by injecting an equal dose of each protein in fasting Ocn-I- mice and by measuring the level of osteocalcin in the serum using an ELISA assay. In fasting animals, the maximum serum concentrations of ucOCN was reduced at 30 min compared to the level reached in fed animals, regardless of glycosylation status. However, the level of glycosylated ucOCN remained higher compare to that of the non-glycosylated form for the following 90 minutes. (FIGs. 15A-B).
Figure imgf000036_0001
Surprisingly, the alignment of mouse and human mature osteocalcin protein sequences revealed that human osteocalcin does not contain any serine or threonine residues, suggesting that it may not be O-glycosylated (FIG. 16A). In particular, the residue corresponding to mouse Ser57 is a tyrosine in the human protein (Tyr63).
The inventors tested whether mutating the tyrosine residue into a serine could potentially be sufficient to create an artificial O-glycosylation site in human osteocalcin. The inventors tested this hypothesis using V5-tagged human osteocalcin constructs transfected in mouse osteoblasts and observed that indeed the Tyr63Ser (Y63S) mutation was sufficient to induce a molecular shift in human osteocalcin suggesting O-glycosylation (FIG. 16B). Since both wild type and Y63S mutant human osteocalcin molecular weight is further increased following treatment with RVKR (SEQ ID NO: 2), the inventors concluded that this mutation does not affect processing by furin (FIG. 16B). Similar results were obtained when the constructs were transfected in a human cell line (i.e., HEK293 cells; data not shown). Of note, there is no known natural variant (SNP) creating an O-glycosylation site at residue 63 (see Table 1 in FIGs. 27A-B). Together, these results show that wild type human osteocalcin is not subjected to O-glycosylation, but that mutation of a single amino acid is sufficient to induce its glycosylation in osteoblasts and in HEK293 cells.
EXAMPLE 1 1 Glycosylated human osteocalcin is more stable in plasma
The circulating levels of osteocalcin in adult humans are 9-42 ng/ml (Mayo Clinic), while they are much higher (200- 500 ng/ml) in adult mice (Ferron et al., 2015; Ferron et al., 2010b). The inventors therefore tested whether O- glycosylation could increase the stability and half-life of human osteocalcin.
To address this hypothesis, the inventors purified the glycosylated (Y63S) and non-glycosylated human osteocalcin, produced in HEK293 cells and bacteria, respectively. The purity of both forms was assessed using Coomassie staining and proteomics analysis (FIGs. 16C; 17A and B). MS/MS spectrum showed a single abundant peak at 5850.78 for the non-glycosylated hOCN (FIG 17A), This peak is shifted to 6809.10 for the glycosylated hOCN.
Because O-glycosylation impacts mouse ucOCN half-life in plasma, the inventors next aimed to determine whether this post-translational modification has a similar effect on human ucOCN.
The stability of the purified proteins was tested in Ocn-I- mouse plasma incubated at 37°C and the concentration of osteocalcin was monitored over time using an ELISA developed by the inventors’ group (see“homemade ELISA” in Example 1 ). As shown in FIG. 19, this assay revealed that while the concentration of wild type non-glycosylated human osteocalcin in ex vivo human plasma declined gradually during the experiment (by 50% within 180 min), the concentration of the artificially O-glycosylated version remains stable throughout the 300 minutes incubation. As observed with the mouse protein, human ucOCN degradation was only inhibited when the plasma was heat inactivated (HI) or incubated at 4°C, suggesting that glycosylation protects mouse and human ucOCN from being degraded through a similar enzymatic mechanism (FIG. 20).
EXAMPLE 12: Engineering DNA constructs and cell lines to produce and purified recombinant glycosylated mouse and human osteocalcin proteins
O-glycosylated osteocalcin can only be produced in mammalian cells, which possess the enzymatic machinery required for the O-glycosylation of proteins. The inventors therefore engineered recombinant DNA constructs to allow the expression and the purification of large amounts of mouse and human O-glycosylated osteocalcin from human cells.
The map of the DNA construct allowing the production and purification of mouse O-glycosylated osteocalcin is presented in FIG. 21 , its nucleotide sequence is shown in FIG. 22A and the protein sequence to be expressed from this construct is shown in FIGs. 22B-C. The map of the DNA construct allowing the production and purification of human O-glycosylated osteocalcin is presented in FIG. 23, its nucleotide sequence is shown in FIG. 24C and the protein sequence to be expressed from this construct is shown in FIGs. 24D-E. These constructs allow the expression under the control of the CMV promoter of a recombinant osteocalcin protein in fusion with the Fc region of human immunoglobulin G1 (hlgG1 Fc). The presence of an upstream signal peptide allows the secretion of the fusion protein in the supernatant. The hlgG1 Fc domain can be used to purify the recombinant protein from the supernatant using protein G- or protein A- Sepharose™ columns. The hlgG1 hinge domain followed by a thrombin cleavage site was included between the Fc region and the mature osteocalcin sequence to allow cleavage by thrombin a peptidase which cleaves peptides containing the sequence“LeuValProArg^GlySer” (SEQ ID NO: 21 ). This feature allows the removal of the Fc domain to release mature glycosylated osteocalcin at the end of the purification process. The FC could be retained to increase the in vivo stability of the rOCN. The construct to produce glycosylated human osteocalcin also contains the Y63S mutation to create the artificial glycosylation site (FIGs. 23 and 24C-E).
Very similar constructs (i.e. enterokinase cleavage site used instead of the thrombin cleavage site shown in e.g., FIGs. 22B-C) were transfected in HEK 293 cells and the presence of the proteins in the supernatant assessed by Western blots using anti-hlgG1 and anti-hOCN antibodies (FIG. 25). The Y63S human osteocalcin Fc fusion protein migrated at a higher molecular weight on the gel compared to the wild type version of the human protein, confirming that it is indeed glycosylated. Similarly, the S57A mutation in mouse osteocalcin Fc fusion reduces the apparent molecular weight compared to the wild type mouse protein. Hence, mouse and human osteocalcin Fc fusions are properly glycosylated in HEK293 cells on S57 and S63 respectively.
The inventors next generated and screened using ELISA assays large number of clones of HEK 293 cells stably transfected with each of the constructs. The inventors could identify several individual clones secreting mouse osteocalcin in the range of 5 to 40 mg/L (FIG. 26A) or human osteocalcin protein in the range of 1-3 mg/L (FIG. 26B). Constructs with a thrombin cleavage site were also generated and used to purify human and mouse glycosylated OCN (see e.g., FIGs. 10A-C and 16C, 17A-B). The clones with the highest level of secretion are selected and adapted to suspension culture in serum free media to further increase the amount of fusion protein secreted. Using large cultures (10-100L) this method allows the routine purification of grams of glycosylated mouse or human osteocalcin. These recombinant proteins are then used in preclinical studies in rodents and primates to further validate the therapeutic potential of glycosylated versus non-glycosylated.
The scope of the claims should not be limited by the embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.
REFERENCES
Al Rifai, 0., J. Chow, J. Lacombe, C. Julien, D. Faubert, D. Susan-Resiga, R. Essalmani, J.W. Creemers, N.G. Seidah, and M. Ferron, Proprotein convertase furin regulates osteocalcin and bone endocrine function. J Clin Invest, 2017. 127(1 1): p. 4104-41 17.
Bonneau, J., G. Ferland, A.D. Karelis, E. Doucet, M. Faraj, R. Rabasa-Lhoret, and M. Ferron, Association between osteocaicin gamma-carboxyiation and insuiin resistance in overweight and obese postmenopausai women. J Diabetes Complications, 2017.
Das, S.K., N.K. Sharma, and S.C. Elbein, Analysis of osteocalcin as a candidate gene for type 2 diabetes (T2D) and intermediate traits in Caucasians and African Americans. Dis Markers, 2010. 28(5): p. 281 -6.
De Toni, L, A. Di Nisio, E. Speltra, M.S. Rocca, M. Ghezzi, D. Zuccarello, N. Turiaco, A. Ferlin, and C. Foresta, Polymorphism rs2274911 of GPRC6A as a Novel Risk Factorfor Testis Failure. J Clin Endocrinol Metab, 2016. 101 (3): p. 953-61.
De Toni, L., D. Guidolin, V. De Filippis, S. Tescari, G. Strapazzon, M. Santa Rocca, A. Ferlin, M. Plebani, and C. Foresta, Osteocalcin and Sex Hormone Binding Globulin Compete on a Specific Binding Site of GPRC6A. Endocrinology, 2016. 157(1 1 ): p. 4473-4486.
De Toni, L., V. De Filippis, S. Tescari, M. Ferigo, A. Ferlin, V. Scattolini, A. Avogaro, R. Vettor, and C. Foresta, Uncarboxyiated Osteocaicin Stimulates 25-Hydroxy Vitamin D Production in Leydig Cell Line Through a GPRC6a- Dependent Pathway. Endocrinology, 2014. 155(1 1 ): p. 4266-74.
Di Nisio, A., M.S. Rocca, G.P. Fadini, L. De Toni, G. Marcuzzo, M.C. Marescotti, M. Sanna, M. Plebani, R. Vettor, A. Avogaro, and C. Foresta, The rs2274911 polymorphism in GPRC6A gene is associated with insuiin resistance in normal weight and obese subjects. Clin Endocrinol (Oxf), 2017. 86(2): p. 185-191.
Ducy, P., C. Desbois, B. Boyce, G. Pinero, B. Story, C. Dunstan, E. Smith, J. Bonadio, S. Goldstein, C. Gundberg, A. Bradley, and G. Karsenty, increased bone formation in osteocalcin-deficient mice. Nature, 1996. 382(6590): p. 448- 52.
Febbraio, M.A. and B.K. Pedersen, Muscle-derived interleukin-6: mechanisms for activation and possible biological roles. FASEB J, 2002. 16(11 ): p. 1335-47.
Febbraio, M.A., N. Hiscock, M. Sacchetti, C.P. Fischer, and B.K. Pedersen, lnterleukin-6 is a novel factor mediating glucose homeostasis during skeletal muscle contraction. Diabetes, 2004. 53(7): p. 1643-8.
Ferron, M., E. Hinoi, G. Karsenty, and P. Ducy, Osteocalcin differentially regulates beta cell and adipocyte gene expression and affects the development of metabolic diseases in wild-type mice. Proc Natl Acad Sci U S A, 2008. 105(13): p. 5266-70.
Ferron, M., J. Lacombe, A. Germain, F. Oury, and G. Karsenty, GGCX and VKORC1 inhibit osteocaicin endocrine functions. J Cell Biol, 2015. 208(6): p. 761 -76.
Ferron, M., J. Wei, T. Yoshizawa, A. Del Fattore, R.A. DePinho, A. Teti, P. Ducy, and G. Karsenty, Insulin signaling in osteoblasts integrates bone remodeling and energy metabolism. Cell, 2010a. 142(2): p. 296-308.
Ferron, M., J. Wei, T. Yoshizawa, P. Ducy, and G. Karsenty, An ELISA-based method to quantify osteocalcin carboxylation in mice. Biochem Biophys Res Commun, 2010b. 397(4): p. 691-6. Ferron, M., M.D. McKee, R.L. Levine, P. Ducy, and G. Karsenty, Intermittent injections of osteocalcin improve glucose metabolism and prevent type 2 diabetes in mice. Bone, 2012. 50(2): p. 568-75.
Gao, J„ T. Bai, L. Ren, Y. Ding, X. Zhong, H. Wang, Y. Guo, J. Li, Y. Liu, and Y. Zhang, The PLC/PKC/Ras/MEK/Kv channel pathway is involved in uncarboxylated osteocalcin-regulated insulin secretion in rats. Peptides, 2016a. 86: p. 72-79.
Gao, J., X. Zhong, Y. Ding, T. Bai, H. Wang, H. Wu, Y. Liu, J. Yang, and Y. Zhang, Inhibition of voltage-gated potassium channels mediates uncarboxylated osteocalcin-regulated insulin secretion in rat pancreatic beta cells. Eur J Pharmacol, 2016b. 777: p. 41 -8.
Gerken, T.A., J. Raman, T.A. Fritz, and O. Jamison, Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases T1 and T2 derived from oriented random peptide substrates. J Biol Chem, 2006. 281 (43): p. 32403-16.
Goth, C.K., H.E. Tuhkanen, H. Khan, J.J. Lackman, S. Wang, Y. Narimatsu, L.H. Flansen, C.M. Overall, H. Clausen, K.T. Schjoldager, and U.E. Petaja-Repo, Site-specific O-Glycosylation by Polypeptide N- Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates beta 1 -Adrenergic Receptor N-terminal Cleavage. J Biol Chem, 2017. 292(1 1): p. 4714-4726.
Gupte, A.A., O.M. Sabek, D. Fraga, L.J. Minze, S.K. Nishimoto, J.Z. Liu, S. Afshar, L. Gaber, C.J. Lyon, A. Osama Gaber, and W.A. Hsueh, Osteocalcin protects against non-alcoholic steatohepatitis in a mouse model of metabolic syndrome. Endocrinology, 2014: p. en20141430.
Haryadi R, Flo S, Kok YJ, Pu FIX, Zheng L, Pereira NA, et al. (2015) Optimization of Fleavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells. PLoS ONE 10(2): e01 16878. doi: 10.1371 /journal . pone.01 16878.
Hill, H.S., J. Grams, R.G. Walton, J. Liu, D.R. Moellering, and W.T. Garvey, Carboxylated and uncarboxylated forms of osteocalcin directly modulate the glucose transport system and inflammation in adipocytes. Horm Metab Res, 2014. 46(5): p. 341-7.
Hinoi, E., N. Gao, D.Y. Jung, V. Yadav, T. Yoshizawa, M.G. Myers, Jr., S.C. Chua, Jr., J.K. Kim, K.H. Kaestner, and G. Karsenty, The sympathetic tone mediates leptin's inhibition of insulin secretion by modulating osteocalcin bioactivity. J Cell Biol, 2008. 183(7): p. 1235-42.
Huang, L., L. Yang, L. Luo, P. Wu, and S. Yan, Osteocalcin Improves Metabolic Profiles, Body Composition and Arterial Stiffening in an Induced Diabetic Rat Model. Exp Clin Endocrinol Diabetes, 2017.
Kajimura, D., H.W. Lee, K.J. Riley, E. Arteaga-Solis, M. Ferron, B. Zhou, C.J. Clarke, Y.A. Hannun, R.A. DePinho, E.X. Guo, J.J. Mann, and G. Karsenty, Adiponectin regulates bone mass via opposite central and peripheral mechanisms through FoxOI . Cell Metab, 2013. 17(6): p. 901 -15.
Kato K, Jeanneau C, Tarp MA, Benet-Pages A, Lorenz-Depiereux B, Bennett EP, Mandel U, Strom TM, Clausen H . Kato K1 , Jeanneau C, Tarp MA, Benet-Pages A, Lorenz-Depiereux B, Bennett EP, Mandel U, Strom TM, Clausen H . Polypeptide GalNAc-transferase T3 and familial tumoral calcinosis. Secretion of fibroblast growth factor 23 requires O- glycosylation. J Biol Chem. 2006 Jul 7;281 (27): 18370-7. Epub 2006 Apr 25. Keller, C., A. Steensberg, H. Pilegaard, T. Osada, B. Saltin, B.K. Pedersen, and P.D. Neufer, Transcriptional activation of the iL-6 gene in human contracting skeletal muscle: influence of muscle glycogen content. FASEB J, 2001. 15(14): p. 2748-50.
Khrimian, L, A. Obri, M. Ramos-Brossier, A. Rousseaud, S. Moriceau, A.S. Nicot, P. Mera, S. Kosmidis, T. Karnavas, F. Saudou, X.B. Gao, F. Oury, E. Kandel, and G. Karsenty, Gpr158 mediates osteocalcin's regulation of cognition. J Exp Med, 2017. 214(10): p. 2859-2873.
Kingsley, D.M., K.F. Kozarsky, L. Hobbie, and M. Krieger, Reversible defects in O-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant. Cell, 1986. 44(5): p. 749-59.
Korostishevsky, M., I. Malkin, S. Trofimov, Y. Pei, H.W. Deng, and G. Livshits, Significant association between body composition phenotypes and the osteocalcin genomic region in normative human population. Bone, 2012. 51 (4): p. 688-94.
Kover, K., Y. Yan, P.Y. Tong, D. Watkins, X. Li, J. Tasch, M. Hager, M. Clements, and W.V. Moore, Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions. Biochem Biophys Res Commun, 2015. 462(1 ): p. 21 -6.
Kunutsor, S.K., T.A. Apekey, and J.A. Laukkanen, Association of serum total osteocalcin with type 2 diabetes and intermediate metabolic phenotypes: systematic review and meta-analysis of observational evidence. Eur J Epidemiol, 2015. 30(8): p. 599-614.
Lacombe, J., G. Karsenty, and M. Ferron, In vivo analysis of the contribution of bone resorption to the control of glucose metabolism in mice. Mol Metab, 2013. 2(4): p. 498-504.
Lee, N.K., H. Sowa, E. Hinoi, M. Ferron, J.D. Ahn, C. Confavreux, R. Dacquin, P.J. Mee, M.D. McKee, D.Y. Jung, Z. Zhang, J.K. Kim, F. Mauvais-Jarvis, P. Ducy, and G. Karsenty, Endocrine regulation of energy metabolism by the skeleton. Cell, 2007. 130(3): p. 456-69.
Liu, C., J. Wo, Q. Zhao, Y. Wang, B. Wang, and W. Zhao, Association between Serum Total Osteocalcin Level and Type 2 Diabetes Mellitus: A Systematic Review and Meta-Analysis. Florm Metab Res, 2015. 47(1 1 ): p. 813-9.
Mera, P., K. Laue, J. Wei, J. M. Berger, and G. Karsenty, Osteocalcin is necessary and sufficient to maintain muscle mass in older mice. Mol Metab, 2016a. 5(10): p. 1042-7.
Mera, P., K. Laue, M. Ferron, C. Confavreux, J. Wei, M. Galan-Diez, A. Lacampagne, S.J. Mitchell, J.A. Mattison, Y. Chen, J. Bacchetta, P. Szulc, R.N . Kitsis, R. de Cabo, R.A. Friedman, C. Torsitano, T.E. McGraw, M. Puchowicz, I. Kurland, and G. Karsenty, Osteocalcin Signaling in Myofibers Is Necessary and Sufficient for Optimum Adaptation to Exercise. Cell Metab, 2016b. 23(6): p. 1078-92.
Mizokami A, Yasutake Y, Gao J, Matsuda M, Takahashi I, Takeuchi H, Hirata M. Osteocalcin induces release of glucagon-like peptide-1 and thereby stimulates insulin secretion in mice. PLoS One. 2013;8(2):e57375. doi: 10.1371 /journal. pone.0057375. Epub 2013 Feb 20.
Mizokami A, Yasutake Y, Higashi S, Kawakubo-Yasukochi T, Chishaki S, Takahashi I, Takeuchi H, Hirata M. Oral administration of osteocalcin improves glucose utilization by stimulating glucagon-like peptide-1 secretion. Bone. 2014 Dec;69:68-79. doi: 10.1016/j.bone.2014.09.006. Epub 2014 Sep 16. Nielsen, A.R., R. Mounier, P. Plomgaard, O.H. Mortensen, M. Penkowa, T. Speerschneider, H. Pilegaard, and B.K. Pedersen, Expression of interleukin-15 in human skeletal muscle effect of exercise and muscle fibre type composition. J Physiol, 2007. 584(Pt 1 ): p. 305-12.
Oury, F., G. Sumara, O. Sumara, M. Ferron, FI. Chang, C.E. Smith, L. Hermo, S. Suarez, B.L. Roth, P. Ducy, and G. Karsenty, Endocrine Regulation of Male Fertility by the Skeleton. Cell, 2011. 144(5): p. 796-809.
Oury, F., L. Khrimian, C.A. Denny, A. Gardin, A. Chamouni, N. Goeden, Y.Y. Huang, FI. Lee, P. Srinivas, X.B. Gao,
S. Suyama, T. Langer, J.J. Mann, T.L. Horvath, A. Bonnin, and G. Karsenty, Maternal and offspring pools of osteocalcin influence brain development and functions. Cell, 2013a. 155(1): p. 228-41.
Oury, F., M. Ferron, W. Huizhen, C. Confavreux, L. Xu, J. Lacombe, P. Srinivas, A. Chamouni, F. Lugani, FI . Lejeune,
T.R. Kumar, I. Plotton, and G. Karsenty, Osteocalcin regulates murine and human fertility through a pancreas-bone- testis axis. J Clin Invest, 2013b. 123(6): p. 2421 -33.
Pedersen, B.K. and M.A. Febbraio, Muscle as an endocrine organ: focus on muscle-derived interleukin-6. Physiol Rev, 2008. 88(4): p. 1379-406.
Pi, M. and L.D. Quarles, Multiligand specificity and wide tissue expression of GPRC6A reveals new endocrine networks. Endocrinology, 2012. 153(5): p. 2062-9.
Pi, M., K. Kapoor, R. Ye, S. Kenneth Nishimoto, J.C. Smith, J. Baudry, and L. Darryl Quarles, Evidence for Osteocalcin Binding and Activation of GPRC6A in beta-Cells. Endocrinology, 2016: p. en20152010.
Pi, M ., L. Chen, M.Z. Huang, W. Zhu, B. Ringhofer, J. Luo, L. Christenson, B. Li, J. Zhang, P.D. Jackson, P. Faber, K.R. Brunden, J.J. Harrington, and L.D. Quarles, GPRC6A null mice exhibit osteopenia, feminization and metabolic syndrome. PLoS One, 2008. 3(12): p. e3858.
Sabek, O.M., S.K. Nishimoto, D. Fraga, N. Tejpal, C. Ricordi, and A.O. Gaber, Osteocalcin Effect on Human beta- Cells Mass and Function. Endocrinology, 2015. 156(9): p. 3137-46.
Steenbergen, R.D., V.E. OudeEngberink, D. Kramer, H .F. Schrijnemakers, R.H. Verheijen, C.J. Meijer, and P.J. Snijders, Down-regulation of GATA-3 expression during human papillomavirus-mediated immortalization and cervical carcinogenesis. Am J Pathol, 2002. 160(6): p. 1945-51.
Steensberg, A., G. van Flail, T. Osada, M. Sacchetti, B. Saltin, and B. Klarlund Pedersen, Production of interleukin- 6 in contracting human skeletal muscles can account for the exercise-induced increase in plasma interleukin-6. J Physiol, 2000. 529 Pt 1 : p. 237-42.
Tanaka, H., H. Shirota, M. Kuwada, K. Terato, and K. Katayama, Plasma and bone osteocalcin levels in rats with type II collagen-induced arthritis. Arthritis Rheum, 1988. 31 (1 1): p. 1413-20.
Wei, J., T. Flanna, N. Suda, G. Karsenty, and P. Ducy, Osteocalcin promotes beta-cell proliferation during development and adulthood through Gprc6a. Diabetes, 2014. 63(3): p. 1021 -31.
Zhang, J., X. Liu, A. Bell, R. To, T.N. Baral, A. Azizi, J. Li, B. Cass, and Y. Durocher, Transient expression and purification of chimeric heavy chain antibodies. Protein Expr Purif, 2009. 65(1): p. 77-82.
Zhou, B., H. Li, J. Liu, L. Xu, W. Zang, S. Wu, and H . Sun, Intermittent injections of osteocalcin reverse autophagic dysfunction and endoplasmic reticulum stress resulting from diet-induced obesity in the vascular tissue via the NFkappaB-p65-dependent mechanism. Cell Cycle, 2013. 12(12): p. 1901-13.

Claims

1. Glycosylated recombinant osteocalcin (gly-rOCN), comprising the sequence of formula (I):
YX2X3X4X5X6X7X8X9VX11X12X13X14X15LEPX19REVCEX25X26X27DCX30X31 LX33DHX36X37FQX40AX42X43X44X45YX47X48V (SEQ ID NO: 15),
wherein X2-X9, Xu -X15, X19, X25-X27, X30-X31 , X33, X36-X37, X40, X42-X45, and X47-X48 are each independently any amino acid; and at least one of X9, X12 and X19 is O-glycosylated.
2. The gly-rOCN of claim 1 , wherein the at least one of X9, X12 or X19 is O-glycosylated S or T.
3. The gly-rOCN of claim 1 , wherein X12 is O-glycosylated S or T.
4. The gly-rOCN of any one of claims 1 to 3, wherein
X2 is L;
X3 is T;
X4 is Q;
X5 is W;
Cb is L;
X7 is G;
Xs is A;
X9 is P;
X11 is P;
Xi3 is P;
Xi4 is D;
Xi5 is P;
Xi9 is R;
X25 is L;
X26 is N;
X27 is P;
X30 is D;
X31 is E;
X33 is A;
X36 is I;
X37 is G;
X4o is E;
X42 is Y;
X43 is R; X44 is R;
X45 is F;
X47 is G; and
X48 is P,
with the proviso that up to 28 of X2-X9, Xu, X13 -X15, Xi 9, X25-X27, X30-X31, X33, X36-X37, X40, X42-X45, and X47-X48 are substituted by their corresponding X’ as follows:
X'2 is P;
X’3 is C;
X'4 is P;
X's is R;
X's is V;
X'7 is E;
X's is P or G;
X'9 is A;
X'ii is S or H;
X'i3 is R or L;
X'i4 is G, Y or E;
X'i5 is T, A or S;
X'i9 is G or S;
X'25 is P;
X'26 is S;
X'27 is L;
X'30 is E;
X'si is K or D;
X'33 is V;
X'36 is R, V or S;
X'37 is S;
X'42 is C;
X'43 is W or Q;
X'44 is C, H or P;
X'45 is Y;
Figure imgf000044_0001
X'48 is S or L.
5. The gly-rOCN of claim 4, wherein up to 20 of X2-X9, Xu, X13 -X15, X19, X25-X27, X30-X31, X33, X36-X37, X40, X42- X45, and X47-X48 as defined in claim 4, are substituted by their corresponding X' defined in claim 4.
6. The gly-rOCN of claim 4, wherein up to 10 of X2-X9, Xu, X13 -X15, X19, X25-X27, X30-X31, X33, X36-X37, X40, X42- X45, and X47-X48 as defined in claim 4, are substituted by their corresponding X’ defined in claim 4.
7. The gly-rOCN of claim 4, wherein up to 5 of X2-X9, Xu, X13 -Xis, X19, X25-X27, X30-X31, X33, X36-X37, X40, X42- X45, and X47-X48 as defined in claim 4, is substituted by its corresponding X’ defined in claim 4.
8. The gly-rOCN of any one of claims 1 to 3, wherein X2-X9, Xn, X13 -X15, X19, X25-X27, X30-X31, X33, X36-X37, X40, X42-X45, and X47-X48 are as follows:
X2 is L or P;
Xs is T or C;
X4 is Q or P;
X5 is W or R;
Cb is L or V;
X7 is G or E;
Xs is A, P or G;
X9 is P or A;
X11 is P, S or H;
Xi3 is P, R or L;
Xi4 is D, G, Y or E;
Xis is P, T, A or S;
Xi9 is R, G or S;
X25 is L or P;
X26 is N or S;
X27 is P or L;
X30 is D or E;
X31 is E, K or D;
X33 is A or V;
X36 is I, R, V or S;
X37 is G or S;
X40 is E, G or D;
X42 is Y or C;
X43 is R, W or Q;
X44 is R, C, H or P;
X45 is F or Y;
X47 is G, S or R; and
X48 is P, S or L.
9. The gly-rOCN of claim 1 , wherein the sequence of formula (I) is
YLYQWLGAPVPX12PDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV, wherein Xi2 is O-glycosylated S or T.
10. The gly-rOCN of any one of claims 1 -9, wherein X12 is O-glycosylated S.
1 1. The gly-rOCN of any one of claims 1 -10, in its circulating form.
12. The gly-rOCN of any one of claims 1 -1 1 , in its active form.
13. An isolated nucleic acid molecule encoding the rOCN defined in any one of claims 1 -12.
14. An expression vector comprising the nucleic acid molecule defined in claim 13.
15. A host cell transformed or transfected with the vector defined in claim 14.
16. A method of producing the gly-rOCN defined in any one of claims 1 -12, comprising culturing the host cell defined in claim 14 under conditions suitable to effect expression of the gly-rOCN and recovering the gly-rhOCN.
17. A method of increasing the stability in blood of osteocalcin (OCN) comprising O-glycosylating at least one residue of the OCN using the method defined in claim 16.
18. A method of preventing or treating an osteocalcin associated disease or condition, or symptom thereof, comprising administering to a subject in need thereof a therapeutically effective amount of the gly-rOCN defined in any one of claims 1 -12.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112472788A (en) * 2020-12-04 2021-03-12 广西医科大学 Application of Furin inhibitor in preparation of medicine for treating osteoporosis
WO2022136238A1 (en) * 2020-12-22 2022-06-30 Sanofi Effectorless fc molecules

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE UNIPROT [online] 1 August 1990 (1990-08-01), OSTCNHUMAN, Database accession no. P02818> *
FERRON, M. ET AL.: "Osteocalcin differentially regulates beta cell and adipocyte gene expression and affects the development of metabolic diseases in wild-type mice", PNAS, vol. 105, April 2008 (2008-04-01), pages 5266 - 5270, XP002559551, ISSN: 1091-6490, DOI: 10.1073/pnas.0711119105 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112472788A (en) * 2020-12-04 2021-03-12 广西医科大学 Application of Furin inhibitor in preparation of medicine for treating osteoporosis
WO2022136238A1 (en) * 2020-12-22 2022-06-30 Sanofi Effectorless fc molecules

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