AU2006289667A1 - Noggin and chimeric molecules thereof - Google Patents

Noggin and chimeric molecules thereof Download PDF

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AU2006289667A1
AU2006289667A1 AU2006289667A AU2006289667A AU2006289667A1 AU 2006289667 A1 AU2006289667 A1 AU 2006289667A1 AU 2006289667 A AU2006289667 A AU 2006289667A AU 2006289667 A AU2006289667 A AU 2006289667A AU 2006289667 A1 AU2006289667 A1 AU 2006289667A1
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noggin
man
glcnac
cnac
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Teresa A. Domagala
Catherine A. Liddell
Glenn R. Pilkington
John D. Priest
Raina J. Simpson
Alan D. Watts
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Apollo Life Science Ltd
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Apollo Life Science Ltd
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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    • C07—ORGANIC CHEMISTRY
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    • 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

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Description

WO 2007/028212 PCT/AU2006/001325 NOGGIN AND CHIMERIC MOLECULES THEREOF BACKGROUND OF THE INVENTION 5 FIELD OF THE INVENTION The present invention relates generally to the fields of proteins, diagnostics, therapeutics and nutrition. More particularly, the present invention provides an isolated noggin or 10 chimeric noggin molecule comprising at least a portion of noggin, wherein the noggin or chimeric noggin molecule has a profile of measurable physiochemical parameters, wherein the profile is indicative of, associated with or forms the basis of one or more pharmacological traits. The present invention further contemplates the use of the isolated noggin or chimeric noggin molecule in a range of diagnostic, prophylactic, therapeutic, 15 nutritional and/or research applications. DESCRIPTION OF THE PRIOR ART Reference to any prior art in this specification is not, and should not be taken as an 20 acknowledgment or any form of suggestion that this prior art forms a part of the common general knowledge. Dorsal-ventral patterning occurs during early embryogenesis of vertebrates and invertebrates. An important family of molecules regulating this body patterning is the bone 25 morphogenetic proteins (BMPs). Expression of BMPs is tissue specific and their actions are dependent on expression of their cell surface receptors. Additionally, three classes of secreted antagonists regulate BMP signalling, these include noggin, the DAN family of proteins and chordin (in vertebrates) or SOG (drosophila). 30 Noggin is predominantly expressed by the dorsal mesoderm during embryogenesis. Human noggin is a 205 amino acid glycoprotein and is secreted as a covalently linked homodimer WO 2007/028212 PCT/AU2006/001325 -2 of approximately 32kDa with a similar structure to BMP-7. Noggin has been shown to bind with high affinity to BMP-4 and with lower affinity to BMP-2 and BMP-7 and specifically inhibits the action of BMPs by preventing interaction with their receptors. BMPs have been shown to promote the differentiation of human embryonic stem cells 5 (hES) into trophoblasts or endoderm cells. This suggests inhibitors of BMP signalling, will be valuable for maintaining hES cells in an undifferentiated state. Human embryonic stem cells have the potential for supplying cells for transplantation therapy, drug screening, toxicology studies and functional genomics applications. 10 However, maintaining hES in an undifferentiated state usually involves the growth on inactivated mouse embryonic fibroblast (MEF) feeder layers, or alternatively supplementing cultures with MEF conditioned medium. .Cells maintained in this way are not suitable for clinical applications because of the possibility of infectious material from animal cells and/or animal-derived reagents contaminating the cultures. Hence the 15 elimination or minimization of xenogeneic proteins from culture conditions is of paramount importance in the development of hES for therapeutic applications. In addition to the risk of infection by non-human pathogens, the presence of non-human sialic acid Neu5Gc on hES grown on MEF feeder layers was shown to evoke an immune response with Neu5G-specific antibodies, which are present in most human sera (Martin et al. Nat 20 Med 11(2):228-32, 2005). Since most available noggins are derived from non-human cells such as prokaryotic cells or lower eukaryotic cells such as yeast or insect cells, there is a need to obtain therapeutic quality noggin. 25 WO 2007/028212 PCT/AU2006/001325 -3 SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the 5 inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 10 (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims. The present invention relates generally to an isolated noggin or chimeric noggin molecule comprising a profile of physiochemical parameters, wherein the profile is indicative of, 15 associated with, or forms the basis of one or more distinctive pharmacological traits. More particularly, the present invention provides an isolated noggin or chimeric noggin molecule comprising a physiochemical profile comprising a number of measurable physiochemical parameters, {[Px]1, [P] 2 ,...[Px],}, wherein P, represents a measurable physiochemical parameter and "n" is an integer 1, wherein each parameter between and including [Pj 1 to 20 [Px], is a different measurable physiochemical parameter, wherein the value of any one or more of the measurable physiochemical characteristics is indicative of, associated with, or forms the basis of, a distinctive pharmacological trait, Ty, or series of distinctive pharmacological traits {[Ty]1, [Ty]2, ... [Ty]m} wherein Ty represents a distinctive pharmacological trait and m is an integer 1 and each of [Ty]i to [Ty]m is a different 25 pharmacological trait. As used herein the term "distinctive" with regard to a pharmacological trait of a noggin or chimeric noggin molecule of the present invention refers to one or more pharmacological traits of a noggin or chimeric noggin molecule which are distinctive for the particular 30 physiochemical profile. In a particular embodiment, one or more of the pharmacological traits of an isolated noggin or chimeric noggin molecule is different from, or distinctive relative to a form of the same protein or chimeric molecule thereof produced in a WO 2007/028212 PCT/AU2006/001325 -4 prokaryotic or lower eukaryotic cell or even a higher eukaryotic cell of a non-human species. In another embodiment, the pharmacological traits of a subject isolated noggin or chimeric noggin molecule contribute to a desired functional outcome. As used herein, the term "measurable physiochemical parameters" or Px refers to one or more measurable 5 characteristics of the isolated noggin or chimeric noggin molecule. In a particular embodiment of the present invention, the measurable physiochemical parameters of a subject isolated noggin or chimeric noggin molecule contribute to or are otherwise responsible for the derived pharmacological trait, Ty. 10 An isolated noggin or chimeric noggin molecule of the present invention comprises physiochemical parameters (P,) which taken as a whole define noggin or chimeric noggin molecule. The physiochemical parameters may be selected from the group consisting of molecular weight (P 1 ), isoelectric point (pI) (P 2 ), number of isoforms (P 3 ), relative intensities of the different number of isoforms (P 4 ), percentage by weight carbohydrate 15 (PS), observed molecular weight following N-linked oligosaccharides deglycosylation (P 6 ), observed molecular weight following N and O-linked oligosaccharides deglycosylation
(P
7 ), percentage acidic monosaccharide content (P 8 ), monosaccharide content (P 9 ), sialic acid content (P 10 ), sulfate and phosphate content (P 1 1), Ser/Thr: GalNAc ratio (P 12 ), neutral percentage of N-linked oligosaccharide content (P 13 ), acidic percentage of N-linked 20 oligosaccharide content (P 1 4 ), neutral percentage of O-linked oligosaccharide content (P 15 ), acidic percentage of O-linked oligosaccharide content (P 1 6 ), ratio of N-linked oligosaccharides (P 1 7 ), ratio of O-linked oligosaccharides (P 18 ), structure of N-linked oligosaccharide fraction (P 19 ), structure of O-linked oligosaccharide fraction (P 20 ), position and make up of N-linked oligosaccharides (P 21 ), position and make up of O-linked 25 oligosaccharides (P 22 ), co-translational modification (P 23 ), post-translational modification
(P
24 ), acylation (P 2 5 ), acetylation (P 26 ), amidation (P 27 ), deamidation (P 28 ), biotinylation
(P
29 ), carbamylation or carbamoylation (P 30 ), carboxylation (P 3 1), decarboxylation (P 32 ), disulfide bond formation (P 33 ), fatty acid acylation (P 34 ), myristoylation (P 35 ), palmitoylation (P36), stearoylation (P 37 ), formylation (P 3 s), glycation (P 3 9 ), glycosylation 30 (P 40 ), glycophosphatidylinositol anchor (P 4 1), hydroxylation (P42), incorporation of selenocysteine (P 43 ), lipidation (P 44 ), lipoic acid addition (P 45 ), methylation (P46), N- or C terminal blocking (P 47 ), N- or C-terminal removal (P48), nitration (P49), oxidation of WO 2007/028212 PCT/AU2006/001325 -5 methionine (P 5 o), phosphorylation (P 5 ), proteolytic cleavage (P 5 2 ), prenylation (P 5 3 ), farnesylation (P 5 4 ), geranyl geranylation (P 55 ), pyridoxal phosphate addition (P 5 6 ), sialylation (P 57 ), desialylation (P 5 8 ), sulfation (P 5 9 ), ubiquitinylation or ubiquitination (P 60 ), addition of ubiquitin-like molecules (P 61 ), primary structure (P 62 ), secondary structure 5 (P 63 ), tertiary structure (P 64 ), quaternary structure (P 65 ), chemical stability (P 66 ), thermal stability (P 67 ). A list of these parameters is summarized in Table 2. In an embodiment, a noggin of the present invention is characterized by a profile of one or more of the following physiochemical parameters (P,) and pharmacological traits (Ty), 10 comprising: - an apparent molecular weight (P 1 ) of 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 15 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 kDa and in one embodiment 23 to 35 kDa; - a pI (P 2 ) of noggin molecule is 2 to 14 such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and in one embodiment 8 to 14; 20 - about 2 to 50 isoforms, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 isoforms and in one embodiment 2 to 16 isoforms; - a percentage by weight carbohydrate (P 5 ) of about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 25 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% and in one embodiment, 0 to 35%; 30 - an observed molecular weight of the molecule after the N-linked oligosaccharides are removed (P 6 ) of about 23 to 29 kDa; WO 2007/028212 PCT/AU2006/001325 -6 - an observed molecular weight of the molecule after the N-linked and 0-linked oligosaccharides are removed (P 7 ) of about 23 to 27 kDa; - one or more N-glycan structures as listed in Table 9 in the N-linked fraction (P 19 ); - one or more 0-glycan structures as listed in Table 10 in the 0-linked fraction (P 20 ); 5 - a site of N-glycosylation (P 2 ) which includes N-62 (numbering from the start of the signal sequence); - an immunoreactivity profile (T 1 3 ) distinct from that of a mammalian noggin expressed in a non-human cell system, and in one embodiment, the protein concentration of the noggin of the present invention is underestimated when assayed 10 using a quantitative immunoassay in comparison to a mammalian noggin expressed in a non-human cell system; - a biological activity that is distinct from that of a human noggin expressed in a non human cell system, and in one embodiment, a greater ability of noggin of the present invention to inhibit BMP-4 mediated alkaline phosphatise production (T 3 1) of 15 MC3T3 pre-osteoblast cells than that of a human noggin expressed in E. coli cells; - an effect on stem cell expansion (T 73 ) that is distinct from that of a human noggin expressed in a non-human cell system, and in one embodiment, a greater ability of noggin of the present invention to inhibit BMP-4 mediated expansion of CD34+ human hematopoietic progenitor cells than that of a human noggin expressed in E. 20 coIl. In one embodiment, the noggin or chimeric noggin molecule of the present invention contains at least one of the following structures in the N-linked fraction. In these representations, "u" or "?" represents that the anomeric configuration is either a or b, 25 and/or the linkage position is 2, 3, 4, and/or 6.
WO 2007/028212 PCT/AU2006/001325 -7 Gal ui-u GlcNRCI U man 1 alFuc Gal ui-u GIcHRAc1 66 GicHRcul- 4 Man bi-4'GlcNRcbi-4 GlcNfic 3 Gal ui-u GlcHRc Han Gal ui-u GlcNRCU M + 3 x Gal(?I-?)GlcNc(?i-?) Glycanstructure Gal(? 1 -?)G1cNAc(? 1 -?)[Gal(? 1 -?)GlcNAc(? 1 -?)]Man(al -3) [Gal (?1 -?)GlcNAc(? 1 -?)[Gal(? 1 -?)GlcNAc(? 1 -?)]Man(al -6)] [GlcNAc (?1-4)]Man(bl-4)GIcNAc(bl-4)[Fuc(?1-6)]GlcNAc+"+ 3 x Gal (?1-?)GlcNAc(?1-?)" Gal ui-u GlcNR , Uhan Fuc Gal ui--u GIcNACUi Ui 6 6 GlcNcul- 4 Han bi-4 GlcNAcbI-4 GlcNAc 3 Gal ui-u GlcNRc/ N a1 U Han Gal ui-u GlcNRcui + 3 x Gal(?1-?)GIcNc{?1-?) + Fuc(?1-?) Glycan structure Gal(? 1-?)GlcNAc(? 1 -?)[Gal(? 1-?)GlcNAc(? 1-?)]Man(al -3)[Gal (?1 -?)GleNAc(? 1 -?)[Gal(? 1 -?)G1cNAc(? 1 -?)]Man(al-6)] [GlcNAc (?1-4)]Man(bl-4)GlcNAc(bl-4)[Fuc(?1-6)]GlcNAc+"+ 3 x Gal (?1-?)GlcNAc(?1-?)+ Fue(?1-?)" WO 2007/028212 PCT/AU2006/001325 -8 Gal bi-4 GkNAlc Gal~~~~~ b- cHo i/ an, Gal bi-4 G]LcNfkP Gal bi-4 GleNflei/ ad + 3 x Gal(bI-41)GlcN~c~bI-3) Glycan structure Gal(bl1 -4)GlcNAc(bl -2)[Gal(b 1 -4)GlcNAc(bl1-4)]Man(al -?)[Gal (b 1 -4)G~cNAc(b 1 -2)[Gal(b 1 -4)G~cNAc(b 1 -6)]Man(al -?)]Man( bl1-4)GlcNAc(bl1-4)GlcNAc+"+ 3 x Gal(bl -4)GLcNAc(b 1-3)" Gal bi-4 GlcNAb Fuc hana al Gal bi-4 GleNflbi U Gal j- GcA bi -4 GlcNcbi-4 Ghcac Gal bi-4 GlCNAhPi + 3 x Gal(bI-4)GlcNAe(bi-3) Glycan structure Gal(b 1 -4)G~cNAc(bl -2) [Gal(b 1 -4)GlcNAc(b 1-4)]Man(al -?)[Gal (bl1-4)GlcNAc(bl1-2)[Gal(b 1 -4)GlcNAc(bl1-6)]Man(al -?)]Man( bl1-4)G~cNAc(bl1-4)[Fuc(al -6)] G~cNAc+ t ± 3 x Gal(bl -4)G~cNAc (bl-3)" Gal bi-4 GleNflbf Galn b±-4 GlccNb-4GlPfl U Gal bi-4 GlcNRcb, \fa kb±-4 /lNci4 l~ Gal bi-4 GlcNRbi/ + 3 x Gal(bI-4)GlcH~c(bI-3) + Gal(bi-S)Glc~nfc(bi-3) Glycan structure Gal(bl -4)GlcNAc(bl -2) [Gal(b 1 -4)GlcNAc(bl1-4)]Man(al -?)[Gal (b 1 -4)G~cNAc(b 1 -2) [Gal(bl -4)G~cNAc(b 1 -6)] Man(al -?)]Man( bl-4)GlcNAc(bl-4)GlcNAc+"+ 3 x Gal(bl-4)GlcNAc(bl-3) + Gal(bl1-3)GlcNAc(bl -3)" WO 2007/028212 PCT/AU2006/001325 -9 Gal bi-4 GleNA% Fuc Gal bi-4 GlcNfA 6 Gal bi- G~cNan bi-4 GlNflcbi-4 G1c0flc Gal bi-4 GlcNRc',/ +' 3 x Gal(bI-4)GlcNfic(bI-3) +4 Gal(bI-3)GlcHf~c(bI-3) Glycan structure Gal(b 1 -4)G~cNAc(b 1 -2) [Gal(b 1 -4)GlcNAc(b 1-4)]Man(al ?)[Gal (b 1 -4) G~cNAc(b 1 -2) [Gal(blI -4)G~cNAc(b 1 -6)]Man(al ?)]Man( bl1-4)GIcNAc(bl1-4)[Fuc(al1-6)]G~cNAc±"+ 3 x Gal(b 1 4)G~cNAc (bl1-3) + Gal(blI-3)G~cNAc(bl1-3)" Gal ui-u GlcNftu y~ Fuc Gal ul-u UIohcI4C U1 GlcNRu- 4 Man bi-4 GlcN~cbl-4 GlCNAC 3 Gal uI--U GlCNflkd /1 U an Gal u-u GIcHACUI +' 4 x Gal (?I-?)GlcNRc (?I-?) Glycan structure Gal(? 1 -?)G~cNAc(? 1 -?)[Gal(? 1 -?)G~cNAc(? 1 -7)] Man(al 3)[Gal (71 -?)G~cNAc(? 1 -?)[Gal(? 1 -?)G~cNAc(? 1 -7)] Man(al 6)] [G~cNAc (71 -4)]Man(bl -4)GlcNAc(bl1-4)[Fuc(? 1-6)1 G~cNAc+"+ 4 x Gal (71 -?)GlcNAc(? 1-?)" WO 2007/028212 PCT/AU2006/001325 -10 Gal ui-u GlcNfe UHan uz ai Fue Gal u1-u GlcNAcUI Ui 6 6 GlcNAc ui- 4 Han bi-4 GIcNAcbi- 4 GlcNAc 3 Gal ui-u GlcNAc Ma d Han1 Gal ui-u GlcNACU/ + 4 x Gal(?i-?)GlcNAc(?i-?) + Fuc(?I-?) Glycan structure Gal(? 1-?)GlcNAc(? 1-?)[Gal(? 1 -?)GlcNAc(? 1-?)]Man(al -3)[Gal (?1 -?)GlcNAc(? 1 -?)[Gal(? 1-?)GIcNAc(? 1 -?)]Man(al 6)][GlcNAc (?1-4)]Man(bl-4)GlcNAc(bl-4)[Fuc(?1-6)]GlcNAc+"+ 4 x Gal (?1-?)GlcNAc(?1-?)+ Fuc(?1-) Gal ui-u GlicNIA Uman at Fuc Gal ui-u GliCu U1 6 6 GlcNcu-- 4 Han bi-4 GleNfcbi--4 GlcNAc 3 Gal ui-u GlcNAe Puan Gal ui-u GlcNRCUt + 5 x Gal(?1-?)GlcNAc(?I-?) Glycan structure Gal(? 1-?)GlcNAc(? 1 -?)[Gal(? 1 -?)GlcNAc(? 1-?)]Man(al-3)[Gal (?1 -?)GIcNAc(?1 -?)[Gal(? 1 -?)GleNAc(? 1 -?)]Man(al 6)][GlcNAc (?1-4)]Man(bl-4)GlcNAc(bl-4)[Fuc(?1-6)]GlcNAc+"+ 5 x Gal (?1-?)GlcNAc(?1-?)" WO 2007/028212 PCT/AU2006/001325 - 11 Gal bi-41GlcNci- 3 Gal bi-43jGlcNFcbi- 2 Hana \ ~Han bi-4 GlcN~cbi-4 GlcNAc Gal bi-4 EGlcNAci- 3 Gal bl-43kGlcNAcbi- 2 Hanai Here j+k='l4 & j,k>=l Glycan structure Gal(b1-4){GlcNAc(b1-3)Gal(bl-4)}kGlcNAc(bl-2)Man(al-3)[ Gal(b1-4){GlcNAc(bl-3)Gal(b1-4)}jGlcNAc(b1-2)Man(al-6)] Man(bl-4)GlcNAc(bl-4)GlcNAc+"Where j+k=14 & j,k>=1" HeuRc a2- u Gal bi-4(GlcNcbi-3 Gal bi-4jGlcNfcbi- 2 Hanai U Hanbi-4 GlcNcbi-4 GlcNflc Gal bi-4EGlcNncbi--3 Gal bi-43kGlcNfcbi- 2 hanai Hhere j+k=14 8 j,k>=1 Glycan structure NeuAc(a2-?)Gal(bl-4){GlcNAc(bl-3)Gal(bl-4)}jGlcNAc(bl-2 )Man(al-?)[Gal(b1-4){GlcNAc(bl-3)Gal(bl-4)}kGlcNAc(b1-2 )Man(al-?)]Man(b1-4)GlcNAc(b1-4)GlcNAc+"Where j+k=14 & j,k>=1" HeuAca2- U Gal bi-4EGlcNcbi- 3 Galbi-4jGlcNRcbi-2 Hanad 9 Hian bi--4 GlcNAc bi-4 GlcNAc Neuflca2- U Gal bi-4EGlcNcbl- 3 Gal bi-4]kGlcNAcbi- 2 Hanai Here j+k=14 a kj>=l Glycan structure NeuAc(a2-?)Gal(bl-4){GlcNAc(bl-3)Gal(bl-4)}kGlcNAc(bl-2 )Man(al-3)[NeuAc(a2-?)Gal(bl-4){GlcNAc(b1-3)Gal(b1 4)}jGlcNAc (b1-2)Man(al-6)]Man(b1-4)GlcNAc(bl-4)GlcNAc+"Where j+k= 14 & kj>=1" Fuc al. Gal bi-4 EGlcNAebi- 3 Gal bl-43jGlcNAcbi-- 2 Hanai 6 Han bi-4 GlcNcbi-4 GlcNRc Gal bi-4[GlcNicbi- 3 Gal bi-43kGlcHAcbI- 2 Hanai Here j+k=14 & j,k>=1 Glycan structure Gal(b1-4){GlcNAc(b1-3)Gal(bl-4)}kGlcNAc(b1-2)Man(al-3)[ Gal(bl-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(b1-2)Man(al-6)] Man(b1-4)GlcNAc(b1-4)[Fuc(al-6)]GIcNAc+"Where j+k=14 & j,k>=1" WO 2007/028212 PCT/AU2006/001325 - 12 Fuc NeuRca2- u Gal b-4 EGlcNncbi- 3 Gal bi-1jlIfci aa al. -43JGobI-4l 2l~~b- Han ac Gal bi-4EG lcM Oc- 3 Gal bi-4Jk~lcNebi- 2 Han al aIi 4Gc fri 4G~ i There j+k=14 a j,k>=i Glycan structure NeuAc(a2-?)Gal(b 1-4)f {GlNAc(bl1-3)Gal(bl -4)}jGlcNAc(b 1-2 )Man(al -?)[Gal(b 1-4) {GlcNAc(bl1-3)Gal(bl1-4)}kGlcNAc(b 1-2 )Man(al -?)]Man(bl -4)G~cNAc(bl -4) [Fuc(al -6)] G~cNAc+"Where j+k=14 & j,k>=1 t Fue Neu~c a2- u Gal b-4 I GleNflb- 3 Gal bI-43 jGldHflcbi- 2 HIand± a6 leu lca2- u Gal bi-46Gct4 bi-3 Gal bI-43kGleNRcbi- 2 lanal Ha i 4Ge cb 4Gefi ~4hore j+k=14 & j,k>=aL Glycan structure NeuAc(a2-?)Gal(b 1-4) {G~cNAc(b 1 -3)Gal(b 1-4)}kGlcNAc(b 1-2 )Man(al -3) [NeuAc(a2-?)Gal(b 1-4) {G~cNAc(bl -3)Gal(b 1 4)}jGlcNAc (bi -2)Man(al -6)]Man(bl1-4)G~cNAc(b 1-4) [Fuc(al -6)]G~cNAc±' t Where j+k=14 & j,k>=1 Gal bi -4 1GkNffibi- 3 Gal bi-43 jGlcNflcbi- 2 Man., GleNeb- Han bi-4 GleNflbi-4 GeNfic Gal Ii-4 [GlefNiebi- 3 Gal bi-43kGlcN~cbi- 2 Hlan a' Where j+k=14 & j,k>=I Glycan structure Gal(b 1-4) {GlcNAc(bl -3)Gal(bl1-4)}kGlcNAc(bl1-2)Man(al -3)[ Gal(b 1-4) {GIcNAc(bl -3)Gal(b 1-4)}j GlcNAc(bl -2)Man(al -6)] [ G~cNAc(bl -4)]Man(bl1-4)G~cNAc(bl -4)G~cNAc+"W here j+k=14 HqeuRo a2--u Gal bI-4 GlcNNibi- 3 Gal bi-43 jGlcNncbi- 2 Hlanai Gal bi-4 (QlcHbi- 3 Gal bI-43kGlcIRcbI- 2 Hlan a- 4 Han bi-4 GlcNRcbi-4 GlcN~c b/ GICtHAC JRhere j+k=14 & j,k>i Glycan structure NeuAc(a2-?)Gal(b 1-4) {GlcNAc(bl1-3)Gal(bl -4)}j GlcNAc(b 1-2 WO 2007/028212 PCT/AU2006/001325 - 13 )Man(al -?)[Gal(b 1-4) {G~cNAc(b 1 -3)Gal(b 1 -4)) kGlcNAc(b 1 -2 )Man(al -?)] [G~cNAc(b 1-4)] Man(b 1 -4)G~cNAc(b 1 4)G~cNAc+"Where j+k=14 &j,k>=l" Neufic a2- U Gal bi-4 (Glclcb- 3 Gal bi-43 jGlclfcbi- 2 H~an a Glclfficbi- Htan bi-4 GlcNficblt-4 GleNfle Reunoca2- u Gal bi-4 (GicNffibI- 3 Gal bI-43kGlcHicbi- 2 tianPl mhere j+k=14 & j,k>=i Glycan structure NeuAc(a2-?)Gal(b 1-4) {GlcNAc(b 1 -3)Gal(b 1-4)) kGlcNAc(b 1-2 )Man(al -3) [NeuAc(a2-?)Gal(b 1-4) { G~cNAc(b 1-3)Gal(b 1 4))jGlcNAc (bl1-2)Man(al -6)] [G~cNAc(b 1-4)] Man(bl1-4)G~cNAc(bl1-4)G~cNAc +"Where j+k=14 & j,k> 1" Gal bi-4[GlcfiAcbi- 3 Gal bI-43jGlcH~cbi- 2 Han al Fuc 66 Glc hI- 4 Han bl-4 GlcNflebi-4 Gleffic 3 Gal b-4 (GlcN~cI-3 Gal bI-43kGlcN~cbI- 2 Han here j+k=14 & jk>=i Glycan structure Gal(bl -4) { G~cNAc(bl1-3)Gal(bl1-4)}kGlcNAc(bl1-2)Man(al -3)[ Gal(b 1-4) { G~cNAc(b 1-3)Gal(b 1 -4)}j G1cNAc(bl -2)Man(al -6)][ G~cNAc(bl1-4)]Man(bl1-4)GIcNAc(b 1-4) [Fuc(al 6)] G~cNAc+" Where j+k=1 4 & j,k>=11 fNeu~c a2- u Gal bI-4 1GloN~cbi- 3 Gal bi-43jGlNlcbi- 2 Han al Fue U6 Gal bi-4 EGlcNcbi- 3 Gal bI-43kGlcH~cbi- 2 H~an al-u Han bi-4 G10061cb-4 GlCRAC bi Glcnnc Here j+k--14 & j,k>=i Glycan structure NeuAc(a2-?)Gal(b 1 -4) f G~cNAc(b 1 -3)Gal(b 1-4) }j G~cNAc(b 1 -2 )Man(alI -?) [Gal(b 1 -4) f{G~cNAc(b 1 -3)Gal(b 1-4)} kGlcNAc(b 1-2 WO 2007/028212 PCT/AU2006/001325 -14 )Man(al -?)] [GlcNAc(b 1 -4)]Man(b 1 -4)G~cNAc(b 1 -4) [Fuc(al -6 )]G~cNAc+"Where j+k=14 & j,k>= 1" Neuflo a2- u Gal bi-4 fGlcNfbi- 3 Gal b~I-43 jGlcN~cbi- 2 Han ai Fue 6 6 GlCHAbi- 4 Han bi-4 GleN~ebi-4 G~cNF~c 3 al Neuflc a2- u Gal bi-4 IGlcN~cb- 3 Gal bI-43kGlcN~cb1- 2 Han JMhere j+k=:14 & j,k>mi Glycan structure NeuAc(a2-?)GaI(b 1-4) {G~cNAc(b 1 -3)Gal(bl -4)}kGlcNAc(bl -2 )Man(al -3)[NeuAc(a2-?)Gal(b 1-4) { G~cNAc(b 1-3)Gal(b 1 4)}j GloNAc (bl1-2)Man(al -6)] [G~cNAc(bl -4)]Man(blI-4)G~cNAc(b 1-4) [Fuc (al -6)] GIeNAc+"Where j+k=14 & j,k>=l" G l c~ l c b l 2 h n a i m a n b i - 4 G l c N~ c b I- 4 G ILc N ~ c Ilanal Glycan structure GlcNAc(bl1-2)Man(al -6) [Man(al -3)]Man(bl -4)GlcNAc(bl 4)G~cNAc Ha .
an bi- 4 GJlc Acbi- 4 GIcN~c 6lcH~ebl--- 4 IHanal Glycan structure GIcNAc(bl -4)Man(al -3) [Man(al -6)]Man(bl -4)G~cNAc(b 1 4)G~cNAc WO 2007/028212 PCT/AU2006/001325 - 15 Fuc al, Han ai Han bi-4 GlcNAc bi-4 GlcNAc GlcNRc bi- 2 Hanai Glycan structure GlcNAc(bl-2)Man(al-3)[Man(al-6)]Man(bl-4)GleNAc(b1-4)[Fuc (al-6)]GlcNAc GlcNAcbi- 2 Hana 1 9 Han bi-4 GlcNAcbi-4 GlcNAc GlcNAcbl- 2 hanai Glycan structure GlcNAc(b1-2)Man(al-3)[GlcNAc(b1-2)Man(al-6)]Man(bl 4)GlcNAc (bl-4)GlcNAc HanaI qHan bi-4 GlcNHiebi-4 GlcNAc Hanal Glycan structure Man(al-3)[Man(al-6)]Man(b1-4)GlcNAc(bl-4)GldNAc Fuc Hanal i Han bi-4 GlcNc bi-4 GlcNAc Hanal Glycan structure Man(al-3)[Man(al-6)]Man(b1-4)GlcNAc(bl-4)[Fuc(al 6)]GlcNAc WO 2007/028212 PCT/AU2006/001325 -16 Man GIcNRcb1- 4Han bi-4 GleNAcbi-4 GlcNAc GlcNcbi- 2 Hana1 Glycan structure GlcNAc(b1-2)Man(al-3)[GlcNAc(b1-4)][Man(al-6)]Man(bl-4) G1cNAc(bl-4)GlcNAc Fucai 6 GlcNAc /4 GlcNAP Glycan structure Fuc(al-6)[GlcNAc(bl-4)]GlcNAc Fuc al 6 Han ai- 6 Han bi-4 GlcNcbi-4 GlcNAc Glycan structure Man(al-6)Man(bl-4)GlcNAc(bl-4)[Fuc(al-6)]GlcNAc Fuc al 6 GlcHRcb- 2 Han a1- 6 Han bi--4 GlcN~fcbi-4 GlcNRc Glycan structure GlcNAc(bl-2)Man(al-6)Man(bl-4)GlcNAc(bl-4)[Fuc(al 6)]GlcNAc Han a1- 3 Han ai- 6 Han bi-4 GlcNRcb1-4 GlcNAc Glycan structure Man(al-3)Man(al-6)Man(bl-4)GlcNAc(b1-4)GlcNAc Heu a2-- u Gal bi-4 GlcNAcbi- 2 Han a- 3 Han bi-4 GlcNAc WO 2007/028212 PCT/AU2006/001325 -17 Glycan structure NeuAc(a2-?)Gal(b1-4)GlcNAc(b1-2)Man(al-3)Man(b1-4)GlcNAc Hlana1 k Han bi- 4 GlN cli-4 GilcNfc HS03 - 4 GalNRcbi-4 GlcNflbi- 2 Hanai Glycan structure HSO3(-4)GalNAc(bl-4)GlcNAc(bl-2)Man(al-3)[Man(al-6)]Man (bl-4)GlcNAc(bl-4)GlcNAc Fuc GlcNfbi- 2 Hanai 6 Shan bi-4 GlcNRcbi-4 GlcNRc GlcNflebi- 2 Mana1 Glycan structure GlcNAc(bl-2)Man(al-3)[GlcNAc(bl-2)Man(al-6)]Man(b1 4)GlcNAc (b1-4)[Fuc(al-6)]GlcNAc GlcNflcbi- 2 Hana1 GlcN bi- H1ian bi-4 GlcNfRcbi-4 GlcNRc GlcNcbi- 2 Hana1 Glycan structure GlcNAc(b1-2)Man(al-3)[GlcNAc(bl-2)Man(al-6)][GlcNAc(bl 4)]Man(bl-4)GlcNAc(bl-4)GlcNAc WO 2007/028212 PCT/AU2006/001325 - 18 Han al 6 GlcNRc bh- 4 Han b-4 GlcNfecb1-4 GlcNHc 3 GlcNA% / Sat Hlan GlcNRcil Glycan structure GlcNAc(bl-2)[GlcNAc(bl -4)]Man(al-3)[GLcNAc(bl -4)][Man(al -6)jMan(bl-4)GIcNAc(bl-4)GlcNAc GlcNAcbhi- 2 Hani GHan bi-4 GlcNAcbi-4 GIclAc HS03 - 4 GalfNfcbi-4 GicHcbi- 2 Hanal GlcNAcbi- 2 Han ai Fuc at 6 6 GlcNfebi-- 4 Han bi-4 GlcNRcbi-4 GlcNAc 3 at GlcNeci- 2 Han Glycan structure GlcNAc(bl-2)Man(al -3)[GlcNAc(b1-2)Man(al -6)][GlcNAc(b 1 4)]Man(bl-4)GlcNAc(b1-4)[Fue(al-6)]GlcNAc GlcNRcbi- 2 Hana1 Fuc 3 Han bi-4 GlcNRcbi-4 GLcNAc GlcNA%7 hana1 GIcNA4P 1 WO 2007/028212 PCT/AU2006/001325 -19 Glycan structure GlcNAc(b1-2)[GlcNAc(b1-4)]Man(al-3)[GlcNAc(b1-2)Man(al 6)]Man(b 1 -4)GlcNAc(b1-4)[Fuc(al -6)]GIcNAc GlcNAcbi- 2 Han 6 GlcNAcbl- 4 Han bi-4 GlcNAcb--4 GlcNAc 3 GlcNArbx Han GlcNAcb Glycan structure GLcNAc(bl-2)[GlcNAc(b1-4)]Man(al-3)[GlcNAc(b1-2)Man(al 6)] [GlcNAc(bl-4)]Man(bl-4)GlcNAc(bl-4)GlcNAc GlcNA% M lana 1 GlcNAkP 6 Han bi-4 GlcNAcbi-4 GleNAc GlcNA // M ana1 GICNAP/ Glycan structure GlcNAc(bl-2)[GlcNAc(bl-4)]Man(al-3)[GIcNAc(bl-2)[GlcNAc (b1-6)]Man(al-6)]Man(bl-4)GlcNAc(b1-4)GlcNAc HS03 - 4 GalNc bi--4 GlcNAc bi- 2 Han m i Han bi--4 GlcNAci-4 GlcNAc H503 -- 4 GalNAcbl-4 GlcNAcbi- 2 Hanal Glycan structure HSO3(-4)GalNAc(b1-4)GlcNAc(b1-2)Man(al-3)[HSO3( 4)GalNAc (bl-4)GlcNAc(bl-2)Man(al-6)]Man(b1-4)GlcNAc(bl-4)GlcNAc WO 2007/028212 PCT/AU2006/001325 -20 Hanai g Han bi-4 GlcNRc NeuAc a2- u Gal bi-4 GlcNAcbI- 2 Hana1 Glycan structure NeuAc(a2-?)Gal(bl-4)GlcNAc(bl-2)Man(al-3)[Man(al-6)]Man (bl-4)GlcNAc IHan a1 Ha an bi--4 GlcNAcbi-4 GlcHAc Gal b1-4 GlcNFccbi- 2 Han1 Glycan structure Gal(b1-4)GlcNAc(b1-2)Man(al-3)[Man(al-6)]Man(bl-4)GlcNAc (bl-4)GlcNAc Gal bi-4 GleN bi-- 2 Hlan a1 i Han b1-4 GlcNAcb1--4 GilcN~c Hanai Glycan structure Gal(bl-4)GlcNAc(b1-2)Man(al-6)[Man(al-3)]Man(bl-4)GlcNAc (bl-4)GlcNAc Fuc Hlana ai | Han bi-4 GlcNAc b-4 GleNflc Gal bi-4 GlcNAcb1- 2 Hanai H Glycan structure Gal(bl-4)GlcNAc(b1-2)Man(al-3)[Man(al-6)]Man(b1-4)GlcNAc (bl-4)[Fuc(al -6)]GleNAc WO 2007/028212 PCT/AU2006/001325 -21 Fucui UG~cHAcui- u iirnu Fue U U Galu± U Nana'4GlHcb- Gc~ Glycan structure Fuc(? 1 -?)[Gal(? 1 -?)]IcNAc(? 1 -?)Man(al -?)[Man(aI -?)]Man (bI1-4)G~cNAc(b 1 -4)[Fuc(? 1-6)] GleNAc G~~cH~~~c bi tan i an bi- 4 GlcNflbl-4 GicH fle Gal bi-4 GloNebi- 2 Hanal Glycan structure Gal(bl -4)GlcNAc(bl1-2)Man(al -3) [GicNAc(bl -2)Man(al -6)]Man (b 1-4) G~cNAc(b 1-4) GloNAc M a n a - 3 M n~ i a n b i - 4 G Jl c N ~ c b i- 4 G l oN A c Manal Glycan structure Man(al -3)Man(al -6)[Man(al -3)]Man(bl1-4)G~cNAc(bl -4)G~cNAc G l c~ ~ ft b i - ~ 2 aH1 g I a n b i - 4 G l c N o e b i - 4 G l c c f ~ c Neufta2- 6 Gal bl-4 GlcNcb- 2 hanai Glycan structure NeuAc(a2-6)Gal(bl1-4)G~cNAc(bl1-2)Man(al -3)[GlcNAc(bl1-2)Man (al -6)]Man(bl1-4) G~cNAc(bl1-4)G~cNAc WO 2007/028212 PCT/AU2006/001325 - 22 Fur, Gal b-4 Gkcflbi- 2 "anal Gloffc bl 2 Mnal an U-4 GlN~eb-4 GlCNAC Glycan structure Gal(b 1-4) G~cNAc(bl -2)Man(al -6) [GIcNAc(bl1-2)Man(al -3)]Man (b 1 -4) GIcNAc(b 1 -4) [Fuc(al -6)] G~cNAc Fuc GlcH~cb- 2 Manal Gal bi-4 GcAcb- 2 Manai ka i 4Gc±b- lfl Glycan structure Gal(bl -4)GlcNAc(bl1-2)Man(al -3)[G~cNAc(b 1 -2)Man(al -6)]Man (bI1-4)G~cNAc(b 1-4) [Fuc(al -6)1 G~cNAc Fue Gal ui-u GlcNAcui- u alI U UU Man Ii-4 GleNflebi-4 GIcH~e GicHRCU- U "anal Glycan structure Gal(? I -?)G~cNAe(? 1 -?)Man(al -?)[GIcNAc(? 1 -?)Man(al -?)]Man (bI -4)G~cNAc(b 1 -4) EFuc(? 1-?)] GIcNAc GleNflb- 2 hlana. GlCNCbL- Mhan bi -4 GleNflibi- 4 GicHAc Gal b- 4 GlctRb- 2 Hanal Glycan structure Gal(bl1-4)G~cNAc(bl1-2)Man(al -3) [G~cNAc(bl1-2)Man(al 6)] [GloNAc (b 1-4)]Man(bl1-4)G~cNAc(bl1-4)G~cNAc WO 2007/028212 PCT/AU2006/001325 - 23 Gal bi-4 Glc~ficbi- 2 Ilana, GlefNiebi- MlIan bi-4 GleNRchi-4 GleNne GlcNAc bi- 2 Man Glycan structure Gal(b 1 -4)G~cNAc(bI1-2)Man(al -6)[G~cNAc(bI1-2)Man(al 3)] [GlcNAc (bi1 -4)] Man(blI-4)G~cNAc(blI-4)G~cNAc Gal bi-4 GlcN~cb- 2 Ilanal GlcNflAl NHna an b-4 GleN~cbi-4 GlecHle GlcHAjbI: Glycan structure Gal(bI1-4)G~cNAc(b 1 -2)Man(al -6)[G~cNAc(b 1-2)[G~cNAc(b 1-4 )]Man(al -3)] Man(b 1-4) G~cNAc(bl -4)G~cNAc Gal i - Gl N~c i - Ha .1 an bi- 4 GlcN flbi-4 G cN~c Neu~ea2-6 GalN~obl-4 GlcNcbI- 2 hlanal Glycan structure NeuAc(a2-6)Ga1NAc(bl1-4)G~cNAc(b 1-2)Man(al -3)[Gal(b 1 4)CTcNAc (bl1-2)Man(al -6)]Man(bl1-4)G~cNAc(bl1-4)G~cNAc N e u ft a 2 - 3 G a l b i - 4 G lc N A c b i- 2 H la n .1Hnal ; c~ l b - l ~ l H503 -4 GaLN[cbl-4 GlcN~cbI- 2 hlanal Glycan structure HSO3 (-4)Ga1NAc(bl1-4)GIcNAc(bl1-2)Man(al -3)[NeuAc(a2-3)Gal (bl1-4)G~cNAc(bl -2)Man(al -6)]Man(bl1-4)G~cNAc(bl1-4) GleNAc WO 2007/028212 PCT/AU2006/001325 - 24 Gal UI-4 GlkNcb- 2 IlanF rGlcNRcb- 4 Man b-4 GlctNfcb-4 GICNRC 3 al. GlefNieb- 2 Man Glycan structure Gal(bl1-4)G~cNAc*bl-2)Man(al -6)[G~cNAc(bl1-2)Man(al 3)] [GlcNAc (bl1-4)]Man(bl -4)GlcNAc(bl1-4)[Fuc(al -6)] GleNAc GlcHflcbi- 2 Han al Fuc 6 6 GlcNFoIb- 4 Han bi-4 GlefNlebi-4 IflcN~c al Gal hi-4 GleNobi- 2 Han Glycan structure Gal(bl -4)G~cNAc(bl1-2)Man(al -3) [GlcNAc(bl1-2)Man(al 6)] [G~cNAc (b 1 -4)]Man(b 1 -4)GIcNAc(b 1-4) [Fuc(al -6)]G~cNAc Gal ui-u GicHAcul- u Man al Fuc U GicN~cu- u Man al- u man bi-4 GlcNRcbi-4 GlcNRc 4 A Glycan structure Gal(? 1 -?)G~cNAc(? 1 -?)Man(al -7) [GlcNAc(? 1 -?)Man(al 7)] G1cNAc (? 1-4)] Man~b 1 -4)G~cNAc~b1 -4)[Fuc(? 1-6)] GleNAc WO 2007/028212 PCT/AU2006/001325 - 25 Fur Neu~c a2- 3 Gal bi-4 Glc~nebi- 2 Ilanal Neufloa2-6 GaN~eb-4 GleNfebi- 2 Hanal Hnb- i~foi4Gc~ Glycan structure NeuAc(a2-3)Gal(b 1-4) G~cNAc(bl1-2)Man(al -6) [NeuAc(a2 6)GaINAc (b 1 -4)GlcNAc(bl -2)Man(al -3)]Man(bl1-4)G~cNAc(b 1 4)[Fuc(al -6)] GloNAc Neue a2- 3 Gal bl.-4 GlcH~cbn- 2 klan, GkcNfbI- Har bi-4 GLetIcbi-4 GlcNRc NeuAa2-6 GalNflebi-4 GlcHAbi- 2 IHanl Glycan structure NeuAc(a2-3)Gal(bl -4)G~cNAc(bl -2)Man(al -6)[NeuAc(a2 6)Ga1NAc (bi -4)G~cNAc(bl -2)Man(al -3)] [GIcNAc(bl -4)]Man(bl1-4)G~cNAc (bI -4)G~cNAc Han.1Han bi-4 GJlcNlcbi-4 GIc Hlanal + 2 x Han Glycan structure Man(al-3) [Man(al -6)]Man(bl -4)GIcNAc(bl1-4)G~cNAc-I"+ 2 x Man" H a n a - 3 a n i a n b l- 4 G l c N fl c b i- 4 G c N~ c Neuflc a2- u Gal bi-4 GlefNiebi- 2 hlanal Glycan structure NeuAc(a2-?)Gal(bl -4)G~cNAc(bl1-2)Man(al -3)[Man(al -3)Man( al -6)]Man(bl1-4)G~cNAc(bl1-4)G~cNAc WO 2007/028212 PCT/AU2006/001325 - 26 N e u c a 2 - 3 G a l bi - 4 G l eN fl e b i- 2 a n a ± S a i - ~ ~ l b l 4 G c~ Neu~c a2- 3 Gal bi-4 GlcNRcbi- 2 al Glycan structure NeuAc(a2-3)Gal(bl1-4)G~cNAc(bl1-2)Man(al -3) [NeuAc(a2-3)Gal (bl1-4)G~cNAc(bl1-2)Man(al -6)]Man(bl1-4)G~cNAc(b 1-4)G~cNAc Fuc Gal bi-4 GlcNfcbi- 2 Mlana.,a Gal i-4 ~cNcbi-2 Maal an bi-4 GlcN~cbI-4 GlcN~c Glycan structure Gal(bl1-4)G~cNAc(bl1-2)Man(al -3) [Gal(bl1-4)GlcNAc(bl1-2)Man (al -6)]Man(bl1-4)G~cNAc(bl1-4)[Fuc(al1-6)]G~cNAc Gal b-4 GleNflebi- 2 Manal S an bi -4 GlcNRc bi-4 Gle~ffl Fue al- 2 Gal b-4 GlcI4Abi- 2 hlanalz Glycan structure Fuc(al -2)Gal(bl1-4)G~cNAc(bl -2)Man(al -3)[Gal(bl1-4)G~cNAc (bi -2)Man(al -6)]Man(bl -4)G~cNAc(bl1-4)GIcNAc UGIcNACui- u Mlana Galui //U an bi-4 GlcHflb-4 GleNfic Gal u-u G cNflu- u tMana Glycan structure Fuc(? 1-?) [Gal(? 1 -?)]G~cNAc(? 1 -?)Man(al -?2)[Gat(? 1 -. ?)GIeNAc (?1 -?)Man(al -?)]Man(bl1-4)G~cNAc(b 1-4)GicNAc WO 2007/028212 PCT/AU2006/001325 - 27 Fuc ai- 2 Gal bi-4 Glo~iebi- 2 Han.a±" 9 an bi-4 GlcNflCbi-4 GicN~c Gal bi- 4 GloHftbl- 2 thanal Glycan structure Fuc(a1 -2)Gal(bl1-4)G~cNAc(bl -2)Man(al -6) [Gal(b 1 -4)G~cNAc (hi -2)Man(al -3)]Mani(bl1-4)G~cNAc(b 1-4) GleNAc N oc a i - 6 G a l b i - 4 G lc N A c b I- 2 Ila n a l h n b iff e i 4 G~~ Neu~c a2- 6 Gal bi-4 GleH~bl- 2 man a Glycan structure Fuc(al -6)Gal(bl1-4)GlcNAc(blI-2)Man(al -6) [NeuAc(a2-6)Gal( bl1-4)G~cNAc(bl1-2)Man(al -3)]Man(bl1-4)G~cNAc(bl -4)G~cNAc H S 0 3 F u c S Gal bi -4 GlcHc bi- 2 Ilanal NeURca UhnbI-4 GlcNHflrb-4 GlcN~c Neufic a2- u Gal bi-4 GlcNflcbi- 2 man" Glycan structure HSO3(-6)[NeuAc(a2-3)] Gal(bl1-4)G~cNAc(bl1-2)Man(al -?) [NeuAc (a2-?)Gal(bl1-4)G~cNAc(bl1-2)Man(al -?)]Man(bl1-4)G~cNAc(b 1 -4)[Fuc(al -6)] GleNAc Ga b , G c~ b -2 Han ., Fuc 33 Gal bi-4 GlcN~cb- 2 tHanal, Glycan structure Fuc(al -3) [Gal(b 1 -4)1G~cNAc(b 1-2)Man(a 1 -6)[Gal(bl 4)G~cNAc (bl1-2)Man(al -3)]Man(bl -4) G~cNAc(bl -4) [Fuc(al -6)1 GleNAc WO 2007/028212 PCT/AU2006/001325 - 28 Ga b GcH cbi- 2 h1art FUC Fucal 3 ilan bi-4 GlcNAcbI-4 GlcNRc Gal bi-4 Gleffiebi- 2 klan~l Glycan structure Fuc(al -3) [Gal(bl -4)]GlcNAc(bl1-2)Man(al -6)[Gal(bl -4)G~cNAc (bl1-2)Man(al -3)]Man(bl1-4)G~cNAc(bl -4)[Fuc(al -6)] GleNAc Gal b-4 GleNflebi- 2 Man., ali 66 Gal,:L\ 4~e~leb- 2kiaal 3 har bi-4 GicN~obi-4 GloNflo Fucai Glycan structure Fuc(al -3)[Gal(bl1-4)]CdcNAc(bl1-2)Man(al -3)[Gal(bl1-4)GIcNAc (bl1-2)Man(al -6)]Man(bl -4)GlcNAc(bl1-4)[Fuc(al -6)] GleNAc Fuc Fuc al- 2 Gal b-4 GlcHNr-b- 2 tHan.1 Gal bi-4 Gccbi- 2 Ha .nal ka i 4G± ~b- ~~i Glycan structure Fuc(al -2)Gal(bl-4)GIcNAc(bl -2)Man(al -6) [Gal(bl -4)G~cNAC (b 1 -2)Man(aI-3)jMan(b 1 -4)G~cNAc(bl -4)[Fuc(al -6)]G~cNAc WO 2007/028212 PCT/AU2006/001325 - 29 Gal b1, 4 GlcNcbi- 2 han ., Fuc 3 at Fuc1 6 Han bi-4 GlcNAcbi-4 GlcNAc NeuRc a2- 6 Gal bi-4 GlcNRobi- 2 Mana1 Glycan structure NeuAc(a2-6)Gal(bl -4)GlcNAc(b 1 -2)Man(al -3)[Fuc(al -3) [Gal (b1-4)]GlcNAc(bl-2)Man(al-6)]Man(bl-4)GleNAc(bl-4)[Fuc( al-6)]G1cNAc Fuc NeuRc a2- 6 Gal bi-4 GlcNRcbi- 2 Hana1 a1 I 6 Han bi-4 GlcNAcbi-4 GlcNnc . Gal b1 4 GlcNAcbi- 2 Nana1 Fue ai/ Glycan structure NeuAc(a2-6)Gal(bl-4)GlcNAc(bl -2)Man(al -6)[Fuc(al -3) [Gal (bl-4)]GlcNAc(bl-2)Man(al -3)]Man(bl-4)GlcNAc(bl -4)[Fuc( al-6)]GlcNAc Galb1 4 GlcNlcbl- 2 Hana1 Fuc 3 at Fuca1 6 \Ua 6 Galbi 2 / an bt-4 GlcNfbl-4 GlcHc 4 GlcNAcbi- 2 Hana1 Fuc a1 Glycan structure Fuc(al-3)[Gal(b1 -4)]GlcNAc(b 1 -2)Man(al -3)[Fuc(al -3) [Gal (bl-4)]GlcNAc(bl-2)Man(al-6)]Man(bl-4)GlcNAc(bl-4)[Fuc( al-6)]GlcNAc WO 2007/028212 PCT/AU2006/001325 -30 Gal bi-4 GlcNAcb- 2 H~an., GICHNoib- 4htan b-4 GlcN~cbI-4 GlcN~lc NeuRc a2- 6 Gal bi-4 GlcNAcb- 2 al Glycan structure NeuAc(a2-6)Gal(bl1-4) G~cNAc(bl -2)Man(al -3) [Gal(b 1 4)G~cNAc (bI1-2)Man(al -6)] [G~cNAc(b 1-4)] Man(b 1 -4) GlcNAc(b 1 4)GlcNAc NeuRc a2- 3 Gal bi-4 GLc~fcbi- 2 HIan, GlcNflcbI- 4Han bi-4 GlcNcbI-4 GlcNflc Neu~c a2- 6 G al bi-4 GlcH~obi- 2 Han"/ Glycan structure NeuAc(a2-3)Gal(bl1-4)G~cNAc(bl -2)Man(al -6) [NeuAc(a2 6)Gal (bi -4)G~cNAc(bl -2)Man(al -3)] [GlcNAc(b 1-4)] Man(bl 4)G~cNAc (b 1-4)G~cNAc G a l b i - 4 G l N c b I - 2 f l a n 1 a b i 4 G c I b - l N c SGal b-4 GlcNRbi- 2 hlanal b- llb- lt~ Glycan structure Fuc(al -2) [GaINAc(al -3)] Gal(bl1-4)G~cNAc(bl1-2)Man(al -3)[Gal (bl1-4) GIcNAc(bl1-2)Man(al -6)]Man(bl1-4)G~cNAc(b 1-4) G~cNAc WO 2007/028212 PCT/AU2006/001325 -31 SGal bi-4 GikHflcbi- 2 blana± Fucai Shan bi-4 GlcNFcbi-4 GIcN~c Gal bi-4 GleNF~bi- 2 11anal Glycan structure Fuc(al -2)[GalNAc(al -3)] Gal(bl1-4)G~cNAc(bl -2)Man(al -6) [Gal (bi -4)GlcNAc(b 1 -2)Man(al -3)]Man(b 1 -4)G~cNAc(b 1-4)G~cNAc Gal Ii- 4 GlcNic bi- 2 kaa GIcAcbl- 4HIan bi-4 GlcNR~cbI-4 GIcNAo Fuc al- 2 Gal b-4 GlcNAcbi- 2 Ilanai Glycan structure Fuc(al -2)Gal(b 1-4)G~cNAc(bl -2)Man(al -3)IiGal(bl1-4)G~cNAc (bi -2)Man(al -6)] [GlcNAc(b 1-4)]Man(bl -4)G~cNAc(bl -4)G~cNAc Fuc A - 2 Gal bi -4 GlcNc bi- 2 Han. GlcN"iob- {hlan bi-4 GlcN~ebi-4 GlcN~c Gal bi-4 GlcNcbI- 2 hlanal Glycan structure Fuc(al -2)Gal(b 1-4)G~cNAc(b 1 -2)Man(al -6)IIGal(b 1 -4)G~cNAc (bl1-2)Man(al -3)] [G~cNAc(bl -4)]Man(bl -4)G~cNAc(bl -4)G~cNAc Gal b-4 GlcH~ebi- 2 Man ai u al 66 GkeN~b- 4 Man bi-4 GlcN~cbi-4 GleNflc Neuflc a2- 6 Gal bi-4 GlcNficil- 2 Mlan Glycan structure NeuAc(a2-6)Gal(bl1-4)G~cNAc(bl1-2)Man(al -3)[Gal(bl 4)GIcNAc WO 2007/028212 PCT/AU2006/001325 - 32 (bl1-2)Man(al -6)1 [GLcNAc(b 1-4)]Man(bl1-4)G~cNAc(bl1-4)[Fuc (al -6)]G~cNAc NeuRc a2- 6 Gal bi-4 GlcNcb- 2 Han Fue al 6 GlcNflcbi- 4 Han b-4 GlctfliA-4 GICHflC 3 Neuflc a2- 6 Gal bi-4 GlcHAcb- 2 Man Glycan structure NeuAc(a2-6)Gal(b 1 -4)G1cNAc(b 1-2)Man(al -3)[NeuAc(a2 6)Gal (b 1-4) G~cNAc(bl -2)Man(aI -6)] [GIcNAc(bl -4)]Man(b 1 4)GIcNAc (b 1-4) [Fuc(al -6)] GloNAc Fuc a- 2 Gal bi-4 GlcNfcbi- 2 Hana, GlCNcbi- han bi-4 GloNAcbl-4 GlcNAc Fuc a- 2 Gal bi-4 GlcHRbi- 2 )lanal Glycan structure Fuc(al -2)Gal(bl1-4)G~cNAc(blI-2)Man(al -3)[Fuc(al -2)Gal(b 1 -4)G~cNAc(b 1-2)Man(al -6)] [G~cNAc(b 1-4)]Man(b 1 4)G~cNAc( bI1-4) GloNAc Gal ul-u Glfc.c U Han A? al Fuc GIcNRCU± U1 U Gal ui-u GleNAcui- u Man al- u Han bi-4 GlcNAbi-4 GlocH~ 4 U! G1cHAC Glycan structure Gal(? 1-?)G~cNAc(? 1 -7)[G~cNAc(? 1 -?)]Man(al -?)Ga1(? 1 ?)G~cNAc (71 -?)Man(al -?)] [G~cNAc(? 1-4)] ManQ,1 -4)G~cNAc(bl - WO 2007/028212 PCT/AU2006/001325 - 33 4)[Fuc (?1 -6)] GlcNAc G al Ui-4 GlefNieb- 2 Hanai Gal bi-4 GICNflc%\ mn an bi-4 GkeNFn Gal. bi-4
GICHACP
1 r INeuflc Glycan structure Gal(bl -4)GlcNAc(b 1 -2)[Gal(bl1-4)G~cNAc(bl1-4)]Man(al 3)[Gal (bl1-4)G~cNAc(bl -2)Man(al -6)]Man(b 1 -4)GlcNAc+'+ NeuAc" G a l b i - 3 G a l b i - 4 G l N f o b i - 2 H a n .m a i 4 G e f o - G N c Neu~c a2- 6 Gal b-4 GlcNflcb- 2 Hanal Glycan structure Gal(bl1-3)Gal(bl1-4)GlcNAc(bl1-2)Man(a1 -6)[NeuAc(a2-6)Gal( bl1-4)GlcNAcb1 -2)Man(al1-3)]Man(bl1-4)GIcNAc(bl -4)G~cNAc Gal bi-4 GlcH~bk- 2 anal Gal bi-4 GL-cN~cP 1 + 2 x Neune Glycan structure Gal(b 1 -4)GlcNAc(b 1 -2) [Gal(bl -4)GIcNAc(b 1 -4)]Man(al -3) [Gal (bi -4)GIcNAc(bl1-2)Man(al -6)] Man(bl1-4)GlcNAc+' t + 2 x NeuAc it WO 2007/028212 PCT/AU2006/001325 -34 Neu~c a2- u Gal bi -4 GlcNAc bi- 2 Ilan al !4eune a2- u Gal bi-4 GlcHNbl Han bi-4 GleN~c ", Hanai-I Neuc a2- u Gal bi-4 GlcNfkPI/ Glycan structure NeuAc(a2-?)Gal(bl1-4)G~cNAc(bl1-2)[NeuAc(a2-?)Gal(b 1 4)G~cNAc (bl1-4)]Man(al -3)[NeuAc(a2-?)Gal(bl -4) G~cNAc(bl1-2)Man(al -6)]Man(bl1-4) GlcNAc Gal bi-4 GlcNflbl- 2 Man., rue 6 Gal~ Galbi- Glciflci- 2marO / an bi-4 GlcNAcbi-4 Glc:Nfc Glycan structure Fuc(al -2) [Gal(a1 -3)]Gal(b 1 -4)G~cNAc(b 1 -2)Man(al -3) [Gal( bl1-4)G~cNAc(b 1 -2)Man(a1 -6)IMan(bl -4)G~cNAc(bl1-4) [Fuc (al -6)]GIcNAc SGal bi -4 ClcNfleb- 2 Man., Fue Fucai6 Phan bi-4 GlcNfcbi-4 GIcH~c Gal 1,1-4 GlcNAcb- 2 Danai Glycan structure Fuc(al -2) [Gal(al -3)] Gal(blI -4)GlcNAc(blI -2)Man(al -6)I[Gal( bi -4)G~cNAc(bl -2)Man(al -3)]Man(bl -4)G~cNAc(bl -4)[Puc (al -6)] G~cNAc WO 2007/028212 PCT/AU2006/001325 - 35 4 U Gal bi-4Go~ Gal b±4 VcI 3 Ilan u an at4/JC bi Gal bl-4 GlcNRtC FUC a- 3 Fucal + Neuilc(?2-6) Glycan structure Gal(b 1-4)G~cNAc(b 1-2)[Gat(bl 4)GIcNAc(bl -4)]Man(al -3)[Fuc (a 1-6) [Gal(b 1-4)] G~cNAc(?t1-2)Man(? 1 6)]Man(? 1-4) [Fuc(al -3)Fuc(al-3)]GIcNAc+"+ NeuAc(?2-6)" Gal bi- 4 GlcNA% nA Gal bi-4 GlcN~iP 1 Man b1-4 Glefhbi- 4 GlcHRfi Gal b-4 GleNflebl- 2 hanal Glycan structure Gal(bl -4)G~cNAc(bl -2)[Gal(bl1-4)G~cNAc(bl -6)]Man(al 6)[Gal (b 1-4) G~cNAc(bl1-2)Man(al -3)]Man(bl1-4)G~cNAc(b1 1 4)GjlcNAc Gal bi- 4 Gl Nflobi- 2 Han a Ma a-4Gctcb -4G C R Gal bJ- 4 GIcNA % \ Manna4 GlN cb- Gcl Gal bl-4 Gld4AJbi/ Glycan structure Gal(b 1 -4)GIcNAc(bl1 -2) [Gal(b 1 -4)GstcNAc(blI -4)]Man(al -3) [Gal (bl1-4)GIcNAc(bl -2)Man(al -6)]Man(bl1-4)G~cNAc(bl1-4)GIcNAc WO 2007/028212 PCT/AU2006/001325 -36 Gal.b i- 4 G lcH cbi- 3 G al b i- 4 G l etf cbI- 2 hIanai g a b - ~ H c I 4 G eii Iialbi-4 GlcAWb-2 hanal Glycan structure Gal(b 1 -4)G~cNAc(b 1 -3)GaI(b 1-4)GIcNAc(b 1 -2)Man(al -6) [Gal (bl1-4)G~cNAc(bl1-2)Man(al -3)]Man(bl1-4)G~cNAc(bl1-4)GlcNAc Manaj Manai Man bi-4 GlefNlobi-4 GICHRC Man al- 2 Ilanal Glycan structure Man(al -3)[Man(al -6)]Man(al -6)[M\an(al -2)Man(al -3)]Man(b 1 -4)G~cNAc(bl1-4)G~cNAc IManal MIan bi-4 GlNcb-4 GlcNflc Gal bi-4 EGcN~bi- 2 Ilanai Glycan structure Gal(b 1 -4)GlcNAc(b 1 -2)Man(al1 -3) [Man(al -3) [Man(a 1 -6)] Man( al -6)]Man(bl1-4)G~cNAc(bl -4)G~cNAc Han ai gMa , Ilanal an bi-4 GlcNlicbi-4 Glc~flc Heufl a2- u Gal bi-4 GlcNflbi- 2 Ilanai Glycan structure NeuAc(a2-?)Gal(bl -4)G~cNAc(b 1 -2)Man(a1 -3)[Man(al -3) [Man (al -6)]Man(al -6)]Man(bl1-4)G~cNAc(bl1-4)G~cNAc WO 2007/028212 PCT/AU2006/001325 -37 Gal b-4 GlcHcbi- 2 klanai Gal bi-4 GlcNAck Man b±-4 Glefiftbi-4 GlcNW~c " Ha na Gal bi-4 GkeNflePi + Fuc(ai-3) Glycan structure Gal(b 1 -4)GlcNAc(bl -2)[Gal(b 1 -4)G~cNAc(b 1 -4)]Man(al -3) [Gal (bl1-4)G~cNAc(bl1-2)Man(al -6)]Man(bl -4)G~cNAc(bl1-4)G~cNAc +"+ Fuc(a1 -3)" Neufca2- u Gal b-4 GlcNfleb- 2 Ilan.± Gal bi-4 GlcNflk klna an b - 4 GlcNciA-4 GlcNilc Gal bi-4 GlcHflcP/ Glycan structure NeuAc(a2-?)Gal(bl1-4)G~cNAc(bl1-2)Man(al -6)[Gal(bl1-4)G~cNAc (b 1-2) [Gal(bl -4)G~cNAc(bl1-4)]Man(al -3)]Man(bl1-4)G~cNAc( bl1-4)GIcNAc Gal bi -4 GlcN~c bi- 2 IManal NeuAc a2- u Gal bi -4 GlcNA%k S an bi-4 GlrcN~cb-4 GlclIAc Gal b - 4 Glc:HAP 1 Glycan structure NeuAc(a2-?)Gal(bl -4)GlcNAc(bl1-4)[Gal(bl1-4)GIcNAc(b 1-2)] Man(al -3) [Gal(bl1-4)G~cNAc(bl1-2)Man(al -6)] Man(b 1-4) GIcNAc (bl1-4)G~cNAc Gal bi-4 GlcNRobi--- 2 hlanal Gal bi-4 GlcNA % 'IhMna an bi-4 GleN~ebi-4 GlcNfic Neu~c a2- u Gal bi -4 GlcNARP 1 Glycan structure NeuAc(a2-?)Gal(bl1-4)G~cNAc(bl1-2)[Gal(b 1 -4)G~cNAc(b 1-4)] WO 2007/028212 PCT/AU2006/001325 -38 Man(al -3)[GaI(bl1-4)G~cNAc(bl1-2)Man(al -6)]Man(bl1-4)GIcNAc (bI1-4)G1cNAc Gal ul-u GlGNA%,,- UU Gal ui-u GlcNflcu- u Ihanal + Neuflc~a2-6) Glycan structure Gal(?1 -?)GlcINAc(?1 -?)[Gal(? 1 -?)GlcNAc(? 1 -?)]Man(al-?)[Gal (?1 -?)GkcNAc(?l1-?)Man(al -?)]Man(bl -4)G~cNAc(bl1-4)G~cNAc +"1+ NeuAc(a2-6)" Neu~c a2- 6 Gal bi-4 GlcHlcbi- 2 Nan.1 Gal bi-4 GlcNAb Man bi-4 GlcN~rbI-4 GlcN~c mHana' tIeugo a2- 6 Gal bI-4 GlcNfIdb 1 Glycan structure NeuAc(a2-6)Gal(bl1-4)G~cNAc(b 1-2) [Gal(bl1-4)G~cNAc(b 1-4)] Man(al -3)[NeuAc(a2-6)GaI(bl1-4)G~cNAc(bl1-2)Man(al -6)]Man (bl-4)GIcNAc(bl -4)GIcNAc Neuflc a2- 6 Gal bi-4 GlcNNflci- 2 Ilan.1 Neue a2- 3 Gal bi-4 GlcHtkIR% anIlSan b-4 GleNReb-4 GlcNRc NeuAc a2- 3 Gal bi-4 GlcNflk~f Glycan structure NeuAc(a2-3)Gal(b 1 -4)GIcNAc(b 1-2) [NeuAc(a2-3)Gal(b 1 4)G~cNAc (bi -4)]Man(al -3)[NeuAc(a2-6)Gal(b 1-4)G~cNAc(bl1-2)Man(aI -6)]Man(b 1-4)G~cNAc(bI1-4)G~cNAc WO 2007/028212 PCT/AU2006/001325 -39 Gal bi-4 GleNflebl- 2 Hianal, Gal bi-4 hl~lb\\ aa an bi -4 GlcNflebi- 4 GlcHFc Gal bi -4 GleNfldP + 3 H NeuRC(a2-?) Glycan structure Gal(bl1-4)G~cNAc(b 1-2) [Gal(bl1-4)G~cNAc(bl1-4)]Man(al 3)[Gal (b 1-4) G~cNAc(bl1-2)Man(al -6)]Man(bl -4)G~cNAc(b 1 4)G~cNAc +I'+ 3 x NeuAc(a2-?)" G al b i- 4 G lcN A b, , M a ,, U Gal b-4 GLCNAal PIan bi -4 GlcNAc bi- 4 GlecHic Gal bi-4 GlcHflobi- 2 Nana' Glycan structure Gal(bI1-4)G~cNAc(b 1 -2)[Gal(bl1-4)G~cNAc(bl1-6)]Man(al 6)[Gal (bi -4)GTIcNAc(bl -2)Man(al -3)]Man(bl1-4)G~cNAc(b 1 4)[Fuc(a 1 -6)] G~cNAc Galbi 4 GICNRij Gal bi-4 GlcNAbi I Uanb- lNbI4GHc Uanb-GlN b-4Gcc Gal b-4 GlcNcbI- 2 Nlana' Glycan structure Fuc(al -3)[Gal(bl -4)] G~cNAc(bl -4)[Gal(bl1-4)G~cNAc(b 1-2)] Man(al -?)[Gal(bl -4)GlcNAc(bl -2)Man(al -?)]Man(bl1-4)G~cNAc (bi -4)GlcNAc WO 2007/028212 PCT/AU2006/001325 - 40 Fue Gal bi-4 GloNob- 2 HIand Gal b-4 GlecHl c.- 3 Gal bi-4 GlcNfi ci- 2 Ifan di/ ga i 4GIH~ i 4Gc Glycan structure GaI(b 1 -4)G~cNAc(bl1-3)Gal(bl1-4)G~cNAc(bl1-2)Man(al -3)[Gal (bl1-4)GlcNAc(b 1 -2)Man(al -6)]Man(b 1-4)GIcNAc(bl -4)[Fuc(al -6)]G~cNAc Gal bi -4 GlcN~c b- 2 ha".. Galbi. \1\ han 41-4 Glct4cbi-4 GlcN~o Fue a,/ han"i Gal bl-4 GktIR4P 1 / Glycan structure Fuc(al -3) [Gal(b 1-4)] GlcNAc(b 1-4) [Gal(bl1-4)G~cNAc(b 1-2)] Man(al -3) [Gal(b 1-4)GlcNAc(bl -2)Man(al -6)]Man(blI-4)GIcNAc (bl-4)G~cNAc Gal b-4 GlcHcbl- 2 than~i Gal b-4 GIcN~rl an1iSan b-4 GlhNftbi-4 GJLcNAc Gal b-4 GcNfibp 1 X + Fuc(ai-2) Glycan structure Gal(bl -4)G~cNAc(b 1 -2) [Gal(b 1 -4)GlcNAc(b 1 -4)]Man(al1 -3) [Gal (bi -4)G~cNAc(bl1-2)Man(al -6)]Maii(bl1-4)GIcNAc(bl1-4)G~cNAc ±"+ Fuc(al-2)' WO 2007/028212 PCT/AU2006/001325 -41 Fuc Gal bi-4 GlcNAcb- 2 IMana Gal i-4 ~cHA Nana / an bi-4 GlCN~cb-4 GIcNAc Gal bi-4 GICNA% 3 +Fuc(a1-3) Glycan structure Gal(bl -4)G~cNAc(b 1 -2) [Gal(b 1 -4)G~cNAc(blI -4)]Man(al1 -3) [Gal (bI -4)G~cNAc(b 1-2)Man(al -6)]Man(bI1-4)G~cNAc(b 1-4) [Fuc(al -6)] GleNAc+"+ Fuc(al1 -3)" Gal b-4 GicNbi- 2 Mana Fuc Neufic a2- 3 Gal bl-4 GlcNR%, a i 4GcHcI46OA hanal Heuc a2- 6 Gal bi- 4 GlcHAcbi/ Glycan structure NeuAc(a2-3)Gal(bl -4) G~cNAc(bl1-4) [NeuAc(a2-6)Gal(bl1-4)G~cNAc (b 1 -2)]Man(al -3) [Gal(b 1 -4)G~cNAc(b 1 -2)Man(al -6)]Man(b 1 4)G~cNAc(bl -4)[Fuc(al -6)] GIcNAc Neufic a2- 3 Gal b-4 GlcNAcb- 2 Mlan Fuc 6 hlan bi-4 G]kN~cbi-4 GICNAC Gal bi-4 GlcNA% an' Neufic a2- 6 Gal bi -4 GlcNfliP 1 Glycan structure NeuAc(a2-6)Gal(b 1-4) GIcNAc(bl -2)[Gal(bl1-4)G~cNAc(bl -4)] Man(al -3)[NeuAc(a2-3)Gal(bl1-4)GIcNAc(bl1-2)Man(al -6)]Man (bI1-4)G~cNAc(bl -4) [Fuc(al -6)] GleNAc WO 2007/028212 PCT/AU2006/001325 - 42 Neu~e a2- 3 Gal bi-4 GLeN~b- 2 Ha. Au 66 Heuo a- 3Galbi-4 Gc~fcb&\ Nan:' 3 an bi-4 GcNAcbi-4 GICHRC Gal bi -4 GUHR iP 1 Glycan structure NeuAc(a2-3)Gal(bl -4)G~cNAc(bl -4) [Gal(b 1-4) GlcNAc(bl -2)] Man(al -3) [NeuAc(a2-3)Gal(bl -4)GlcNAc(bl -2)Man(al -6)]Man (bl-4)G~cNAc(bI -4)[Fuc(al-6)]GlcN'Ac Fuc Gal Ul-4 IGleNflb- 2Ma. 6 Han bi-4 GlcNflcb±-4 GlcNAo Gal bi-4 GlcNR Nna Gal bi-4 GlcNAkP 1 / + NeuRC(a2-3I) + NeuflC(a2-6) Glycan structure Gal(bl1-4)GIcNAc(bl -2)[Gal(bl1-4)G~cNAc(bl -4)]Man(a 1-3) [Gal (bl1-4)GlcNAc(bl1-2)Man(al -6)]Man(bl1-4)G~cNAc(b 1 -4)[Fuc(al -6)] GlcNAc+"+ NeuAc(a2-3) + NeuAc(a2-6)" Neuc a2- 6 Gal bI-4 Glct4Abi- 2Na, Galb \1 6hanb-4 GlcNAcbI-4 GleNflc 4 G~N Fucalt Naz Neu~c a2- 6 Gal b - 4 GlcN~c? 1 Glycan structure NeuAc(a2-6)Gal(bI1-4)GIcNAc(b 1 -2)[Fuc(al -3) [Gal(b 1 4)] G~cNAc (bi -4)]Man(al -3) [NeuAc(a2-6)Gal(bl -4)GIcNAc(bl1-2)Man(al -6)]Man(bI-4)GIcNAc(bl -4)G~cNAc WO 2007/028212 PCT/AU2006/001325 - 43 Gal bi-4 GlcNA% Gal bi-4 GlcNACuI8 Han bi-4 GlcNAcb1-4 GIcNAc Gal bi-4 GlcHNAcui- u Han at + Fuc + 2 x NeuAc(a2-?) Glycan structure Gal(bl-4)GIcNAc(?1-?)[Gal(b1-4)GlcNAc(? 1 -?)]Man(al ?)[Gal (b1 -4)GIcNAc(? 1-?)Man(al-?)]Man(bl-4)GleNAc(b1 4)GlcNAc +"+ Fuc + 2 x NeuAc(a2-?)" NeuAca2- 6 Gal bi-4 GlcNFIcbi- 2 Han Fue at at 6 Pan b1-4 GlcNA cbi-4 GlcNAc NeuAc a2- 3 Gal bi-4 GIcNcb H SHana 1 Neufc a2- 6 Gal bi--4 GlcNAdb1 Glycan structure NeuAc(a2-3)Gal(bl -4)GlcNAc(b1-4)[NeuAc(a2-6)Gal(b1 4)GIcNAc (b1-2)]Man(al -3)[NeuAc(a2-6)Gal(b1 -4)G1cNAc(b 1 2)Man(al -6)]Man(b1 -4)GlcNAc(bl -4)[Fuc(al -6)]GlcNAc NeuAc a2- 6 Gal b1-4 GlcNfc bi- 2 hana1 NeuAc a2- 3 Galbi 4 Glc3 Han b--4 GlcNAcb-4 GleNAc 3GlcN NeuAc a2- 6 Gal bi-4 GlcNIUAt Glycan structure NcuAc(a2-3)Gal(bl-4)[Fuc(al-3)]GlcNAc(bl-4)[NeuAc(a2 6) Gal(b1 -4)GlcNAc(bl-2)]Man(al-3)[NeuAc(a2-6)Gal(b1 4)GLcNAc (b1-2)Man(al-6)]Man(bl-4)GlcNAc(bl-4)GlcNAc WO 2007/028212 PCT/AU2006/001325 - 44 Gal bi-4 Glcnncb- 2 Ha".. Galbi '\ Gan bi- 4 Glodlicbl- 4 GlefNic Fucai z ,I ana± Gal b-4 GlctI~bP 1 + 3 H Neuflc(a2-?) Glycan structure Fuc(al -3) [Gal(b 1-4)] GIcNAc(b 1-4)[Gal(bl1-4)G~cNAc(b 1-2)] Man(al-3) [Gal(b 1-4) GlcNAc(bl1-2)Man(al -6)]Man(bl -4)G~cNAc (bl-4)G~cNAc±"+ 3 x NeuAc(a2-?)' Gal bl-4 GleNflb- 2 iiari Fal 66 Gal b-4 GcNR~k kan3'IPan bi-4 GlcNFcbI-4 GlcNfle Gal bi-4 GlcNAb + H503(-6) + 2 x Neu~c(a2-3) + Neuflc(a2-6) Glycan structure Gal(bl -4)GlcNAc(bl -2) [Gal(bl -4)G~cNAc(bl1 -4)]Man(al -3) [Gal (bi -4)G~cNAc(bl -2)Man(al -6)]Man(bl1-4)G~cNAc(bl -4)[Fuc(al -6)]GTcNAc±"+ HSO3(-6) + 2 x NeuAc(a2-3) + NeuAc(a2-6)' Gal bi-4 GlcH~cbi- 2 ktanai Fuc "6a6 3 han bi-4 GlcNAr-bi-4 GlcHAc Gal b-4 GlcNARj, ma" Gal bi-4 GlcNfkP 1 + 2 x H303(-6) + 2 x Neuflc(a2-3) + Neuflc(a2-6) Glycan structure Gal(b 1 -4)G~cNAc(b 1 -2) [Gal(bl1 -4)G~cNAc(blI -4)]Man(al -3) [Gal (bi -4) G~cNAc(bl1-2)Man(al -6)IIMan(bl1-4)G~cNAc(b 1-4) [Fue(al -6)]G~cNAc+"+ 2 x HSO3(-6) + 2 x NeuAc(a2-3) + NeuAc(a2 -6)" WO 2007/028212 PCT/AU2006/001325 -45 4obi\ bi 2Han Fuc 6 ian bi-4 Glclffcbi-4 GlClflC Neuflc a2- 6 Gal b-4 GleNabi- 3 Ga1 bi-4 GlcN~cbi- 2 Hanl Glycan structure NeuAc(a2-6)Gal(b I-4)GIcNAc(bl -3)Gal(bl -4)GIcNAc(b 1-2)Man (al -3)[Fuc(al -3)[GaI(b 1-4)] G~cNAc(bl1-2)Man(al -6)JMan(bI -4)G~cNAc(b 1-4)[Fuc(al-6)]GtcNAc Gal bi-4 GleNflobi- 2 hlanai Gal b-4 GlcNR~ %'-g an bi- 4 GicHRC Gal b-4 Glcffli ,/ + Gal~bi-2)Gkt4AC(b1-3) + 3 x tNeu~e Glycan structure Gal(bl1-4)GlcNAc(b 1-2) [Gal(b 1 -4)G~cNAc(b 1 -4)]Man(al -3) [Gal (b 1 -4)G~cNAc(b 1 -2)Man(al -6)]Man(bl1-4)G~cNAc+'+ Gal(b 1-2 )GlcNAc(bl -3) + 3 x NeuAc" Gal a- 3 Gal bi-4 Glchflcbi- 2 Man. Fal 66 Gal i-4Glcfl%,-" ma" / 3 6 hMan bi-4 GlcHFcbI-4 Glot4Ac Gal bi-4 GIcNFI% / + Neuflc(a2-?) Glycan structure Gal(al -3)Gal(bl1-4)GlcNAc(bl1-2)Man(al -6)[Gal(b 1-4)GIcNAc (b 1-2) [Gal(b 1 -4)G~cNAc(bl1-4)]Man(al -3)]Man(b 1-4)G~cNAc( bi -4) [Fuc(al -6)]GlcNAc+"+ NeuAc(a2-?)' WO 2007/028212 PCT/AU2006/001325 - 46 Gal al- 3 Gal b-4 GleNflb- 2Ma. Fuc 66 Gal i-4 ~cNRbk\, Ha6 I an bi-4 GleNflcW.-4 GlcNRc Gal bi -4 GIcNCAP + Neuflc(a2-3) +1 Neufic(a2-6) Glycan structure Gal(al -3)Gal(bl -4)G~cNAc(bl -2)Man(al -6)[GaI(b1 -4)G~cNAc (bi -2)[Gal(bl -4)GlcNAc(bl -4)]Man(al -3)]Man(bl -4)GlcNAc( b I-4)[Fuc(al -6)] G~cNAc+"+ NeuAc(a2-3) ± NeuAc(a2-6)" Gal a- 3 Gal bi-4 GlcHflb- 2 Man.,u 6mnb±-4 GlcNlcbi-4 GlcN~ic Gal b-4 GlcNA% + 1S03-6) + 2 Hc Neu~c(a2-?) Glycan structure Gal(al -3)Gal(bl1-4)GIcNAc(bl1-2)Man(al-6) [Gal(bl1-4)GlcNAc (b 1 -2)[Gal(bl1-4)G~cNAc(bl1-4)]Man(al -3)]Man(bl1-4)G~cNAc( bl -4)[Fuc(al-6)]G~cNAc±"+ T{S03(-6) + 2 x NeuAc(a2-?)" Gal bi-4 GlcHRb' Ga b-4GlNA 1 6ha bi -4 GkcNRc bi- 4 GlecHlo Gal bi- 4 GlctR %c j " a a Gal bi-4 GlcNAcbi/ Glycan structure Gal(bl -4)GlcNAc(b I1-2) [Gal(b I -4)GIcNAc(bl -4)]Man(aI-3) [Gal (bl-4)GIcNAc(bl1-2)[Gal(b i-4)GlcNAc(bl -6)]Man(al -6)]Man( b 1-4) GtcNAc(b 1-4) GIcNAc WO 2007/028212 PCT/AU2006/001325 - 47 Gal bi-4 GlcNRc Galbit 4GlCNACbi- 26Hiana Fucaal 6an bi- 4 GloMRcbl-4 GlcNc Gal bi-4 GlcNACb, 64ana Galbi 4 bi. 3GlcNAc Fuca1 Glycan structure Fuc(al-3)[Gal(bl-4)]GIcNAc(b1-4)[Gal(bI-4)GIcNAc(bl-6)] Man(al-3)[Fuc(al-2)[Gal(bl-4)]GlcNAc(b1-2)[Gal(b1-4)GlcNAc (b1-6)]Man(al-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc Gal bi-4 GlcNRc ( Hana Gal bi--4 GlcNIFRP GH6an bi-4 GleNRebi-4 GlcNRc Gal bi-4 GlcNRAb i' Hanai Gal bi-4 GlciRcP' + 3 x Neuflc(a2-?) Glycan structure Gal(b 1 -4)GlcNAc(b 1 -2)[Gal(b 1 -4)GlcNAc(b 1 -4)]Man(al -3)[Gal (bl-4)GlcNAc(bl -2)[Gal(bl-4)GlcNAc(b1 -6)]Man(al-6)]Man( b 1 -4)GlcNAc(b 1 -4)GlcNAc+"+ 3 x NeuAc(a2-?)" Gal bi-4 GlcNHc bim Gal bi-4 GlcNflcbir-'a Galbi 6HMan bi-4 GlcNFcbI-4 GlcNfc Fucai 42 b/ Gal bi-4 GlcNAc Glycan structure Fuc(al-3)[Gal(bl-4)]GlcNAc(b1-4)[Gal(b1-4)GlcNAc(b1-2)] WO 2007/028212 PCT/AU2006/001325 - 48 Man(al -3)[Gal(bI1-4)G~cNAc(b 1 -2)[Gal(b 1 -4)GIcNAc(b 1 6)]Man (al -6)IJMan(bl1-4)G~cNAc(bl1-4)GIcNAc Galbi G c A Fua/3 "1-, .2 an, Gal b-4 GlcNFIP 1 / 6i\ Snb- l~lb- lfi Galla bl-4 GlcONC6bl- I Han' /k Gal bl-4 GlcNR~b,/ Glycan structure Fuc(al -3)lGal(b 1 -4)]GlcNAc(b 1-6) [Gal(bl -4)G~cNAc(b 1-2)] Man(al -6)[Gal(b 1-4)G1cNAc(b 1-2) [Gal(bl -4)G~cNAc(b 1-4)]Man (al -3)]Man(bl -4)G~cNAc(bl1-4)GlcNAc Gal b-4 ClcH~o Galbi6 \I4GlcNAob1--'- 2 Han, FUCaII 6 Ilan bl-4 GJlcNflcbl-4 GleNAc 3 Gal b-4 GlcNAcbhaa / Gal b-4 GlcNAb 1 Glycan structure Fuc(al -3)[Gal(bl -4)] G~cNAc(b 1 -2)[Gal(b 1-4)G~cNAc(b 1-6)] Man(al -6)[Gal(bl1-4)GIcNAc(bl -2)[Gal(bl1-4)G~cNAc(bl -4)]Man (al -3)]Man(b 1-4) G~cNAc(bl -4)GIcNAc WO 2007/028212 PCT/AU2006/001325 - 49 Gal bi-4 GicHR c Gal b-4 GlcNftb± 2"aa Gal,,, 3 kan bi-4 GlcN~cbl-4 GIcHFc b/ Gal b-4 GlcNnlc 43 m~ NeURC(a2-?) Glycan structure Fuc(al -3)[Gal(b 1-4)] GlcNAc(bl -4)[Gal(b 1 -4)G~cNAc(b 1-2)] Man(al -3)[Gal(bl -4)G~cNAc(bl -2)[Gal(bl1-4)GIcNAc(bl -6)]Man (al -6)]Man(bl -4)G~cNAc(bl -4)GlcNAc±"+ 3 x NeuAc(a2-?)" Gal bl- 4 G UcN R% k ' u Gal bl-4 GlcNRcP 1 : Il~an bl-4 GlcHAcbI-4 GlcNfic Gal b-4 GIcNR% /3" h tana' 1 Gal b-4 GlcN~cbi± + 3 mc Neuflc~a2-?) Glycan structure Gal(bl1-4)GlcNAc(bl -2)[Gal(bl1-4)G~cNAc(bl1-4)]Man(al -3)[Gal (blI-4)GlcNAc(bl1-2)[Gal(bl -4)G~cNAc(bl -6)]Man(al -6)]Man( hi -4)G~cNAc(bl -4)[Fuc(al -6)] G1&NAc±"± 3 x NeuAc(a2-?)" NeuAc a2- 3 Gal bi- 4 GkNeb" NeuAc a2- 3 Gal bl- 4 GlCNbI~P 3an bIo-4 GlctIcbl-4 GICNRC NeuRc a2- 3 Gal bl- 4 GictiRc%- 3 Neu~c a2- 6 Gal b- 4 GlcNRJbi Glycan structure NeuAc(a2-3)Gal(bl1-4)G~cNAc(bl -2) [NeuAc(a2-3)Gal(bl 4)G~cNAc (hi -6)]Man(al -6)[NeuAc(a2-3)Gal(bl1-4)G~cNAc(bl -4) [NeuAc (a2-6)Gal(bl1-4)G~cNAc(bl1-2)]Man(al -3)]Man(bl -4)GlcNAc(b 1 -4) [Fuc(a 1-6)] GloNAc WO 2007/028212 PCT/AU2006/001325 -50 Heuflc a2- 14 Gal b - 4 GcNAC Han Fue A al. Heu~c a2- u Gal bl-4 GlcNIJ ,/ 5 an bi-4 GlcNmcbi-4 GlcNflc Houflc 2- U Gal bi-4 GlcNR % aa S tNeue a2- u Gal bi- 4 GlcNRcP 1 / Glycan structure NeuAc(a2-?)Gal(b 1-4)G~cNAc(b 1-2) [NeuAe(a2-?)Gal(b 1 4)GIcNAc (bl1-4)]Man(al -3)[NeuAc(a2-?)Gal(b 1 -4)G~cNAc(b 1-2)[NeuAc (a2-?)Gal(bl -4)GlcNAc(bl -6)]Man(al -6)]Man(bl1-4)GlcNAc(b 1 -4) [Fuc(al -6)]G~cNAc Gal bI-4 GleNflc bi Gal bi-4 Glc~flcbvl
\
6 la Galbi 3 Han bi-4 GloHAcbI-4 GlcN~c 4 GIONRcbl- ,Haa Fu4tcal Gal bl-4 GlcNAc + 4 m teufc~a2-?) Glycan structure Fuc(al -3)[Gal(bl1-4)]G~cNAc(b 1-4) [Gal(b 1 -4)G~cNAc(b 1-2)1 Man(al -3)[Gal(bl1-4)G~cNAc(b 1-2) [Gal(b 1 -4)G~cNAc(bI1-6)]Man (al1-6)]Man(b 1 -4)G~cNAc(b 1 -4)G~cNAc+"+ 4 x NeuAc(a2-?)' Gal bl-4 GlcNflG\ Gal bi-4 GlcN U an,: Gal~~~~ Mi4GCiI~ Ian bi-4 GloN~cbI-4 GloNfic Gal bi-4 GIcNH% Gal bi-4 GlcNflcU + 2 xc Fuc Glycan structure Gal(bl -4)GIcNAc(? 1-?) [Gal(b 1 -4)G~cNAc(? 1 -?)]Man(al -3)[Gal (bl1-4)G~cNAc(? 1I-?) [Gal(bl1 -4)GlcNAc(? 1 -?)]Man(al1 -6)] Man( bl1-4)G~cNAc(bl1-4)G~cNAc+"± 2 x Fuc" WO 2007/028212 PCT/AU2006/001325 -51 Gal ut-u GlcNA u Han a, Fuc Gal ut-u GlCNACUt Ut 6 6 GlcNAcu- 4 Han bi-4 GlcNAcbi-4 GlcNlc 3 Gal ui-u GlcNA% / at N an Gal ui-u GloNAcut Glycan structure Gal(? 1 -?)GlcNAc(? 1 -?)[Gal(? 1-?)GlcNAc(? 1-?)]Man(al-3)[Gal (?1 -?)GLcNAc(? 1-?)[Gal(? 1 -?)GlcNAc(?1-?)]Man(al-6)] [GlcNAc (?1 -4)]Man(bl-4)GlcNAc(bl -4)[Fuc(? 1-6)]GlcNAc Gal ui-u GlcNA% UHan z aFuc Gal ul-u GlcNRcui U1 6 GleNAcu1- 4 Han bi-4 GlcNAcbi-4 GlcNfAc 3 Gal ui-u GlcNA% / 8 Han Gal ui-u GlCNRCu + Fuc Glycan structure Gal(? 1 -?)GlcNAc(? 1 -?)[Gal(? 1 -?)GlcNAc(? 1 -?)]Man(al-3)[Gal (?1 -?)GlcNAc(? 1 -?)[Gal(? 1 -?)GlcNAc(? 1 -?)]Man(al -6)] [GlcNAc (?1 -4)]Man(bl -4)GlcNAc(bl -4)[Fuc(? 1-6)]GlcNAc+"+ Fuc" Gal bi-4 GlcNA% i Han 1 Gal bi-4 GlcNfbi- 3 Gal bi-4 GlcNRP t GHan bi-4 GlcNc bt-4 GlcNf/c Gal bi-4 GlcND% ( Haa Gal bi--4 GlcNAiP WO 2007/028212 PCT/AU2006/001325 - 52 Glycan structure Gal(b 1-4)GlcNAc(b 1-4) [Gal.(b 1 -4)GlcNAc(bl1-6)IJMan(al -3)[Gal (bl1-4)G~cNAc(blI-3)Gal(bl -4)GlcNAc(bl1-2)[Gal(bl1-4)GIcNAc (bl1-6)IMan(al -6)]Man(bl1-4)G~cNAc(bl1-4)GIcNAc Gal bi-4 G~cHAebi- 3 Gal bi-4 GlcR%I\ Gal bi-4 GleNflb Ha i4GP~lb-4GcR Gal bI-4 GlIcHfl%j\ 1 aa 3 Gal bi-4 G~cNHdR Glycan structure Gal(b 1 -4)G~cNAc(b 1-4) [Gal(b 1 -4)G~cNAc(b I -6)]Man(al -3) [Gal (bl1-4)G~cNAc(bl1-2)[Gal(bl1-4)G~cNAc(bl1-3)Gal(bl1-4)GIcNAc (bl1-6)]Man(al -6)]Man(b 1-4) G~cNAc(b 1-4)GIcNAc WO 2007/028212 PCT/AU2006/001325 - 53 Gal ai- 3 Gal bi-4 Glc~Nfcbi- 2 hanad Fuc Neuflc a2- u Gal bi - 4 GlcNfl%\ ai/ 3anI-Gccb 4Gcc Gal al- 3 Gal bi-4 GlcNflcP 1 Glycan structure Gal(al -3)Gal(bl1-4)G~cNAc(b 1-2) [NeuAc(a2-?)Gal(bl1-4)G~cNAc (bl1-4)]Man(al -3)[Gal(al -3)Gal(bl1-4)GlcNAc(bl1-2)Man(al -6 )]Man(bl-4)G~cNAc(bl -4)[Fuc(al-6)]G~cNAc Galal- 3Gal bi-4 Glclcbi-2 Man. Fue 66 Gal al- 3 Gal b-4 GloNA Mnl/ 3anb- j~lei4Gc~ lNeuRe a2- u Gal bI - 4 GlcNAP 1 / Glycan structure Gal(al -3)Gal(bl1-4)GIcNAc(b 1-4) [NeuAc(a2-?)Gal(bl1-4)G~cNAc (bl1-2)]Man(al -3)[Gal(al -3)Gal(bl1-4)G~cNAc(bl1-2)Man(al -6 )]Man(bl -4)G~cNAc(bl1-4)[Fuc(al -6)] GleNAc Gal bi-4 GlcNAcbI- 2 Ilanal Gal bi-4 GlcNAl%, h aa an bi -4 GlefHic Gal b-4 GlcNAP 1 b +2 x Gal(bi-4)GlcNAc(bI-3) *1 2 x HeuAc Glycan structure Gal(bl -4)G~cNAc(bl1-2)[Gal(bl1-4)G~cNAc(bl -4)]Man(al 3)[Gal (bl-4)GlcNAc(bl -2)Man(al -6)]Man(bl1-4)G~cNAc+'+ 2 x Gal( bl-4)G~cNAc(bl-3) + 2 x NeuAc" WO 2007/028212 PCT/AU2006/001325 - 54 Gal bi-4 GlcNA U Han Gal b-4 GlCNACU a Han bi-4 GlcNcbi-4 GlcNAc Gal bi-4 GlcNR/ S Hana1 Gal bi-4 GlcNACUI + Gal(bl-4)GlcNfc(?1-?) + 4 x NeuAc(a2-?) Glycan structure Gal(b1 -4)GLcNAc(? 1-?)[Gal(b 1-4)GlcNAc(? 1 -?)]Man(al -3)[Gal (b 1 -4)GlcNAc(? 1 -?)[Gal(b 1 -4)GlcNAc(? 1 -?)]Man(al -6)]Man( b1-4)GlcNAc(b1-4)GlcNAc+"+ Gal(b1-4)GlcNAc(?1-?) + 4 x NeuAc(a2-?)" Gal bi-4 GlcNRc bi- u Gal bi-4 GlcNR% (Hanai Gal b1--4 GlcNA b1 G Han bi-4 GlcNRcbi-4 GlcNfic Gal bi-4 GlcNA% / Gal bi-4 GlcNAJ 1 + 5 x NeuFc(a2-?) Glycan structure Gal(bl-4)GlcNAc(bl-?)Gal(bl-4)GlcNAc(b1-6)[Gal(bl-4)GlcNAc (b1-2)]Man(al-6)[Gal(bl-4)GleNAc(b1-2)[Gal(b1-4)GlcNAc( b1-4)]Man(al-3)]Man(bl-4)GlcNAc(b1-4)GlcNAc+"+ 5 x NeuAc (a2-?)" WO 2007/028212 PCT/AU2006/001325 - 55 Gal bi-4 GIcNAbI Gal bi-4 GlcNRcb 0 6 Ilar bi-4 GlcNflcbi-4 GicNik Gal b-4 GlcNl%, " I Man
"
1 Gal b-4 GlcNR kP' + Gal(bi-4)GlCNRC(bl-3) Glycan structure Gal(b 1 -4)G~cNAc(b 1 -2)[Gal(b 1 -4)GIcNAc(b 1 -4)]Man(al -3) [Gal (bl1-4)GlcNAc(b 1 -2)[Gal(bl1-4)GlcNAc(bl1-6)]Man(al -6)]Man( bl1-4)G~cNAc(bl1-4)[Fuc(a 1-6)] G~cNAc+"+ Gal(bl1-4)G~cNAc(b 1 -3)11 Gal b - 4 GIcNRc , U Han U al Gal bi-4 Gilulf anb-GHcb 4lNc Gal bi-4 GloNAGlou-1 u //n. 3 Gal bi-4 GlcNiCUi +42 x Fue + Ga]l(bi-4)GlcN~c('?i-?) Glycan structure Gal(bl1-4)G~cNAc(? 1 -?)[Gal(bl1-4)G~cNAc(? 1 -?)]Man(al -3)[Gal (bi -4)G~cNAc(? 1-?)[Gal(b 1 -4)G~cNAc(? 1 -?)]Man(al -6)]Man( bl-4)GIcNAc(bl-4)GlcNAc+"+ 2 x Fuc + Gal(bl-4)G~cNAc(?1 Gal bi-4 GlcNAcb- 2 Ilanai Gal bi-4 GlcNfle U man bi-4 GlcNAcbI-4 GlcHAc, I\ Imana 1 Gal bi-4 GlcNACP' + 2 x Gal(bI-4)GlcHc(bI-31) + Gal~hl-3)GlcN~c(bI-3) Glycan structure Gal(bl1-4)GlcNAc(bl1-2)[Gal(bl1-4)G~cNAc(bl -4)]Man(al ?)[Gal (b 1-4) G~cNAc(bl -2)Man(al -?)]Man(bI -4)G~cNAc(b 1 4)GIcNAc +"± 2 x Gal(bl-4)GlcNAc(bl-3) + Gal(bl-3)GIoNAc(bl-3)" WO 2007/028212 PCT/AU2006/001325 -56 In one embodiment, the noggin or chimeric noggin molecule of the present invention contains at least one of the following structures in the O-linked fraction, In these representations, "u" or "?" represents that the anomeric configuration is either a or b, 5 and/or the linkage position is 2, 3, 4, and/or 6. Fuc Glycan structure Fuc Glc ul- u Fuc Glycan Glc(?l-?)Fue structure GlCNAc Glycan GlcNAc structure GalNfAc Glycan GalNAc structure NeuAc a2-6 GalNAec Glycan NeuAc(a2-6)GalNAc structure GlcNAc bl-3 GalNAc Glycan GlcNAc(b1-3)GalNAc structure WO 2007/028212 PCT/AU2006/001325 -57 NeuAc XGaNA c 3 GlcnAld Glycan GlcNAc(b1-3)[NeuAc(a2-6)]GaINAc structure Gal bi-3 GalNAc Glycan Gal(bl-3)GalNAc structure Gal Glycan structure Gal NeuAc a2- 3 Gal Glycan structure NeuAc(a2-3)Gal Xyl u- U Gic Glycan structure Xyl(? 1 -?)Glc Neufl a2- 3 Gal bi- 4 Xql Glycan structure NeuAc(a2-3)Gal(b 1 -4)Xyl Xyl ui- u Glc Glycan structure Xyl(? 1 -?)Glc Xyl ui- u Gic + xyl Glycan structure Xyl(?1-?)Glc+"+ Xyl" WO 2007/028212 PCT/AU2006/001325 - 58 Neuc a2- 3 Gal bi-3 GalNAc Glycan structure NeuAc(a2-3)Gal(b I -3)GaINAc NeuAc 3 6 GalNHc NeuAc a2- 3 Galbi Glycan structure NeuAc(a2-3)Gal(bl-3)[NeuAc(a2-6)]GalNAc NeuAc 6GalNAc Galbi Glycan structure Gal(b 1-3) (NeuAc(a2-6)]GalNAc Fue a1- 2 Gal bi--3 GaiNAc Glycan structure Fuc(al-2)Gal(b1-3)GalNAc Neufic 3 GalNAc Fuc i- 2 Galbi Glycan structure Fue(al-2)Gal(b1-3)[NeuAc(a2-6)]GalNAc Neufcu2- uGalui uGalNic U Fuc ai Glycan structure NeuAc(?2-?)Gal(? 1-?)[Fuc(al-?)]GaINAc WO 2007/028212 PCT/AU2006/001325 -.59 delta4 , 5GlcA b--3 GalNAc bl-4 GICA bi-3Gal b--3 Galbl-4 Xyl Glycan structure delta4,5GlcA(b1 -3)GalNAc(bl-4)GlcA(b1-3)Gal(b1 -3)Gal(b1 -4)Xyl HSO3 H GalNAcbi-4GlCR bl-3 Galbi- 3 Gal bi- 4Xyl delta4,5G1cA b Glycan structure delta4,5GlcA(bl-3)[HSO3(-4)]GalNAc(bl-4)GlcA(bl-3)Gal(b1 -3)Gal(bl-4)Xyl NeuAca2 uGlcNA% HSO3 6 GalINAc 3 NeuAc a2- 3 Galbi Glycan structure HSO3(-?)[NeuAc(a2-?)]GlcNAc(bl-6)[NeuAc(a2-3)Gal(bl-3)] GaINAc GlcNA% 6 GalNAc Galbi Glycan structure Gal(bl-3)[GlcNAc(bl-6)]GalNAc Fuc a1-4 GlcNA%\ 6GalNAc Galbi Glycan structure Fuc(al-4)GlcNAc(bl-6)[Gal(bl-3)]GalNAc WO 2007/028212 PCT/AU2006/001325 - 60 Fuc ai-4 GleNR%\ 3 3 GalNAc GlcNAcbi- 6 Galbi Glycan structure Fuc(al-4)GlcNAc(b1-6)[GlcNAc(bl-6)Gal(bl-3)]GalNAc Fuc ai--4 GlcNA>e 6 GaINIc Fuc a1-4 GlcNAcbi- 6 GaliI Glycan structure Fuc(al -4)GlcNAc(bl-6)Gal(bl-3)[Fuc(al-4)GlcNAc(bl-6)]GalNAc Gal bi--4 GlcNAc% 6 SGalNAc Galbi Glycan structure Gal(bl-4)GlcNAc(bl-6)[Gal(bl-3)]GalNAc Fue al- 2 Gal bi-3 GlcNAcbi-3 GalNfc Glycan structure Fuc(al-2)Gal(bl -3)GlcNAc(b1-3)GaLNAc Galbi a b GlcNHfcbi- 3 GalNAc Fuc al Glycan structure Fuc(al-3)[Gal(bl-4)]GlcNAc(bl-3)Ga1NAc Fuc al- 2 Galbi SGlcNlcbi-3 GalNc Fuc ai WO 2007/028212 PCT/AU2006/001325 -61 Glycan structure Fuc(al-2)Gal(bl-4)[Fuc(al-3)]GlcNAc(bl-3)GalNAc Gal bi-4 GlcNA l\ 3 GalNAc GlcNAIkP Glycan structure Gal(b1-4)GlcNAc(bl-6)[GlcNAc(b1-3)]GalNAc GlcNA\ 6 GalNAc Gal b-3 GlcNA Glycan structure Gal(bl-3)GlcNAc(b1-3)[G1cNAc(b1-6)]GalNAc WO 2007/028212 PCT/AU2006/001325 -62 Gal b1 4 GIcNFcbi-6 GalNAc 3 Fuc aibi GlcNAc Glycan structure Fuc(al-3)[Gal(bl-4)]GlcNAc(b1-6)[GIcNAc(bl-3)]GaINAc Gal bi-4 GlcNAc bi- 3 Gal bi-3 GalNAc Glycan structure Gal(bl-4)GlcNAc(bl-3)Gal(bl-3)GalNAc GalNA% 9 Gal bi--3 GalNAc Neufica Glycan structure GalNAc(bl-4)[NeuAc(a2-3)]Gal(bl-3)GalNAc NeuAc GalNAS a2 i Gal bi-3 GaINAc NeuR0c Glycan structure GalNAc(b1-4)[NeuAc(a2-3)]Gal(b1-3)[NeuAc(a2-6)]GalNAc NeuAc u2- u Gal ui-u GalNAcul-u GaINflc Glycan structure NeuAc(?2-?)Gal(?1-?)GalNAc(?l-?)GaINAc WO 2007/028212 PCT/AU2006/001325 - 63 NeuAc a2- 3 Gal bi-4 GlcNA9\ 6 GalNAc 3 Neufc a2- 3 Galbi Glycan structure NeuAc(a2-3)Gal(b1 -4)GlcNAc(b1 -6)[NeuAc(a2-3)Gal(b1 3)]GaINAc Gal bi-u Gic NA\ 3 GalNAc NeuAc a2- 3 Galbi Glycan structure Gal(b1-?)GlcNAc(bl-6)[NeuAc(a2-3)Gal(bl-3)]GalNAc Neuc a2- 3 Gal bi-u GlcNAcbi-6 GaINAc 3 Gal Glycan structure NeuAc(a2-3)Gal(b1 -?)G1cNAc(b1-6)[Gal(b1 -3)]GalNAc NeuAc a2- u Gal bi-u GlcNflbi- u Gal ui-u GaliNAc Glycan structure NeuAc(a2-?)Gal(bl -?)GlcNAc(b1-?)Gal(? 1-?)GalNAc NeuAc a2- 3 Gal bi-4 GlcNAr. 6GalNAc NeuAc a2- 3 Galbi Glycan structure NeuAc(a2-3)Gal(b1-4)GlcNAc(bl-6)[NeuAc(a2-3)Gal(bl 3)]GalNAc WO 2007/028212 PCT/AU2006/001325 - 64 NeuRc a2- 3 Gal bi-4 GlcNfcbi-6 GalNFc 3 bi Gal Glycan structure NeuAc(a2-3)Gal(bl-4)GlcNAc(bl-6)[Gal(bl-3)]GalNAc Gal bi-4 GlcNR 9 %I\ 6 alN~c '3 Fuc al- 2 Galb 1 Glycan structure Fue(al-2)Gal(bl-3)[Gal(b1-4)GlcNAc(bl-6)]GalNAc Galbi GlcNRi Fuc a1 3 GalNAc Neuc a2- 3 Galb1 Glycan structure Fuc(al-3)[Gal(bl -4)]GlcNAc(bl-6)[NeuAc(a2-3)Gal(bl-3)]GaINAc Galbi 4 GlcNebl-6 GalNAc 3 3 Fucal Gal Glycan structure Fuc(al-3)[Gal(bl-4)]GlcNAc(bl-6)[Gal(b1-3)]GalNAc HSOS -6 GlcNH \ Galal 6 3 GalNRc 3Galbi Fuct Glycan structure Fuc(al-2)[Gal(al -3)]Gal(b1-3)[HSO3(-6)GlcNAc(b1-6)]GalNAc WO 2007/028212 PCT/AU2006/001325 - 65 Galbi Fucai GalNAc NeuAc a2- 3 Galbi Glycan structure Fuc(al-3)[Gal(bl -4)]GlcNAc(bl-6)[NeuAc(a2-3)Gal(bl 3)]GalNAc Neule a2- 3 Galbi 4 GlcNi1cfbi-6 GalNflc .33 Fucal b bi Gal Glycan structure NeuAc(a2-3)Gal(b1-4)[Fuc(al-3)]GIcNAc(bl-6)[Gal(b1 3)]GalNAc Fuca1 4 GlcNAcbi-6 GalNfc 1/ 3 3 Fuc al- 2 Galb1 Gal Glycan structure Fuc(al-2)Gal(bl-3)[Fuc(al-4)]GlcNAc(bl-6)[Gal(bl-3)]GaINAc Fuc al- 2 Galbi 4 GlcNAcb-6 GalNAc 3 Fucal b bi Gal Glycan structure Fuc(al-2)Gal(bl-4)[Fuc(al-3)]GlcNAc(b1-6)[Gal(b1-3)]GalNAc WO 2007/028212 PCT/AU2006/001325 - 66 Galbi 4 GlcNAc bi- 6 GaiNflc 3 Fucai b bi Gal +Fuc(al-2) Glycan structure Fuc(al -3)[Gal(bl-4)]GlcNAc(bl-6)[Gal(b1-3)]GaINAc+"+Fuc (al-2)" Fuc al- 2 Galbi 3 GIcNA%\ Fucal 6GalNnc NeuAc a2- 3 Galbi Glycan structure Fuc(al-2)Gal(bl-4)[Fuc(al -3)]GlcNAc(bl-6)[NeuAc(a2-3)Gal (bl-3)]GalNAc Gal bi-4 GlcNl%\ 3 3 GalNAc Gal bi-4 GleNA 1 Glycan structure Gal(bl-4)GlcNAc(bl-3)[Gal(b1-4)GlcNAc(bl-6)]GalNAc Fue ai- 2 Gal bi-4 GlcNel% 6 GalN~c 3 Neufnc a2- 3 Galbi Glycan structure Fuc(al-2)Gal(bl-4)GlcNAc(bl-6)[NeuAc(a2-3)Gal(bl-3)]GalNAc Fucui 4GlcNfc u-- 3 Gal ui-3 GalNAc NeuA u2- 3 GalU 1 WO 2007/028212 PCT/AU2006/001325 - 67 Glycan structure NeuAc(?2-3)Gal(?1-3)[Fuc(? 1-4)]GlcNAc(? 1-3)Gal(?1 -3)Ga1NAc Fuc at--- 2 Gal bi -GlcNAcbi- 3 Gal bi-3 GalNAc Fucal Glycan structure Fuc(al-2)Gal(bl-4)[Fuc(al-3)]GIcNAc(bl-3)Gal(bl-3)GaLNAc Fuc at- 2 Galbi 3 GlcNAg Fuca1 6GalNAc NeuAca2- 3 Gal 1 Glycan structure Fuc(al-2)Gal(b1-4)[Fuc(al-3)]GLcNAc(b1-6)[NeuAc(a2-3)Gal (bl-3)]GaINAc NeuAc a2- 3 Galbi 3 GlcNASy Fucai 6GalNAc Neufl a2- 3 Galbi Glycan structure NeuAc(a2-3)Gal(bl -4)[Fuc(al-3)]GLcNAc(b1-6)[NeuAc(a2-3) Gal(bl-3)]GalNAc Gal bi-3 GlcNbi-- 3 Gal bi-4 GlcNfcbi-6 GalNAc 3 Gal Glycan structure Gal(bl-3)GlcNAc(bl-3)Gal(bl-4)GlcNAc(bl-6)[Gal(bl 3)]GalNAc WO 2007/028212 PCT/AU2006/001325 - 68 Gal bi-4 GlcNAcbi- 3 Gal bi-4 GlcNA%\ 6 GalNA c 3 NeuAca2- 3 Galbi Glycan structure Gal(bl-4)GlcNAc(b1-3)Gal(b1 -4)GlcNAc(bl-6)[NeuAc(a2-3)Gal (bl-3)]GalNAc Gal bi-4 GlcNA%\ 6 GalNHc ,3 Fuc al- 2 Gal bi-3 GlcNAcbi- 3 Galb 1 Glycan structure Fuc(al-2)Gal(b1-3)GlcNAc(b1-3)Gal(bl-3)[Gal(b1-4)GlcNAc (bl-6)]GalNAc Fue al- 2 Gal bi-3 GlcNRcbi- 3 Gal bi-4 GlcNRcbi-6 GaINAc 3 b1 Gal Glycan structure Fuc(al -2)Gal(bl -3)GlcNAc(b 1 -3)Gal(b 1 -4)GlcNAc(b 1-6) [Gal (bl-3)]GalNAc Gal bi-3 GlcNAcbi- 3 Galbi 3 GlcNfcb-6 GalNAc / 3 FUCaiI Gal Glycan structure Gal(bl-3)GIcNAc(bl-3)Gal(b1-4)[Fuc(al -3)]GlcNAc(bl-6)[Gal (bl-3)]GalNAc Fue a1- 2 Gal bi-3 GlcNAchbi- 3 Gal bi-4 GlcNAb\ 6 GalN~c 3 Neufl a2- 3 Galb 1 Glycan structure Fuc(al-2)Gal(bl-3)GlcNAc(bl-3)Gal(bl-4)GlcNAc(bl-6)[NeuAc WO 2007/028212 PCT/AU2006/001325 - 69 (a2-3)Gal(b 1 -3)]GalNAc Gal bi-3 GlcNcbl- 3 Galbi OGlcNA%\ Fuca1 6GaINAc Neuflc a2- 3 Gal 1 Glycan structure Gal(b1 -3)GlcNAc(b1 -3)Gal(b1 -4)[Fuc(al-3)]GlcNAc(b1 6)[NeuAc (a2-3)Gal(b1-3)]GalNAc Gal bi-4 GlcNAcb, Gal bi-4 GlcNA% 6 GalNAc \N Galbl Gal bi-4 GIcNAcP 1 Glycan structure Gal(b1-4)GlcNAc(b1-3)[Gal(bl -4)G1cNAc(bl-6)]Gal(b1 -3)[Gal (b1-4)GlcNAc(b1-6)]GalNAc Gal bi-4 GcN lcNA 9 Gal bi--4 GleNAb Gal bi-3 GlcNHAdb 6 GalNAc Neul a2- 3 Galbi Glycan structure Gal(bl-3)GlcNAc(bl-3)[Gal(b1-4)GlcNAc(bl-6)]Gal(b 1 4)GlcNAc (bl-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc NeuAc a2- 3 Gal bi-4 GlcHAcbi- 3 Gal bi-4 GlcNA%\ 6GaINRc 3 NeuAc a2- 3 Galbi Glycan structure NeuAc(a2-3)Gal(bl-4)GlcNAc(b1-3)Gal(bl-4)GlcNAc(bl 6)[NeuAc WO 2007/028212 PCT/AU2006/001325 -70 (a2-3)Gal(bl-3)]GalNAc Gal ui-4 GlcHA%\ 5 GalNAc y3 NeuAc u2- 3 Gal ul-u GlcNcui--- 3 Galul + Fuc Glycan structure NeuAc(?2-3)Gal(?1-?)GlcNAc(?1-3)Gal(?1 -3)[Gal(?1-4)GlcNAc (?1-6)]GalNAc+"+ Fuc" Gal bi-u GlcNAc bi- u Gal bi 4 GlcNAb Fucal 6 GalNAc Neuflc a2- 3 GalI Glycan structure Gal(bl-?)GlcNAc(bl-?)Gal(bl-4)[Fuc(al-3)]GIcNAc(bl-6)[NeuAc (a2-3)Gal(bl-3)]GalNAc Gala1 u FUCai U b U Gal bi--4 GlcNA%\ 3 GalNRc Neul a2- 3 Galbi Glycan structure Fuc(al-?)[Gal(b1-?)]GlcNAc(b1-?)Gal(bl-4)GlcNAc(bl-6)[NeuAc (a2-3)Gal(bl-3)]GalNAc Fuc ui- u Galui uGlcNAcui- u Gal ui-u GlcNR\ Fucui UGalNAC Neufc u2- u Galul Glycan structure Fuc(? 1 -?)Gal(? 1 -?)[Fuc(? 1-?)] GlcNAc(? 1 -?)Gal(? 1 -?)GlcNAc (?1-?)[NeuAc(?2-?)Gal(?1-?)]GalNAc WO 2007/028212 PCT/AU2006/001325 -71 Gal ui-u GlcNcui- u Galui ~GlcNAq\ Fucui uGalN~c FUCUI NeuAcu2- u Gal 1 +Fuc Glycan structure Gal(? 1-?)GlcNAc(? 1 -?)Gal(? 1-?)[Fuc(?1-?)]GlcNAc(?1-?)[NeuAc (?2-?)Gal(? 1-?)] GaINAc+"+ Fuc" Fuc u1- u Galui UGlcNAcu- u GaluI Fucui UGleNcu Fucui GalNRc NeuAc u2- u Galu1 Glycan structure Fuc(? 1 -?)Gal(? 1 -?)[Fuc(? 1-?)]GlcNAc(? 1 -?)Gal(? 1-?)[Fuc(? 1-?)]GlcNAc(?1-?)[NeuAc(?2-?)Gal(?1-?)]GalNAc Gal ui-u GlcNAcui- u Gal ui-u GlcNAcui- u Gal ui-u GlcHA% UGaINAC Neufc 2- U Galu1 Glycan structure Gal(?1-?)GlcNAc(? 1 -?)Gal(? 1 -?)GlcNAc(? 1 -?)Gal(? 1 -?)GlcNAc (?1-?)[NeuAc(?2-?)Gal(?1-?)]GalNAc WO 2007/028212 PCT/AU2006/001325 - 72 Galbi Gl1cNikjb Fucai 6 GalNAc /3 Gal bi-3 GlcHNAcI Glycan structure Fuc(al -3)[Gal(bl-4)]GlcNAc(bl-6)[Gal(bl -3)GlcNAc(bl-3)] GalNAc Gal bi-4 GIcNR%\ Galbi 6GalNAc 4 GkNAP 1 T Fueai'/ Glycan structure Fuc(al-3)[Gal(bl-4)]GlcNAc(bl-3)[Gal(b1 -4)GlcNAc(b1-6)] GalNAc NeuRc Galai a2 6 F Gal bi-u GlcNAcbi- 3 Gal bi-3 GalNRc Fuc a Glycan structure Fuc(a1-2)[Gal(al-3)]Gal(bl -?)GIcNAc(b1 -3)Gal(b1-3)[NeuAc (a2-6)]GalNAc WO 2007/028212 PCT/AU2006/001325 - 73 Gal bI-U GlcNfiku U Gal bi - U l H C \ Gal bi GlcNA Neu~la2- 3 GaPb /3Gl~ Glycan structure Gal(bl1-?)G~cNAc(?l1-?)[Gal(bl1-?)G~cNAc(? I-?)]Gal(b 1 ?)GlcNAc (bi -6)[NeuAc(a2-3)Gal(bl -3)]Ga1NAc Gal bi-4 GlcNAcbi- 3 Gal bi-4 GlcN~cbl- 3 Gal bL-4 GlcNAcb-6 GaiHfic 3 bi Gal Glycan structure Gal(bl1-4)GlcNAc(bl1-3)Gal(bl1-4)GlcNAc(bl1-3)Gal(bl 4)G~cNAc (bi -6)[Gal(bl-3)]Ga1NAc Neufic a2- 3 Gal bi-4 GlcN~ebi- 3 Gal bi-4 GlfcA%\ GIc 3 Neufla2- 3Ga Glycan structure NeuAc(a2-3)Gal(bl1-4)G~cNAc(bl1-3)Gal(bl1-4)G~cNAc(b 1 6)[NeuAc (a2-3)Gal(b 1 -3)] GalNAc Neufic a2- 3 Gal bi 4~ H c i a l 4G~ N e Heufl a2- 3 Gal 1 Glycan structure NeuAc(a2-3)Gal(b 1-4) [Fuc(al -3)]G~cNAc(bl1-3)Gal(bl 4)G~cNAc (bi -6) [NeuAc(a2-3)Gal(bl -3)] GaINAc WO 2007/028212 PCT/AU2006/001325 - 74 Galbi GlcNAc bi- u Gal bi--4 GlcNHAl Fuca1 6 GalNAc Neuc a2- 3 Galbi Glycan structure Fuc(al-3)[Gal(b1 -4)]GlcNAc(b1-?)Gal(b1-4)GLCNAc(b1 6)[NeuAc (a2-3)Gal(b1-3)]GaINAc Neu nl a2 - 6 Galbi 43GcN~hi-u Gal bi--4GlN Fuc ai 3\G6alNflc NeuAc a2- 3 GalbI Glycan structure NeuAc(a2-6)Gal(b1-4)[Fuc(al-3)]GlcNAc(b1-?)Gal(bl 4)GlcNAc (b1-6)[NeuAc(a2-3)Gal(bl-3)]GalNAc NeuRc a2- 6 Gal bi-4 GlcNAcbi- u Gal bi-4 GlcNbi- u Gal b1--4 GlcHAcbi-6 GaiNHc 3 bI Gal Glycan structure NeuAc(a2-6)Gal(b1-4)G1cNAc(b1 -?)Gal(bl -4)GlcNAc(bl -?)Gal (bl-4)GlcNAc(bl-6)[Gal(bl-3)]GalNAc Gal bi--4 GICNflcNl Gal bi-4 GlcNHi 3GalNA , Galbi Fuc a1- 2 Gal bi--3 GlcNRP Glycan structure Fuc(al -2)Gal(b 1 -3)GlcNAc(b 1-3) [Gal(bl -4)G1cNAc(b 1-6)] Gal (b1 -3)[Gal(bl -4)GlcNAc(bl-6)]GalNAc WO 2007/028212 PCT/AU2006/001325 - 75 Fucai $61cN ci--- 3 Gal bi-4 GlcNcbi- 3 Gal bi-4 GIcNAl% Fuc a- 2 Galbi 6 GalNA c Neufla2- 3 Galbi Glycan structure Fuc(al-2)Gal(bl-3)[Fuc(al-4)]GlcNAc(bl-3)Gal(bl-4)GlcNAc (bl-3)Gal(bl-4)GlcNAc(bl-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc Fucai 4GlcNRcbi- 3 Gal bi-4 GlcNHcbi-- 3 Galbi Galbi 4GlcNfrb Fucai 6 GalNAc NeuRe a2- 3 Galbi Glycan structure Fuc(al-4)[Gal(b1-3)]GlcNAc(b1-3)Gal(bl-4)GlcNAc(b1 -3)Gal (bl-4)[Fuc(al-3)]GlcNAc(b1 -6)[NeuAc(a2-3)Gal(b 1 3)]GalNAc Galbi 3GlcNAeb Fuca Gal bi-4 GlNArb 4 GlcNAk~ 3GalNAc 33 Fue a1- 2 Galbi NeuAc a2- 3 Gal bi Glycan structure Fuc(al -2)Gal(b 1 -3)[Fuc(al -4)] GlcNAc(b 1 -3)[Fuc(al -3) [Gal (bl-4)]GlcNAc(bl-6)]Gal(b1-4)GlcNAc(b1-6)[NeuAc(a2 3)Gal (bl-3)]GalNAc WO 2007/028212 PCT/AU2006/001325 - 76 Gal bi-4 GlCHReb Fuc .1 \ G o n i Galb± \ G~ H e Fuc a- 2Galbi Fucai 6GaJlInc Heuo a2- 3 Gl Glycan structure Fuc(al -2)Gal(bl -3)[Fuc(al -4)]GlcNAc(bl1-3)[Gal(b 1 4)G~cNAc (bi -6)] Gal(b 1-4) [Fuc(al -3)] G~cNAc(b 1-6) [NeuAc(a2-3)Gal( bi -3)]GaINAc Neulc a2- 3 Gal bi-4 GcHlcbi- 3 Gal bi-4 GlcNFcb- 3 Gal bi-4 GUMN% \ 6a~i NeuRc a2- 3 Gal bI Glycan structure NeuAc(a2-3)Gal(bl -4)G~cNAc(b 1 -3)Gal(bI1-4)G~cNAc(bl -3)Gal (bI -4)GlcNAc(bl -6) [NeuAc(a2-3)Gal(bl-3)] GaINAc Galal '11\ j~ al i -U GlcHRGal bl- 4 GlcNR% \6 al~ Fucal 6 Ga1 b-4 GleNfbi- 3 Gal bi- 3 Galb 1 Gal± bi 3Gal bi-u GlcNRc FucalI/ Glycan structure Fuc(al -2)[Gal(al -3)] Gal(b 1 -?)G~cNAc(b 1-3) [Fuc(al -2)[Gal (al -3)] Gal(bl1-?)G~cNAc(b 1-6)] Gal(bl -4)G~cNAc(bI1-3)Gal(bl -3)Gal(bl -3)[Gal(bl -4)G~cNAc(bl -6)] GaINAc WO 2007/028212 PCT/AU2006/001325 - 77 Galai SGal li-u GlcNA Gal bi-4 GlcNA G Gl 6 Gal bi-4 G cAcbi- 3 Gal 6i- 3 abi 3 Ga3a bi Fucal Glycan structure Fuc(al -2)[Gal(al -3)] Gal(b 1 -?)G~cNAc(b 1-3) [Fuc(al -2)[Gal (al -3)]Gal(b-?)GcNAc(b l-6)] Gal(bli-4)GcNAc(b-3)Gal(b -3)Gal(bl -3) [Gal(bl1-4)G~cNAc(b 1-6)] GaINAc Galal jGal bi- U GC Neufic a2- 3 Gal b1-4 GICN %>\ a~l Fucal 6 al bi-4 lNebi- 3 Gal bi- 3 Gabi/ Gal a - bi Fucail Glycan structure Fuc(al -2) [Gal(al -3)] Gal(b 1 -?)GIcNAc(b 1-3) [Fuc(al -2)[Gal (al -3)]Gal(bl-?)GcNAc(bl-6)]Gal(b1 -4)G1cNAc(b1-3)Gal(b1 -3)Gal(b1-3)[NeuAc(a2-3)Gal(bl -4)GcNAc(b-6)]Ga1NAc WO 2007/028212 PCT/AU2006/001325 - 78 Galai Gaa iGal bi-u GlcNRhj Fucal z Gl bl-4alGN 9 c GGlaa bi jGal bi-u GIcNAc Fucal Glycan structure Fuc(al -2)[Gal(al -3)] Gal(b 1 -?)GlcNAc(bl -3)[Fuc(al -2) [Gal (al -3)] Gal(bl -?)GlcNAc(bl1-6)]Gal(bl -4)GlcNAc(bl -3)Gal(b 1 -3) [Fuc(al -2) [Gal(al -3)] Gal(b 1-4) G~cNAc(b 1-6)] GalNAc NeuAc a2- 3 Galbbi GlcNcc b 3allb Fuca1 Gal3i GlcNlcbi----33Galbi 3 bi Gal Glycan structure NeuAc(a2-3)Gal(bl-4)[Fuc(al-3)]GlcNAc(bl-3)Gal(b1-4)[Fuc (al-3)]GlcNAc(bl -3)Gal(b 1-4) [Fuc(al -3)] GlcNAc(b 1-6) [Gal (bl-3)]GalNAc Gal bi-4 GloNAbi- 3 Gal bi-4 GlcNAcbi- 3 Gal bi-4 GlcNRbi- 3 Gal bi-4 GlcNnebi-5 GalNfc 3 bi Gal Glycan structure Gal(bl-4)GlcNAc(bl-3)Gal(b1-4)GlcNAc(bl-3)Gal(bl-4)GlcNAc (b1-3)Gal(bl -4)GlcNAc(bl-6)[Gal(bl-3)]GalNAc WO 2007/028212 PCT/AU2006/001325 - 79 Gal b---4 Glab--- 3 Gal b--4 Glct4R bl- 3 Gal bl-4 Glonbi-- 3 Gal b±-4 Glae~nbi- 3 Gal bi-4 GlaNflbi-G CalNn, Glycan structure Gal(bl-4)GlcNAc(bl-3)Gal(bl -4)GlcNAc(bl-3)Gal(bl-4)GlcNAc (bl-3)Gal(b1 -4)GlcNAc(bl-3)Gal(b1-4)GlcNAc(bl-6)[Gal(b1 -3)]GaINAc Gal bi-4 GlcNhIcbi- 3 Gal bi-4 GlcNAcbI- 3 Gal bi-4 GlcNAr GalNAc NeuAc a2- 3 Galbi + Fucai-3) Glycan structure Gal(bl-4)GlcNAc(bl-3)Gal(bl-4)GlcNAc(b1-3)Gal(bl-4)GlcNAc (b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc+"+ Fuc(al-3)" Gal bi-4 GlcNAcbi- 3 Gal bi-4 GlcNAc bi- 3 Gal b-4 GlcNAq9\ 6 GalNFc NeuAc a2- 3 Galbi + 2 x Fuc(a1-3) Glycan structure Gal(b1-4)GleNAc(b1-3)Gal(bl-4)GlcNAc(bl-3)Gal(b1-4)GlcNAc (b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc+"+ 2 x Fuc(al-3)" Galbi Fuca bl 4 GlcNcbi- 3 Galbi Fucai 3GlcNACb Fuca1 GalNAc Neuc a2- 3 Galbi Glycan structure Fuc(al-3)[Gal(b1 -4)]GlcNAc(bl-3)Gal(bl-4)[Fuc(al 3)]GlcNAc (bl-3)Gal(b1-4)[Fuc(al-3)]GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1 -3)]GalNAc WO 2007/028212 PCT/AU2006/001325 - 80 Galbi 43GlcNAc bi- u Gal bi-4 GleNRc bi- u Gal bi-4 GlcNfcbi-6 GalNAc 3 Fuca1 I1 bi Gal Glycan structure Fuc(al-3)[Gal(b1-4)]GlcNAc(bl-?)Gal(bl-4)GleNAc(bl ?)Gal (bl-4)GlcNAc(b1 -6)[Gal(bl-3)]GalNAc Galb1 4 GlecNAcbi- u Gal bi-4 GlcNRbi- u Gal bi-4 GlcNHb Fucai 6 GalNAc NeuR a2- 3 Galbi Glycan structure Fuc(al -3)[Gal(b 1-4)]GlcNAc(bl-?)Gal(b 1 -4)GlcNAc(bl ?)Gal (bl -4)GlcNAc(bl -6)[NeuAc(a2-3)Gal(b 1 -3)]GalNAc In a particular embodiment, the present invention contemplates an isolated form of noggin or chimeric noggin molecule. An isolated noggin or chimeric noggin molecule of the 5 present invention comprises distinctive pharmacological traits selected from the group comprising or consisting of therapeutic efficiency (Ti), effective therapeutic dose (TCID 5 o)
(T
2 ), bioavailability (T 3 ), time between dosages to maintain therapeutic levels (T 4 ), rate of absorption (T 5 ), rate of excretion (T 6 ), specific activity (T 7 ), thermal stability (T 8 ), lyophilization stability (T9), serum/plasma stability (Ti 0 ), serum half-life (Tii), solubility in 10 blood stream (T 12 ), immunoreactivity profile (T1 3 ), immunogenicity (T 14 ), inhibition by neutralizing antibodies (T14A), side effects (T 15 ), receptor/ligand binding affinity (T1 6 ), receptor/ligand activation (T1 7 ), tissue or cell type specificity (Ti 8 ), ability to cross biological membranes or barriers (i.e. gut, lung, blood brain barriers, skin etc) (Ti 9 ), tissue uptake (T 20 ), stability to degradation (T 2 1), stability to freeze-thaw (T 2 2 ), stability to 15 proteases (T 23 ), stability to ubiquitination (T 24 ), ease of administration (T 25 ), mode of administration (T 26 ), compatibility with other pharmaceutical excipients or carriers (T 27
),
WO 2007/028212 PCT/AU2006/001325 -81 persistence in organism or environment (T 28 ), stability in storage (T 29 ), toxicity in an organism or environment and the like (T 30 ). In addition, noggin or chimeric noggin molecule of the present invention may have altered 5 biological effects on different cells types (T 31 ), including without being limited to human primary cells, such as lymphocytes, erythrocytes, retinal cells, hepatocytes, neurons, keratinocytes, endothelial cells, endodermal cells, ectodermal cells, mesodermal cells, epithelial cells, kidney cells, liver cells, bone cells, bone marrow cells, lymph node cells, dermal cells, fibroblasts, T-cells, B-cells, plasma cells, natural killer cells, macrophages, 10 granulocytes, neutrophils, Langerhans cells, dendritic cells, eosinophils, basophils, mammary cells, lobule cells, prostate cells, lung cells, oesophageal cells, pancreatic cells, Beta cells (insulin secreting cells), hemangioblasts, muscle cells, oval cells (hepatocytes), mesenchymal cells, brain microvessel endothelial cells, astrocytes, glial cells, various stem cells including adult and embryonic stem cells, various progenitor cells; and other human 15 immortal, transformed or cancer cell lines. The biological effects on the cells include effects on proliferation (T 32 ), differentiation
(T
33 ), apoptosis (T 34 ), growth in cell size (T 35 ), cytokine adhesion (T 36 ), cell adhesion
(T
37 ), cell spreading (T 38 ), cell motility (T 39 ), migration and invasion (T 4 0 ), chemotaxis 20 (T 41 ), cell engulfment (T 42 ), signal transduction (T 43 ), recruitment of proteins to receptors/ligands (T 44 ), activation of the JAK/STAT pathway (T 45 ), activation of the Ras erk pathway (T 4 6 ), activation of the AKT pathway (T 47 ), activation of the PKC pathway
(T
48 ), activation of the PKA pathway (T 49 ), activation of src (T 50 ), activation of fas (T 5 ), activation of TNFR (T 5 2 ), activation of NFkB (T 53 ), activation of p38MAPK (T 54 ), 25 activation of c-fos (T 55 ), secretion (T 56 ), receptor internalization (T 57 ), receptor cross-talk
(T
58 ), up or down regulation of surface markers (T 59 ), alteration of FACS front/side scatter profiles (T 6 o), alteration of subgroup ratios (T 61 ), differential gene expression (T 62 ), cell necrosis (T 63 ), cell clumping (T 64 ), cell repulsion (T 65 ), binding to heparin sulfates (T 66 ), binding to glycosylated structures (T 67 ), binding to chondroitin sulfates (T 68 ), binding to 30 extracellular matrix (such as collagen, fibronectin) (T 69 ), binding to artificial materials (such as scaffolds) (T 70 ), binding to carriers (T 71 ), binding to co-factors (T72) the effect alone or in combination with other proteins on stem cell proliferation, differentiation WO 2007/028212 PCT/AU2006/001325 - 82 and/or self-renewal (T 73 ) and the like. These are summarized in Table 3. The present invention further provides a chimeric molecule comprising an isolated noggin having a profile of measurable physiochemical parameters indicative of or associated with 5 one or more distinctive pharmacological traits of the isolated noggin, said noggin or a fragment thereof, linked to the constant (Fc) or framework region human immunoglobulin via one or more protein linker. Such a chimeric molecule is also referred to herein as noggin-Fc. Other chimeric molecules contemplated by the present invention include the noggin or noggin-Fc or a fragment thereof, linked to a lipid moiety such as a 10 polyunsaturated fatty acid molecule. Such lipid moieties may be linked to an amino acid residue in the backbone of the molecule or to a side chain of such an amino acid residue. Accordingly, the present invention provides an isolated polypeptide encoded by a nucleotide sequence selected from the list consisting of SEQ ID NOs: 23, 25, 27, 29, 31, or 15 a nucleotide sequence having at least about 90% identity to any one of the above-listed sequence or a nucleotide sequence capable of hybridizing to any one of the above sequences or their complementary forms under high stringency conditions. Another aspect of the present invention provides an isolated polypeptide comprising an 20 amino acid sequence selected from the list consisting of SEQ ID NOs: 24, 26, 28, 30, 32, or an amino acid sequence having at least about 90% similarity to one or more of the above sequences. The present invention further contemplates a pharmaceutical composition comprising at 25 least part of the noggin or chimeric noggin molecule, together with a pharmaceutically acceptable carrier, co-factor and/or diluent. With respect to the primary structure, the present invention provides an isolated noggin or chimeric noggin molecule, or a fragment thereof, encoded by a nucleotide sequence 30 selected from the list consisting of SEQ ID NOs: 23, 25, 27, 29, 31, or a nucleotide sequence having at least about 90% identity to any one of the above-listed sequence or a WO 2007/028212 PCT/AU2006/001325 - 83 nucleotide sequence capable of hybridizing to any one of the above sequences or their complementary forms under low stringency conditions. Still, another aspect of the present invention provides an isolated nucleic acid molecule 5 encoding noggin or chimeric noggin molecule or a functional part thereof comprising a sequence of nucleotides having at least 90% similarity selected from the list consisting of SEQ ID NOs: 23, 25, 27, 29, 31, or after optimal alignment and/or being capable of hybridizing to one or more of SEQ ID NOs: 23, 25, 27, 29, 31, or their complementary forms under low stringency conditions. 10 In a particular embodiment, the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a noggin or chimeric noggin molecule, or a fragment thereof, an amino acid sequence substantially as set forth in one or more of SEQ ID NOs: 24, 26, 28, 30, 32, or an amino acid sequence having at least about 15 90% similarity to one or more of SEQ ID NOs: 24, 26, 28, 30, 32, after alignment. The present invention further provides a chimeric molecule comprising an isolated noggin having a profile of measurable physiochemical parameters indicative of or associated with one or more pharmacological traits of the isolated noggin, the noggin linked to the constant 20 (Fc) or framework region of a mammalian immunoglobulin via one or more protein linker. In another aspect, the mammal Fc or framework region of the immunoglobulin is derived from a mammal selected from the group consisting of primates, including humans, marmosets, orangutans and gorillas, livestock animals (e.g. cows, sheep, pigs, horses, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits, 25 companion animals (e.g. cats, dogs) and captured wild animals (e.g. rodents, foxes, deer, kangaroos). In another embodiment the Fc or framework region is a human immunoglobulin. In a particular embodiment the mammal is a human. Such a chimeric molecule is also referred to herein as noggin-Fc. Other chimeric molecules contemplated by the present invention include the noggin or noggin-Fc or a fragment thereof linked to a 30 lipid moiety such as a polyunsaturated fatty acid molecule. Such lipid moieties may be linked to an amino acid residue in the background of the molecule or to a side chain of such an amino acid residue.
WO 2007/028212 PCT/AU2006/001325 - 84 In another aspect, the present invention provides an isolated nucleic acid molecule encoding a noggin, or a fragment thereof, comprising a sequence of nucleotides selected from the group consisting of SEQ ID NOs: 23, 25, 27, linked directly or via one or more 5 nucleotide sequences encoding protein linkers known in the art to nucleotide sequences encoding the constant (Fc) or framework region of a human immunoglobulin, substantially as set forth in one or more of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17 or 19. In another aspect, the present invention provides an isolated noggin, or a fragment thereof, 10 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 26, 28, linked directly or via one or more protein linkers known in the art, to the constant (Fe) or framework region of a human immunoglobulin, substantially as set forth in one or more of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18 or 20. 15 The present invention further extends to uses of an isolated noggin or chimeric noggin molecule or nucleic acid molecules encoding same in diagnostic, prophylactic, therapeutic, nutritional and/or research applications. More particularly, the present invention extends to a method of treating or preventing a condition or ameliorating the symptoms of a condition in an animal subject, said method comprising administering to said animal subject an 20 effective amount of an isolated noggin or chimeric noggin molecule. In addition, the present invention extends to uses of a noggin or chimeric noggin molecule for screening small molecules, which may have a variety of diagnostic, prophylactic, therapeutic, nutritional and/or research applications. 25 The present invention further contemplates using an isolated noggin or chimeric noggin molecule as immunogens to generate antibodies for therapeutic or diagnostic applications. The subject invention also provides the use of a noggin or chimeric noggin molecule in the 30 manufacture of a formulation for diagnostic, prophylactic, therapeutic, nutritional and/or research applications.
WO 2007/028212 PCT/AU2006/001325 - 85 The subject invention also provides a human derived noggin or chimeric noggin molecule for use in culturing hES, reducing the potential risks of transferring animal-derived infectious agents to hES, thus allowing hES to be applicable for therapeutic applications. 5 The subject invention also provides a human derived noggin or chimeric noggin molecule for use as a standard protein in an immunoassay and kits thereof. The subject invention also extends to a method for determining the level of human cell-expressed human noggin in a biological preparation. 10 WO 2007/028212 PCT/AU2006/001325 -86 TABLE 1 Sequence Identifier Sequence Identifier Sequence SEQ ID NO:1 Human IgG1 Fc nucleotide sequence SEQ ID NO:2 Human IgG1 Fe amino acid sequence SEQ ID NO:3 Human IgGI Fe nucleotide sequence (variant) SEQ ID NO:4 Human IgG1 Fc amino acid sequence (variant) SEQ ID NO:5 Human IgG2 Fc nucleotide sequence SEQ ID NO:6 Human IgG2 Fc amino acid sequence SEQ ID NO:7 Human IgG3 Fc nucleotide sequence SEQ ID NO:8 Human IgG3 Fc amino acid sequence SEQ ID NO:9 Human IgG4 Fc nucleotide sequence SEQ ID NO:10 Human IgG4 Fe amino acid sequence SEQ ID NO:1 1 Human IgA1 Fc nucleotide sequence SEQ ID NO:12 Human IgAl Fe amino acid sequence SEQ ID NO:13 Human IgA2 Fe nucleotide sequence SEQ ID NO:14 Human IgA2 Fc amino acid sequence SEQ ID NO:15 Human IgM Fe nucleotide sequence SEQ ID NO:16 Human IgM Fc amino acid sequence SEQ ID NO:17 Human IgE Fc nucleotide sequence SEQ ID NO:18 Human IgE Fe amino acid sequence SEQ ID NO:19 Human IgD Fe nucleotide sequence SEQ ID NO:20 Human IgD Fc amino acid sequence SEQ ID NO: 21 Noggin forward primer (nucleotide sequence) SEQ ID NO: 22 Noggin reverse primer (nucleotide sequence) SEQ ID NO: 23 Noggin nucleotide sequence for signal peptide SEQ ID NO: 24 Noggin amino acid sequence for signal peptide SEQ ID NO: 25 Noggin nucleotide sequence for mature peptide SEQ ID NO: 26 Noggin amino acid sequence for mature peptide SEQ ID NO: 27 Noggin nucleotide sequence for signal peptide + mature peptide WO 2007/028212 PCT/AU2006/001325 -87 Sequence Identifier Sequence SEQ ID NO: 28 Noggin amino acid sequence for signal peptide + mature peptide SEQ ID NO: 29 Noggin-Fc nucleotide sequence for whole construct (signal peptide + mature peptide + linker IgG1 Fc) SEQ ID NO: 30 Noggin-Fc amino acid sequence for whole construct (signal peptide + mature peptide + linker IgGI Fc) SEQ ID NO: 31 Noggin-Fc nucleotide sequence for whole construct (signal peptide + mature peptide + linker IgG1 Fc(variant)) SEQ ID NO: 32 Noggin-Fc amino acid sequence for whole construct (signal peptide + mature peptide + linker IgG1 Fc (variant)) SEQ ID NO: 33 Human IgG1 Fc forward primer (nucleotide sequence) SEQ ID NO: 34 Human IgG1 Fc reverse primer (nucleotide sequence) WO 2007/028212 PCT/AU2006/001325 - 88 TABLE 2 List of physiochemical parameters. P, Physiochemical Parameter Noggin PI Molecular weight 23 to 35 kDa
P
2 Isoelectric point (pI) 8 to 14
P
3 Number of isoforms 2 to 16
P
4 Relative intensities of the different number of isoforms
P
5 Percentage by weight carbohydrate 0 to 35%
P
6 Observed molecular weight following N- 23 to 29 kDa linked oligosaccharides deglycosylation
P
7 Observed molecular weight following N- 23 to 27 kDa linked oligosaccharide deglycosylation and 0 linked oligosaccharide deglycosylation Ps Percentage acidic monosaccharide content
P
9 Monosaccharide content
P
1 0 Sialic acid content PH Sulfate and phosphate content
P
1 2 Ser/Thr : GalNAc ratio
P
1 3 Neutral percentage of N-linked oligosaccharide content
P
1 4 Acidic percentage of N-linked oligosaccharide content
P
15 Neutral percentage of 0-linked oligosaccharide content
P
16 Acidic percentage of 0-linked oligosaccharide content
P
1 7 Ratio of N-linked oligosaccharides
P
18 Ratio of 0-linked oligosaccharides WO 2007/028212 PCT/AU2006/001325 - 89 P Physiochemical Parameter Noggin Pi 9 Structure of N-linked fraction Comprises one or more N glycan structures listed in Table 9
P
20 Structure of O-linked fraction Comprises one or more N glycan structures listed in Table 10
P
21 Position and make up of N-linked Includes N-62, numbered oligosaccharides from the start of the signal sequence.
P
22 Position and make up of O-linked oligosaccharides
P
23 Co-translational modification
P
2 4 Post-translational modification
P
2 s Acylation
P
26 Acetylation
P
27 Amidation
P
28 Deamidation
P
29 Biotinylation
P
30 Carbamylation or carbamoylation
P
31 Carboxylation
P
32 Decarboxylation
P
33 Disulfide bond formation
P
34 Fatty acid acylation
P
35 Myristoylation
P
36 Palmitoylation
P
37 Stearoylation
P
38 Formylation
P
39 Glycation
P
40 Glycosylation
P
4 1 Glycophosphatidylinositol anchor WO 2007/028212 PCT/AU2006/001325 -90 Px Physiochemical Parameter Noggin
P
42 Hydroxylation
P
43 Incorporation of selenocysteine
P
44 Lipidation
P
4 s Lipoic acid addition
P
46 Methylation
P
47 N or C terminal blocking
P
48 N or C terminal removal
P
49 Nitration
P
5 o Oxidation of methionine
P
51 Phosphorylation
P
52 Proteolytic cleavage
P
5 3 Prenylation
P
54 Farnesylation
P
55 Geranyl geranylation
P
5 6 Pyridoxal phosphate addition
P
5 7 Sialylation
P
58 Desialylation
P
5 9 Sulfation
P
60 Ubiquitinylation or ubiquitination
P
6 1 Addition of ubiquitin-like molecules
P
62 Primary structure
P
63 Secondary structure
P
6 4 Tertiary structure
P
65 Quaternary structure
P
66 Chemical stability
P
67 Thermal stability WO 2007/028212 PCT/AU2006/001325 -91 TABLE 3 List of Pharmacological traits. Ty Pharmacological trait Noggin Ti Therapeutic efficiency
T
2 Effective therapeutic dose (TCID 5 o)
T
3 Bioavailability T4 Time between dosages to maintain therapeutic levels
T
5 Rate of absorption T6 Rate of excretion T7 Specific activity
T
8 Thermal stability
T
9 Lyophilization stability Tio Serum/plasma stability Til Serum half-life T12 Solubility in blood stream
T
1 3 Immunoreactivity profile Distinct from that of a mammalian noggin expressed in non-human system. T14 Immunogenicity T14A Inhibitable by neutralizing antibodies
T
15 Side effects
T
16 Receptor/ligand binding affinity
T
17 Receptor/ligand activation Ti8 Tissue or cell type specificity
T
19 Ability to cross biological membranes or barriers (i.e. gut, lung, blood brain barriers, skin etc) WO 2007/028212 PCT/AU2006/001325 - 92 Pharmacological trait Noggin T20 Tissue uptake
T
21 Stability to degradation T22 Stability to freeze-thaw
T
23 Stability to proteases T24 Stability to ubiquitination
T
25 Ease of administration T26 Mode of administration T27 Compatibility with other pharmaceutical excipients or carriers
T
28 Persistence in organism or environment
T
29 Stability in storage T30 Toxicity in an organism or environment and the like
T
3 1 Altered biological effects on A greater ability of noggin of the different cells types present invention to inhibit BMP-4 mediated alkaline phosphatise production of MC3T3 pre osteoblast cells than that of a human noggin expressed in E. coli cells
T
32 Proliferation
T
33 Differentiation T34 Apoptosis
T
35 Growth in cell size
T
36 Cytokine adhesion
T
37 Cell adhesion
T
38 Cell spreading
T
39 Cell motility
T
40 Migration and invasion WO 2007/028212 PCT/AU2006/001325 - 93 Ty Pharmacological trait Noggin T41 Chemotaxis T42 Cell engulfment
T
43 Signal transduction T44 Recruitment of proteins to receptors/ligands
T
4 5 Activation of the JAK/STAT pathway T46 Activation of the Ras-erk pathway T47 Activation of the AKT pathway
T
48 Activation of the PKC pathway and PKA pathway T49 Activation of the PKA pathway
T
5 o Activation of src
T
51 Activation of fas
T
5 2 Activation of TNFR
T
53 Activation of NFkB T54 Activation of p38MAPK
T
55 Activation of c-fos T56 Secretion
T
57 Receptor internalization T58 Receptor cross-talk T59 Up or down regulation of surface markers T60 Alteration of FACS front/side scatter profiles
T
6 1 Alteration of subgroup ratios T62 Differential gene expression T63 Cell necrosis T64 Cell clumping
T
65 Cell repulsion WO 2007/028212 PCT/AU2006/001325 - 94 T Pharmacological trait Noggin
T
66 Binding to heparin sulfates
T
67 Binding to glycosylated structures
T
68 Binding to chondroitin sulfates
T
69 Binding to extracellular matrix (such as collagen, fibronectin)
T
70 Binding to artificial materials (such as scaffolds)
T
71 Binding to carriers T72 Binding to co-factors
T
73 The effect alone or in combination A greater ability of noggin of the with other proteins on stem cell present invention to inhibit BMP-4 proliferation, differentiation and/or mediated expansion of CD34* self-renewal. human hematopoietic progenitor cells than that of a human noggin expressed in E. coli. A list of abbreviations commonly used herein is provided in Tables 4 and 5. TABLE 4 5 Abbreviations Abbreviation Description AAA Amino Acid Analysis AFC Affinity Chromatography bFGF Basic Fibroblast Growth Factor, FGF2 BSA Bovine Serum Albumin cDLC Combinatorial Dye Ligand Chromatography CSF Colony Stimulating Factor DCS Donor Calf Serum DeoxGlc 2-deoxyglucose DLC Dye Ligand pseudoaffinity Chromatography WO 2007/028212 PCT/AU2006/001325 - 95 Abbreviation Description DSC Differential Scanning Calorimetry EGF Epidermal Growth Factor ELISA Enzyme-Linked Immunosorbent Assays EPO Erythropoietin Fc Fragment Crystallizable or Immunoglobulin constant region FCS Fetal Calf Serum FGF2 Basic Fibroblast Growth Factor, bFGF FTIS Fourier Transform Infrared Spectroscopy Fuc Fucose G-CSF Granulocyte Colony Stimulating Factor Gal Galactose GalNAc, galactosamine 2-deoxy, 2 amino galactose GFC Gel Filtration Chromatography GlcA Glucuronic acid GlcNAc, glucosamine 2-deoxy, 2 amino glucose Glc Glucose GM-CSF Granulocyte-Macrophage Colony Stimulating Factor HBS Hepes Buffered Saline hES Human Embryonic Stem Cells HIC Hydrophobic Interaction Chromatography HPAEC-PAD High-pH anion-exchange chromatography with pulsed amperometric detection HPLC High Pressure Liquid Chromatography or High Performance Liquid Chromatography HSA Human Serum Albumin HTS High Throughput Screening IdoA Iduronic acid IEC Ion Exchange Chromatography IEF Isoelectric focussing IFN Interferon Ig Immuno globulin WO 2007/028212 PCT/AU2006/001325 - 96 Abbreviation Description IL Interleukin LC Liquid Chromatography MALDI-TOF Matrix-Assisted Laser Desorption Ionization - Time of Flight Man Mannose MCC Metal Chelating Chromatography MS Mass Spectroscopy NacSial, NeuAc or N-acetyl neuraminic acid NeuNAc NGlySial, NeuGc or N-glycolyl neuraminic acid NeuGly OPD O-phenylenediamine PBS Phosphate Buffered Saline PCS Photon Correlation Spectroscopy PDGF-AA Platelet Derived Growth Factor A homodimer PNGase Peptide-N4-(N-acetyl-p-D-glucosaminyl) Asparagine Amidase RMLP Receptor Mediated Ligand Chromatography RPC Reversed Phase Chromatography SDS PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SEC Size Exclusion Chromatography Sia Sialic acid TCA Trichloroacetic acid TFF Tangential flow filtration TGF Transforming Growth Factor TNF Tumor Necrosis Factor TNFR Tumor Necrosis Factor Receptor Xyl Xylose WO 2007/028212 PCT/AU2006/001325 - 97 TABLE 5 Abbreviations for amino acids Amino Acid 3 Letter 1 Letter Code Code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V WO 2007/028212 PCT/AU2006/001325 - 98 TABLE 6 Stem cell list Cell type General Stem Cell Types Embryonic stem cells Somatic stem cells Germ stem cells Human embryonic stem cells (hES) Human epidermal stem cells Adipose derived stem cells Brain Adult neural stem cells Human neurons Human astrocytes Human oligodendrocytes Epidermis Human keratinocyte stem cells Human keratinocyte transient amplifying cells Human melanocyte stem cells Human melanocytes Skin Human foreskin fibroblasts Pancreas Human duct cells Human pancreatic islets Human pancreatic p-cells Kidney Human adult renal stem cells Human embryonic renal epithelial stem cells Human kidney epithelial cells Liver WO 2007/028212 PCT/AU2006/001325 - 99 Cell type Human hepatic oval cells Human hepatocytes Human bile duct epithelial cells Human embryonic endodermal stem cells Human adult hepatocyte stem cells ( existence controversial) Breast Human mammary epithelial stem cells Lung Bone marrow-derived stem cells Human lung fibroblasts Human bronchial epithelial cells Human alveolar type II pneumocytes Muscle Human skeletal muscle stem cells (satellite cells) Heart Human cardiomyocytes Bone marrow mesenchymal stem cells Simple Squamous Epithelial cells Descending Aortic Endothelial cells Aortic Arch Endothelial cells Aortic Smooth Muscle cells Eye Limbal stem cells Comeal epithelial cells Bone-related CD34+ hematopoietic stem cells Mesenchymal stem cells Osteoblasts (precursor is mesenchymal stem cell) Peripheral blood mononuclear progenitor cells (hematopoietic stem cells) Osteoclasts (precursor is above cell type) Stromal cells WO 2007/028212 PCT/AU2006/001325 -100 Cell type Spleen Human splenic precursor stem cells Human splenocytes Immune cells Human CD4+ T-cells Human CD8+ T-cells Human NK cells Human monocytes Human macrophages Human dendritic cells Human B-cells Nose Goblet cells (mucus secreting cells of the nose) Pseudostriated ciliated columnar cells (located below olfactory region in the nose) Pseudostratified ciliated epithelium (cells that line the nasopharangeal tubes) Trachea Stratified Epithelial cells (cells that line and structure the trachea) Ciliated Columnar cells (cells that line and structure the trachea) Goblet cells (cells that line and structure the trachea) Basal cells (cells that line and structure the trachea) Oesophagus Cricopharyngeus muscle cells Reproduction Female primary follicles Male spermatogonium WO 2007/028212 PCT/AU2006/001325 - 101 BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a diagrammatic representation of the cloning process for inserting cDNA encoding noggin into the pIRESbleo3 or pIRESbleo3-Fc vector. 5 Figure 2(a) is a graphical representation showing the LC-MS Base Peak Chromatogram of N-glycans released from the noggin of the present invention. Figure 2(b) is a graphical representation showing the LC-MS Base Peak Chromatogram of 10 0-glycans released from the noggin of the present invention. Figure 3(a) is a graphical representation showing the MS Scan of N-glycans of noggin of the present invention. 15 Figure 3(b) is a graphical representation showing the MS Scan of 0-glycans of noggin of the present invention. Figure 4(a) is a graphical representation showing the MS/MS spectra of the N-glycan ion 1079 present in the noggin of the present invention. 20 Figure 4(b) is a graphical representation showing the MS/MS spectra of the N-glycan ion 986 present in the noggin of the present invention. Figure 4(c) is a graphical representation showing the MS/MS spectra of the N-glycan ion 25 1183 present in the noggin of the present invention. Figure 4(d) is a graphical representation showing the MS/MS spectra of the 0-glycan ion 966 present in the noggin of the present invention. 30 Figure 4(e) is a graphical representation showing the MS/MS spectra of the 0-glycan ion 1331 present in the noggin of the present invention.
WO 2007/028212 PCT/AU2006/001325 - 102 Figure 5(a) is a graphical representation comparing the ability of noggin of the present invention and human noggin expressed using non-human systems in the inhibition of BMP-4 mediated alkaline phosphatase production in MC3T3 pre-osteoblast cells. BMP-4 only (crosses); BMP-4 + recombinant human noggin expressed in E. coli (filled triangles); 5 BMP-4 + noggin of the present invention (filled diamonds); noggin of the present invention only (filled squares); recombinant human noggin expressed in E. coli only (open triangles) cells only (open circles). Figure 5(b) is a bar chart comparing the abilities of Noggin of the present invention and 10 recombinant human noggin expressed in E.coli to inhibit a BMP-4 mediated expansion of CD34+ human hematopoietic progenitor cells after 4 days of culture. Error bars represent standard deviations. Figure 6 is a graphical representation showing the in vitro comparison of 15 immunoreactivity profiles between noggin of the present invention and both human and mouse noggin expressed using non-human systems. OD-concentration plots for the noggin of the present invention (open squares), a recombinant mouse noggin expressed in mouse NSO cells (filled triangles), a recombinant human noggin expressed in E. coli (open circles) and a no noggin control (filled circles). Error bars represent standard error of the mean. 20 WO 2007/028212 PCT/AU2006/001325 - 103 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It is to be understood that unless otherwise indicated, the subject invention is not limited to specific formulations, manufacturing methods, diagnostic methods, assay protocols, 5 nutritional protocols, or research protocols or the like as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the subject specification, the singular forms "a", "an" and 10 "the" include plural aspects unless the context already dictates otherwise. Thus, for example, reference to "a protein", "a cytokine" or "a chimeric molecule" or "a receptor" includes a single protein, cytokine or receptor or chimeric molecule as well as two or more proteins, cytokines or receptors or chimeric molecules; a "physiochemical parameter" includes a single parameter as well as two or more parameters and so forth. 15 The terms "compound", "active agent", "chemical agent", "pharmacologically active agent", "medicament", "active" and "drug" are used interchangeably herein to refer to a chemical compound and in particular a noggin or chimeric noggin molecule that induces a desired pharmacological and/or physiological effect. The terms also encompass 20 pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "compound", "active agent", "chemical agent" "phannacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that this includes the active agent per se as well 25 as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. Reference to a "compound", "active agent", "chemical agent" "pharmacologically active agent", medicamentt", "active" and "drug" includes combinations of two or more actives 30 such as two or more cytokines. A "combination" also includes multi-part such as a two part composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation.
WO 2007/028212 PCT/AU2006/001325 -104 For example, a multi-part pharmaceutical pack may have two or more noggins or chimeric noggin molecules separately maintained. 5 The terms "effective amount" and "therapeutically effective amount" of an agent as used herein mean a sufficient amount of the noggin or chimeric noggin molecule alone or in combination with other agents to provide the desired therapeutic or physiological effect or outcome. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the 10 potential risks in determining what is an appropriate "effective amount". The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using only routine 15 experimentation. By "pharmaceutically acceptable" carrier, excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without 20 causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like. Similarly, a "pharmacologically acceptable" salt, ester, amide, prodrug or derivative of a 25 compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable. The terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms of the condition being treated, elimination of symptoms and/or 30 underlying cause, prevention of the occurrence of symptoms of the condition and/or their underlying cause and improvement or remediation or amelioration of damage following a condition.
WO 2007/028212 PCT/AU2006/001325 - 105 "Treating" a subject may involve prevention of a condition or other adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by ameliorating the symptoms of the condition. 5 A "subject" as used herein refers to an animal, in a particular embodiment, a mammal and in a further embodiment, human who can benefit from the pharmaceutical formulations and methods of the present invention. There is no limitation on the type of animal that could benefit from the presently described pharmaceutical formulations and methods. A 10 subject regardless of whether a human or non-human animal may be referred to as an individual, patient, animal, host or recipient. The compounds and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry. 15 As indicated above, in a particular embodiment, animals are humans or other primates such as orangutans, gorillas, marmosets, livestock animals, laboratory test animals, companion animals or captive wild animals, as well as avian species. Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. 20 Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model. Livestock animals include sheep, cows, pigs, goats, horses and donkeys. Non-mammalian animals such as avian species, fish, and amphibians including Xenopus spp prokaryotes and non-mammalian eukaryotes. 25 The term "cytokine" is used in its most general sense and includes any of various proteins secreted by cells to regulate the immune system, modulate the functional activities of individual cells and/or tissues, and/or induce a range of physiological responses. As used herein the term "cytokine" should be understood to refer to a "complete" cytokine as well as fragments, derivatives or homologs or chimeras thereof comprising one or more amino 30 acid additions, deletions or substitutions, but which substantially retain the biological activity of the complete cytokine. A "cytokine receptor" is a cell membrane associated or soluble portion of the cytokine receptor involved in cytokine signalling or regulation. As WO 2007/028212 PCT/AU2006/001325 - 106 used herein the term "cytokine receptor" should be understood to refer to a "complete" cytokine receptor as well as fragments, derivatives or homologs or chimeras thereof comprising one or more amino acid additions, deletions or substitutions, but which substantially retain the biological activity of the complete cytokine receptor. The term 5 "protein" is used in its most general sense and includes cytokines and cytokine receptors. As used herein, the term "protein" should be understood to refer to a "complete" protein as well as fragments, derivatives or homologs or chimeras thereof comprising one or more amino acid additions, deletions or substitutions, but which substantially retain the biological activity of the complete protein. 10 The present invention contemplates an isolated noggin or chimeric noggin molecule having a profile of measurable physiochemical parameters (Px), wherein the profile is indicative of, associated with or forms the basis of one or more distinctive pharmacological traits (Ty). As used herein, the term noggin includes reference to the whole polypeptide as well 15 as fragments thereof. More particularly, the present invention provides an isolated noggin or chimeric noggin molecule having a physiochemical profile comprising an array of measurable physiochemical parameters, {[Px]i, [Px] 2 ,...[Px]n,}, wherein P, represents a measurable 20 physiochemical parameter and "n" is an integer >1, wherein each of [P] 1 to [Px]n is a different measurable physiochemical parameter, wherein the value of any one or more of the measurable physiochemical characteristics is indicative of, associated with, or forms the basis of, a distinctive pharmacological trait, Ty, or a number of distinctive pharmacological traits {[Ty] 1 , [Ty] 2 , ....[Ty]m} wherein Ty represents a distinctive 25 pharmacological trait and m is an integer 1 and each of [Ty]1 to [Ty]m is a different pharmacological trait. As used herein, the term "measurable physiochemical parameters" (Px) refers to one or more measurable characteristics of an isolated noggin or chimeric noggin molecule. 30 Exemplary "distinctive measurable physiochemical parameters" include, but are not limited to molecular weight (P 1 ), isoelectric point (pI) (P 2 ), number of isoforms (P 3 ), relative intensities of the different number of isoforms (P 4 ), percentage by weight WO 2007/028212 PCT/AU2006/001325 - 107 carbohydrate (P 5 ), observed molecular weight following N-linked oligosaccharides deglycosylation (P 6 ), observed molecular weight following N and 0-linked oligosaccharides deglycosylation (P 7 ), percentage acidic monosaccharide content (P 8 ), monosaccharide content (P 9 ), sialic acid content (Pio), sulfate and phosphate content (Pi), 5 Ser/Thr:GalNAc ratio (P 12 ), neutral percentage of N-linked oligosaccharide content (P 13 ), acidic percentage of N-linked oligosaccharide content (P 14 ), neutral percentage of O-linked oligosaccharide content (P 15 ), acidic percentage of O-linked oligosaccharide content (P 16 ), ratio of N-linked oligosaccharides (P 17 ), ratio of 0-linked oligosaccharides (Pis), structure of N-linked oligosaccharide fraction (P 19 ), structure of O-linked oligosaccharide fraction 10 (P 20 ), position and make up of N-linked oligosaccharides (P 2 1), position and makeup of 0 linked oligosaccharides (P 22 ), co-translational modification (P 23 ), post-translational modification (P 24 ), acylation (P 25 ), acetylation (P 2 6 ), amidation (P 27 ), deamidation (P 28 ), biotinylation (P 29 ), carbamylation or carbamoylation (P 30 ), carboxylation (P 3 1), decarboxylation (P32), disulfide bond formation (P 33 ), fatty acid acylation (P34), 15 myristoylation (P 35 ), palmitoylation (P36), stearoylation (P 37 ), formylation (P 38 ), glycation (P39), glycosylation (P 40 ), glycophosphatidylinositol anchor (P 4 1), hydroxylation (P 42 ), incorporation of selenocysteine (P 43 ), lipidation (P44), lipoic acid addition (P45), methylation (P46), N or C terminal blocking (P 47 ), N or C terminal removal (P48), nitration (P49), oxidation of methionine (P 50 ), phosphorylation (P 5 ), proteolytic cleavage (P52), 20 prenylation (P53), farnesylation (P 5 4 ), geranyl geranylation (P 55 ), pyridoxal phosphate addition (P56), sialylation (P57), desialylation (P 58 ), sulfation (P59), ubiquitinylation or ubiquitination (P 60 ), addition of ubiquitin-like molecules (P 61 ), primary structure (P62), secondary structure (P 63 ), tertiary structure (P 64 ), quaternary structure (P65), chemical stability (P 66 ), thermal stability (P 67 ). A summary of these parameters is provided is Table 25 2. The term "distinctive pharmacological traits" would be readily understood by one of skill in the art to include any pharmacological or clinically relevant property of the noggin or chimeric noggin molecule of the present invention. Exemplary "pharmacological traits" 30 which in no way limit the invention include: therapeutic efficiency (Ti), effective therapeutic dose (TCID 5 o) (T 2 ), bioavailability (T 3 ), time between dosages to maintain therapeutic levels (T 4 ), rate of absorption (T 5 ), rate of excretion (T 6 ), specific activity (T 7
),
WO 2007/028212 PCT/AU2006/001325 - 108 thermal stability (T 8 ), lyophilization stability (T 9 ), serum/plasma stability (Tio), serum half life (Ti I), solubility in blood stream (T 1 2 ), immunoreactivity profile (Ti 3 ), immunogenicity (T1 4 ), inhibition by neutralizing antibodies (T14A), side effects (Ti 5 ), receptor/ligand binding affinity (T 1 6 ), receptor/ligand activation (T1 7 ), tissue or cell type specificity (Ti 8 ), 5 ability to cross biological membranes or barriers (i.e. gut, lung, blood brain barriers, skin etc) (T1 9 ), tissue uptake (T 20 ), stability to degradation (T 2 1 ), stability to freeze-thaw (T 22 ), stability to proteases (T 23 ), stability to ubiquitination (T 24 ), ease of administration (T 25 ), mode of administration (T 26 ), compatibility with other pharmaceutical excipients or carriers (T 27 ), persistence in organism or environment (T 28 ), stability in storage (T 29 ), 10 toxicity in an organism or environment and the like (T 30 ). In addition, the noggin or chimeric noggin molecule of the present invention may have altered biological effects on different cells types (T 3 1), including but not limited to human primary cells, such as lymphocytes, erythrocytes, retinal cells, hepatocytes, neurons, 15 keratinocytes, endothelial cells, endodermal cells, ectodermal cells, mesodermal cells, epithelial cells, kidney cells, liver cells, bone cells, bone marrow cells, lymph node cells, dermal cells, fibroblasts, T-cells, B-cells, plasma cells, natural killer cells, macrophages, neutrophils, granulocytes Langerhans cells, dendritic cells, eosinophils, basophils, mammary cells, lobule cells, prostate cells, lung cells, oesophageal cells, pancreatic cells, 20 Beta cells (insulin secreting cells), hemangioblasts, muscle cells, oval cells (hepatocytes), mesenchymal cells, brain microvessel endothelial cells, astrocytes, glial cells, various stem cells including adult and embryonic stem cells, various progenitor cells; and other human immortal, transformed or cancer cell lines. The biological effects on the cells include effects on proliferation (T 32 ), differentiation (T 33 ), apoptosis (T 34 ), growth in cell size (T 35 ), 25 cytokine adhesion (T 36 ), cell adhesion (T 37 ), cell spreading (T 38 ), cell motility (T 39 ), migration and invasion (T 40 ), chemotaxis (T 41 ), cell engulfment (T 42 ), signal transduction (T43), recruitment of proteins to receptors/ligands (T 44 ), activation of the JAK/STAT pathway (T 4 5 ), activation of the Ras-erk pathway (T 46 ), activation of the AKT pathway
(T
47 ), activation of the PKC pathway (T48), activation of the PKA pathway (T49), activation 30 of src (T 50 ), activation of fas (T 51 ), activation of TNFR (T 52 ), activation of NFkB (T 53 ), activation of p38MAPK (T54), activation of c-fos (T 55 ), secretion (T 56 ), receptor internalization (T 57 ), receptor cross-talk (T 58 ), up or down regulation of surface markers WO 2007/028212 PCT/AU2006/001325 -109
(T
5 9 ), alteration of FACS front/side scatter profiles (T 60 ), alteration of subgroup ratios
(T
61 ), differential gene expression (T 62 ), cell necrosis (T 63 ), cell clumping (T 64 ), cell repulsion (T 65 ), binding to heparin sulfates (T 66 ), binding to glycosylated structures (T 67 ), binding to chondroitin sulfates (T 68 ), binding to extracellular matrix (such as collagen, 5 fibronectin) (T 69 ), binding to artificial materials (such as scaffolds) (T 7 0 ), binding to carriers (T 71 ), binding to co-factors (T 72 ), the effect alone or in combination with other proteins on stem cell proliferation, differentiation and/or self-renewal (T 73 ) and the like. A summary of these traits is provided in Table 3. 10 As used herein the term "distinctive" with regard to a pharmacological trait of a noggin or chimeric noggin molecule of the present invention refers to one or more pharmacological traits of the noggin or chimeric noggin molecule which are distinctive for the particular physiochemical profile. In a particular embodiment, one or more of the pharmacological traits of the isolated noggin or chimeric noggin molecule is different from, or distinctive 15 relative to a form of the same noggin or chimeric noggin molecule produced in a prokaryotic or lower eukaryotic cell or even a higher non-human eukaryotic cell. In a particular embodiment, the pharmacological traits of the subject isolated noggin or chimeric noggin molecule are substantially similar to or functionally equivalent to a naturally occurring protein. 20 As used herein the term "prokaryote" refers to any prokaryotic cell, which includes any bacterial cell (including actinobacterial cells) or archaeal cell. The meaning of the term "non-mammalian eukaryote", as used herein is self-evident. However, for clarity, this term specifically includes any non-mammalian eukaryote including: yeasts such as 25 Saccharomyces spp. or Pichea spp.; other fungi; insects, including Drosophila spp. and insect cell cultures; fish, including Danio spp.; amphibians, including Xenopus spp.; plants and plant cell cultures. Reference to a "stem cell" includes embryonic or adult stem cells and includes those stem 30 cells listed in Table 6. A noggin or chimeric noggin molecule of the present invention may be used alone or in a cocktail of proteins to induce one or more of stem cell proliferation, differentiation or self-renewal.
WO 2007/028212 PCT/AU2006/001325 -110 Primary structure of a noggin or chimeric noggin molecule may be measured as an amino acid sequence. Secondary structure may be measured as the number and/or relative position of one or more protein secondary structures such as a-helices, parallel p-sheets, 5 antiparallel p-sheets or turns. Tertiary structure describes the folding of the polypeptide chain to assemble the different secondary structure elements in a particular arrangement. As helices and sheets are units of secondary structure, so the domain is the unit of tertiary structure. In multi-domain proteins, tertiary structure includes the arrangement of domains relative to each other. Accordingly, tertiary structure may be measured as the presence, 10 absence, number and/or relative position of one or more protein "domains". Exemplary domains which in no way limit the present invention include: lone helices, helix-turn-helix domains, four helix bundles, DNA binding domains, three helix bundles, Greek key helix bundles, helix-helix packing domains, p-sandwiches, aligned p-sandwiches, orthogonal p sandwiches, p-barrels, up and down antiparallel p-sheets, Greek key topology domains, 15 jellyroll topology domains, p-propellers, p-trefoils, p-Helices, Rossman folds, a/p horseshoes, a/p barrels, a+p topologies, disulphide rich folds, serine proteinase inhibitor domains, sea anemone toxin domains, EGF-like domains, complement C-module domain, wheat plant toxin domains, Naja (Cobra) neurotoxin domains, green mamba anticholinesterase domains, Kringle domains, mucin like region, globular domains, spacer 20 regions. Quaternary structure is described as the arrangement of different polypeptide chains within the protein structure, with each chain possessing individual primary, secondary and tertiary structure elements. Examples include either homo- or hetero oligomeric multimerization (e.g. dimerization or trimerization). 25 With respect to the primary structure, the present invention provides an isolated noggin or chimeric noggin molecule, or a fragment thereof, encoded by a nucleotide sequence selected from the list consisting of SEQ ID NOs: 23, 25, 27, 29, 31, or a nucleotide sequence having at least about 90% identity to any one of the above-listed sequence or a nucleotide sequence capable of hybridizing to any one of the above sequences or their 30 complementary forms under low stringency conditions.
WO 2007/028212 PCT/AU2006/001325 - 111 Still, another aspect of the present invention provides an isolated nucleic acid molecule encoding noggin or chimeric noggin molecule or a functional part thereof comprising a sequence of nucleotides having at least 90% similarity selected from the list consisting of SEQ ID NOs: 23, 25, 27, 29, 31, or after optimal alignment and/or being capable of 5 hybridizing to one or more of SEQ ID NOs: 23, 25, 27, 29, 31, or their complementary forms under high stringency conditions. In a particular embodiment, the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a noggin or chimeric noggin 10 molecule, or a fragment thereof, an amino acid sequence substantially as set forth in one or more of SEQ ID NOs: 24, 26, 28, 30, 32, or an amino acid sequence having at least about 90% similarity to one or more of SEQ ID NOs: 24, 26, 28, 30, 32, after optimal alignment. In another aspect, the present invention provides an isolated nucleic acid molecule 15 encoding a noggin, or a fragment thereof, comprising a sequence of nucleotides selected from the group consisting of SEQ ID NOs: 23, 25, 27, linked directly or via one or more nucleotide sequences encoding protein linkers known in the art to nucleotide sequences encoding the constant (Fc) or framework region of human immunoglobulin, substantially as set forth in one or more of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19. 20 In another aspect, the present invention provides an isolated noggin, or a fragment thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 26, 28, linked directly or via one or more protein linkers known in the art, to the constant (Fc) or framework region of human immunoglobulin, substantially as set forth in 25 one or more of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20. Another aspect of the present invention provides an isolated noggin or chimeric noggin molecule, or a fragment thereof, comprising an amino acid sequence selected from the list consisting of SEQ ID NOs: 24, 26, 28, 30, 32, or an amino acid sequence having at least 30 about 90% similarity to one or more of the above sequences.
WO 2007/028212 PCT/AU2006/001325 - 112 In a particular embodiment, percentage amino acid similarity or nucleotide identity levels include at least about 61% or at least about 62% or at least about 63% or at least about 64% or at least about 65% or at least about 66% or at least about 67% or at least about 68% or at least about 69% or at least about 70% or at least about 71% or at least about 5 72% or at least about 73% or at least about 74% or at least about 75% or at least about 76% or at least about 77% or at least about 78% or at least about 79% or at least about 80% or at least about 81% or at least about 82% or at least about 83% or at least about 84% or at least about 85% or at least about 86% or at least about 87% or at least about 88% or at least about 89% or at least about 90% or at least about 91% or at least about 10 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% similarity or identity. A "derivative" of a polypeptide of the present invention also encompasses a portion or a 15 part of a full-length parent polypeptide, which retains partial transcriptional activity of the parent polypeptide and includes a variant. Such "biologically-active fragments" include deletion mutants and small peptides, for example, for at least 10, in a particular embodiment at least 20 and in a further embodiment at least 30 contiguous amino acids, which exhibit the requisite activity. Peptides of this type may be obtained through the 20 application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard which is included in a publication entitled "Synthetic Vaccines" edited by Nicholson and published by Blackwell Scientific 25 Publications. Alternatively, peptides can be produced by digestion of an amino acid sequence of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. Any such fragment, irrespective of its means of generation, is to be understood as being encompassed by the term 30 "derivative" as used herein.
WO 2007/028212 PCT/AU2006/001325 - 113 The term "variant" refers, therefore, to nucleotide sequences displaying substantial sequence identity with reference nucleotide sequences or polynucleotides that hybridize with a reference sequence under stringency conditions that are defined hereinafter. The terms "nucleotide sequence", "polynucleotide" and "nucleic acid molecule" may be used 5 herein interchangeably and encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference nucleotide sequence whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide or 10 the encoded polypeptide. The term "variant" also includes naturally occurring allelic variants. The nucleic acid molecules of the present invention may be in the form of a vector or other nucleic acid construct. 15 In one embodiment, the vector is DNA and may optionally comprise a selectable marker. Examples of selectable markers include genes conferring resistance to compounds such as antibiotics, genes conferring the ability to grow on selected substrates, genes encoding 20 proteins that produce detectable signals such as luminescence. A wide variety of such markers are known and available, including, for example, antibiotic resistance genes such as the neomycin resistance gene (neo) and the hygromycin resistance gene (hyg). Selectable markers also include genes conferring the ability to grown on certain media substrates such as the tk gene (thymidine kinase) or the hprt gene (hypoxanthine 25 phosphoribosyltransferase) which confer the ability to grow on HAT medium (hypoxanthine, aminopterin and thymidine); and the bacterial gpt gene (guanine/xanthine phosphoribosyltransferase) which allows growth on MAX medium (mycophenolic acid, adenine and xanthine). Other selectable markers for use in mammalian cells and plasmids carrying a variety of selectable markers are described in Sambrook et al. Molecular 30 Cloning - A Laboratory Manual, Cold Spring Harbour, New York, USA, 1990.
WO 2007/028212 PCT/AU2006/001325 - 114 The selectable marker may depend on its own promoter for expression and the marker gene may be derived from a very different organism than the organism being targeted (e.g. prokaryotic marker genes used in targeting mammalian cells). However, it is preferable to replace the original promoter with transcriptional machinery known to function in the 5 recipient cells. A large number of transcriptional initiation regions are available for such purposes including, for example, metallothionein promoters, thymidine kinase promoters, p-actin promoters, immunoglobulin promoters, SV40 promoters and human cytomegalovirus promoters. A widely used example is the pSV2-neo plasmid which has the bacterial neomycin phosphotransferase gene under control of the SV40 early promoter 10 and confers in mammalian cells resistance to G418 (an antibiotic related to neomycin). A number of other variations may be employed to enhance expression of the selectable markers in animal cells, such as the addition of a poly(A) sequence and the addition of synthetic translation initiation sequences. Both constitutive and inducible promoters may be used. 15 The genetic construct of the present invention may also comprise a 3' non-translated sequence. A 3' non-translated sequence refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is 20 characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon. Accordingly, a genetic construct comprising a nucleic acid molecule of the present 25 invention, operably linked to a promoter, may be cloned into a suitable vector for delivery to a cell or tissue in which regulation is faulty, malfunctioning or non-existent, in order to rectify and/or provide the appropriate regulation. Vectors comprising appropriate genetic constructs may be delivered into target eukaryotic cells by a number of different means well known to those skilled in the art of molecular biology. 30 The term "similarity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, WO 2007/028212 PCT/AU2006/001325 - 115 "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to -each other at the structural, functional, 5 biochemical and/or conformational levels. In a particular embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity. Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", 10 "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide 15 sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 20 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, 25 FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al. (Nucl Acids Res 30 25:389, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (In: Current Protocols in Molecular Biology, John Wiley & Sons Inc. 1994 1998).
WO 2007/028212 PCT/AU2006/001325 The terms "sequence similarity" and "sequence identity" as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. 5 Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched 10 positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software Engineering 15 Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity. Reference herein to a low stringency includes and encompasses from at least about 0 to at 20 least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30*C to about 421C, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42'C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency 25 conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30% and from at least about 0.5 M to at least about 0.9 M salt, such as 0.5, 0.6, 0.7, 0.8 or 0.9 M for hybridization, and at least about 0.5 M to at least about 0.9 M salt, such as 30 0.5, 0.6, 0.7, 0.8 or 0.9 M for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide, such as 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50% and from at WO 2007/028212 PCT/AU2006/001325 - 117 least about 0.01 M to at least about 0.15 M salt, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 and 0.15 M for hybridization, and at least about 0.01 M to at least about 0.15 M salt, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 and 0.15 M for washing conditions. In general, 5 washing is carried out Tm = 69.3 + 0.41 (G+C)% (Marmur and Doty, J Mol Biol 5:109, 1962). However, the Tm of a duplex DNA decreases by 1'C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur J Biochem 46:83, 1974. Formamide is optional in these hybridization conditions. Accordingly, in a particular embodiment, levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 10 0.1% w/v SDS at 25-42'C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20'C to 65 0 C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65'C. As used herein, the terms "co- or post-translational modifications" refer to covalent 15 modifications occurred during or after translation of the peptide chain. Exemplary co- or post-translational modifications include but are not limited to acylation (including acetylation), amidation or deamidation, biotinylation, carbamylation (or carbamoylation), carboxylation or decarboxylation, disulfide bond formation, fatty acid acylation (including myristoylation, palmitoylation and stearoylation), formylation, glycation, glycosylation, 20 hydroxylation, incorporation of selenocysteine, lipidation, lipoic acid addition, methylation, N- or C-terminal blocking, N- or C-terminal removal, nitration, oxidation of methionine, phosphorylation, proteolytic cleavage, prenylation (including farnesylation, geranyl geranylation), pyridoxal phosphate addition, sialylation or desialylation, sulfation, ubiquitinylation (or ubiquitination) or addition of ubiquitin-like proteins. 25 Acylation involves the hydrolysis of the N-terminus initiator methionine and the addition of an acetyl group to the new N-termino amino acid. Acetyl Co-A is the acetyl donor for acylation. 30 Amidation is the covalent linkage of an amide group to the carboxy terminus of a peptide and is frequently required for biological activity and stability of a protein. Deamidation is the hydrolytic removal of an amide group. Deamidation of amide containing amino acid WO 2007/028212 PCT/AU2006/001325 - 118 residues is a rare modification that is performed by the organism to re-arrange the 3D structure and alter the charge ratio/pI. Biotinylation is a technique whereby biotinyl groups are incorporated into molecules, 5 either that catalyzed by holocarboxylase synthetase during enzyme biosynthesis or that undertaken in vitro to visualise specific substrates by incubating them with biotin-labeled probes and avidin or streptavidin that has been linked to any of a variety of substances amenable to biochemical assay. 10 Carbamylation (or carbamoylation) is the transfer of the carbamoyl from a carbamoyl containing molecule (e.g., carbamoyl phosphate) to an acceptor moiety such as an amino group. Carboxylation of glutamic acid residues is a vitamin K dependent reaction that results in 15 the formation of a gamma carboxyglutamic acid (Gla residue). Gla residues within several proteins of the blood-clotting cascade are necessary for biological function of the proteins. Carboxylation can also occur to aspartic acid residues. Disulfide bonds are covalent linkages that form when the thiol groups of two cysteine 20 residues are oxidized to a disulfide. Many mammalian proteins contain disulfide bonds, and these are crucial for the creation and maintenance of tertiary structure of the protein, and thus biological activity. Protein synthesis in bacteria involves formylation and deformylation of N-terminal 25 methionines. This formylation/deformylation cycle does not occur in cytoplasm of eukaryotic cells and is a unique feature of bacterial cells. In addition to the hydroxylation that occurs on glycine residues as part of the amidation process, hydroxylation can also occur in proline and lysine residues catalysed by prolyl and lysyl hydroxylase (Kivirikko et al. FASEB Journal 3:1609-1617, 1989). 30 Glycation is the uncontrolled, non-enzymatic addition of glucose or other sugars to the amino acid backbone of protein.
WO 2007/028212 PCT/AU2006/001325 -119 Glycosylation is the addition of sugar units to the polypeptide backbone and will be discussed in the section below. 5 Hydroxylation is a reaction which is dependent on vitamin C as a co-factor. Adding to the importance of hydroxylation as a post- translation modification is that hydroxy-lysine serves as an attachment site for glycosylation. Selenoproteins are proteins which contain selenium as a trace element by the incorporation 10 of a unique amino acid, selenocysteine, during translation. The tRNA for selenocysteine is charged with serine and then enzymatically selenylated to produce the selenocysteinyl tRNA. The anticodon of selenocysteinyl-tRNA interacts with a stop codon in mRNA (UGA) instead of a serine codon. An element in the 3' non-translated region (UTR) of selenoprotein mRNAs determines whether UGA is read as a stop codon or as a 15 selenocysteine codon. Lipidation is a generic term that encompasses the covalent attachment of lipids to proteins, this includes fatty acid acylation and prenylation. 20 Fatty acid acylation involves the covalent attachment of fatty acids such as the 14 carbon Myristic acid (Myristoylation), the 16 carbon Palmitic acid (Palmitoylation) and the 18 carbon Stearic acid (Stearoylation). Fatty acids are linked to proteins in the pre-Golgi compartment and may regulate the targeting of proteins to membranes (Blenis and Resh Curr Opin Cell Biol 5(6):984-9, 1993). Fatty acid acylation is, therefore, important in the 25 functional activity of a protein (Bernstein Methods Mol Biol 237:195-204, 2004). Prenylation involves the addition of prenyl groups, namely the 15 carbon famesyl or the 20 carbon geranyl-geranyl group to acceptor proteins. The isoprenoid compounds, including farnesyl diphosphate or geranylgeranyl diphosphate, are derived from the cholesterol 30 biosynthetic pathway. The isoprenoid groups are attached by a thioether link to cysteine residues within the consensus sequence CAAX, (where A is any aliphatic amino acid, except alanine) located at the carboxy terminus of proteins. Prenylation enhances proteins WO 2007/028212 PCT/AU2006/001325 -120 ability to associate with lipid membranes and all known GTP-binding and hydrolyzing proteins (G proteins) are modified in this way, making prenylation crucial for signal transduction. (Rando Biochim Biophys Acta 1300(1):5-16, 1996; Gelb et al. Curr Opin Chem Biol 2(1):40-8, 1998). 5 Lipoic acid is a vitamin-like antioxidant that acts as a free radical scavenger. Lipoyl-lysine is formed by attaching lipoic acid through an amide bond to lysine by lipoate protein ligase. 10 Protein methylation is a common modification that can regulate the activity of proteins or create new types of amino acids. Protein methyltransferases transfer a methyl group from S-adenosyl-L-methionine to nucleophilic oxygen, nitrogen, or sulfur atoms on the protein. The effects of methylation fall into two general categories. In the first, the relative levels of methyltransferases and methylesterases can control the extent of methylation at a particular 15 carboxyl group, which in turn regulates the activity of the protein. This type of methylation is reversible. The second group of protein methylation reactions involves the irreversible modification of sulfur or nitrogen atoms in the protein. These reactions generate new amino acids with altered biochemical properties that alter the activity of the protein (Clarke Curr Opin Cell Biol 5:977-983, 1993). 20 Protein nitration is a significant post-translational modification, which operates as a transducer of nitric oxide signalling. Nitration of proteins modulates catalytic activity, cell signalling and cytoskeletal organization. 25 Phosphorylation refers to the addition of a phosphate group by protein kinases. Serine, threonine and tyrosine residues are the amino acids subject to phosphorylation. Phosphorylation is a critical mechanism, which regulates biological activity of a protein. A majority of proteins are also modified by proteolytic cleavage. This may simply 30 involve the removal of the initiation methionine. Other proteins are synthesized as inactive precursors (proproteins) that are activated by limited or specific proteolysis. Proteins destined for secretion or association with membranes (preproteins) are synthesized with a WO 2007/028212 PCT/AU2006/001325 - 121 signal sequence of 12-36 predominantly hydrophobic amino acids, which is cleaved following passage through the ER membrane. Pyridoxal phosphate is a co-enzyme derivative of vitamin B6 and participates in 5 transaminations, decarboxylations, racemizations, and numerous modifications of amino acid side chains. All pyridoxal phosphate-requiring enzymes act via the formation of a Schiff base between the amino acid and coenzyme. Most enzymes responsible for attaching the pyridoxal-phosphate group to the lysine residue are self activating. 10 Sialylation refers to the attachment of sialic acid to the terminating positions of a glycoprotein via various sialyltransferase enzymes; and desialylation refers the removal of sialic acids. Sulfation occurs at tyrosine residues and is catalyzed by the enzyme tyrosylprotein 15 sulfotransferase which occurs in the trans-Golgi network. It has been determined that 1 in 20 of the proteins secreted by HepG2 cells and 1 in 3 of those secreted by fibroblasts contain at least one tyrosine sulfate residue. Sulfation has been shown to influence biological activity of proteins. Of particular interest is that the CCR5, a major HIV co receptor, was shown to be tyrosine-sulfated and that sulfation of one or more tyrosine 20 residues in the N-terminal extracellular domain of CCR5 are required for optimal binding of MIP-lo/CCL3, MIP-1P/CCL4, and RANTES/CCL5 and for optimal HIV co-receptor function (Moore J Biol Chem 278(27):24243-24246, 2003). Sulfation can also occur on sugars. 25 Post-translational modifications can encompass protein-protein linkages. Ubiquitin is a 76 amino acid protein which both self associates and covalently attaches to other proteins in mammalian cells. The attachment is via a peptide bond between the C-terminus of ubiquitin and the amino group of lysine residues in other proteins. Attachment of a chain of ubiquitin molecules to a protein targets it for proteolysis by the proteasome and is an 30 important mechanism for regulating the steady state levels of regulatory proteins e.g. with respect to the cell cycle (Wilkinson Annu Rev Nutr 15:161-89, 1995). In contrast, mono ubiquitination can play a role in direct regulation of protein function. Ubiquitin-like WO 2007/028212 PCT/AU2006/001325 -122 proteins that can also be attached covalently to proteins to influence their function and turnover include NEDD-8, SUMO-1 and Apg12. Glycosylation is the addition of sugar units in the polypeptide backbone. These sugar units 5 can attach to the polypeptide back bones in at least seven ways, namely, (1) via an N-glycosidic bond to the R-group of an asparagine residue in the consensus sequence Asn-X-Ser; Asn-X-Thr; or Asn-X-Cys (N-glycosylation). (2) via an 0-glycosidic bond to the R-group of serine, threonine, hydroxyproline, tyrosine or hydroxylysine (0-glycosylation). 10 (3) via the R-group of tyrosine in C-linked mannose; (4) as a glycophosphatidylinositol anchor used to secure some proteins to cell membranes; (5) as a single monosaccharide attachment of GleNAc to the R-group of serine or threonine. This linkage is often reversibly associated with attachment of inorganic 15 phosphate (Yin-o-Yang); (6) attachment of a linear polysaccharide to serine, threonine or asparagine (proteoglycans); (7) via a S-glycosidic bond to the R-group of cysteine. 20 The glycosylation structure can comprise one or more of the following carbohydrate antigenic determinants in Table 7. TABLE 7 25 List of carbohydrate antigenic determinants Antigenic Name Antigenic Glycan Structure Blood group H(O), Fuc(al-2)Gal(p1-3)GlcNAc-R type 1 Blood group H(O), Fuc(al-2)Gal(Pl-4)GlcNAc-R type 2 Blood group A, type 1 GalNAc(al-3)[Fuc(al-2)]Gal(j1-3)GlcNAc-R Blood group A, type 2 GalNAc(al-3)[Fuc(al-2)]Gal(l-4)GlcNAc-R WO 2007/028212 PCT/AU2006/001325 - 123 Antigenic Name Antigenic Glycan Structure Blood group B, type 1 Gal(al-3)[Fuc(al-2)]Gal(p1-3)GlcNAc-R Blood group B, type 2 Gal(al-3)[Fuc(al-2)Gal(p1 -4)GlcNAc-R Blood group i [Gal(p1-4)GIcNAc(p1-3)].Gal(p1-R Blood group I Gal(p1-4)GlcNAc(p1-3)[Gal(p1-4)GlcNAc(pl 6)]Gal(p1-4)GlcNAc(p1-3)Gal(p1-R Lewis a (Lea) Gal(p1-3)[Fuc(al-4)]GlcNAc-R Sialyl Lewis a (sLea) NeuAc(a2-3)Gal(p1-3)[Fuc(al-4)]GlcNAc-R Lewis b (Leb) Fuc(al -2)Gal(p 1-3) [Fuc(al 1-4)] GlcNAc-R Lewis x (Lex) Gal(P1l-4)[Fuc(al-3)]GlcNAc-R Sialyl Lewis x (sLex) NeuAc(a2-3)Gal(p1-4)[Fuc(al-3)]GlcNAc-R Lewis y (Ley) Fuc(al-2)Gal(p1-4)[Fuc(al-3)]GlcNAc-R Forssman GalNAc(al-3)GalNAc(p1-3)Gal-R Thomsen-Friedenreich Gal(P1-3)GaINAc(al-0)-Ser/Thr (TF or T) Sialyl-TF (sTF) or Gal(p1 -3)[NeuAc(a2-6)]GalNAc(al -0)-Ser/Thr Sialyl-T (sT) Tn GaINAc(al-0)-Ser/Thr Sialyl Tn (sTn) NeuAc(a2-6)GalNAc(al-0)-Ser/Thr The carbohydrates will also contain several antennary structures, including mono, bi, tri and tetra outer structures. 5 Glycosylation may be measured by the presence, absence or pattern of N-linked glycosylation, O-linked glycosylation, C-linked mannose structure, and glycophosphatidylinositol anchor; the percentage of carbohydrate by mass; Ser/Thr GalNAc ratio; the proportion of mono, bi, tri and tetra sugar structures. 10 WO 2007/028212 PCT/AU2006/001325 - 124 The apparent molecular weight of a protein includes all elements of a protein complex (cofactors and non-covalently bonded domains) and all co- or post-translational modifications (addition or removal of covalently bonded groups to and from peptide). Apparent molecular weight is often affected by co- or post-translational modifications. A 5 protein's molecular weight may be determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), which is also the second dimension on its two dimensional counterpart, 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis). It may be determined more accurately, however, by mass spectrometry (MS)- either by Matrix-Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) MS, which 10 produces charged molecular ions or the more sensitive Electrospray Ionization (ESI) MS, which produces multiple-charged peaks. The molecular weights of the noggin or chimeric noggin molecule may be within the range of 1 to 1000 kDa. Accordingly, the isolated noggin or chimeric noggin molecule of the present invention has a molecular weight of 1, 2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 15 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130,131,132,133,134, 135, 136, 20 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149,150,151,152,153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200,201,202,203,204,205,206,207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 25 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 30 317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370, WO 2007/028212 PCT/AU2006/001325 -125 371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 5 443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460, 461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515,516,517,518,519,520,521,522, 523,524,525, 526, 527,528,529,530,531, 532, 10 533,534,535,536,537,538,539,540,541,542, 543, 544, 545,546,547,548,549,550, 551, 552,553,554, 555,556, 557, 558,559,560,561, 562,563,564,565,566,567, 568, 569,570,571,572,573,574, 575, 576, 577,578,579, 580, 581, 582, 583, 584, 585, 586, 587,588, 589,590,591, 592, 593,594, 595,596,597, 598,599,600,601,602,603,604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 15 623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652,653,654,655,656,657,658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 20 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 25 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824,825, 826, 827, 828, 829,830,831, 832,833, 834, 835, 836, 837, 838, 839,840, 841,842,843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854,855, 856, 857, 858, 859,860,861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872,873, 874, 875, 876, 877,878,879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890,891, 892, 30 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, WO 2007/028212 PCT/AU2006/001325 -126 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000 kDa. The molecular weight or molecular mass of a protein may be determined by any 5 convenient means such as electrophoresis, mass spectrometry, gradient ultracentrifugation. The isoelectric point (or pI) of a protein is the pH at which the protein carries no net charge. This attribute may be determined by isoelectric focusing (IEF), which is also the first dimension of 2D-PAGE. Experimentally determined pI values are affected by a range 10 of co- or post-translational modifications and therefore the difference between an experimental pI and theoretical pI may be as high as 5 units. Accordingly, an isolated noggin or chimeric noggin molecule of the present invention may have a pI of 0, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 15 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, or 14.0. 20 As used herein, the term "isoform" includes a glycoform and encompasses a noggin or chimeric noggin molecule having a constant primary structure but differing at the level of secondary or tertiary structure or co-or post-translational modification such as different glycosylation forms. 25 Chemical stability of a protein may be measured as the "half-life" of the protein in a particular solvent or environment. Typically, proteins with a molecular weight of less than 50 kDa have a half-life of approximately 5 to 20 minutes. An isolated noggin or chimeric noggin molecule of the present invention has an in vivo half-life of for about 5 minutes to 30 about 60 minutes such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 minutes. However, isoforms of noggin or WO 2007/028212 PCT/AU2006/001325 -127 chimeric noggin molecule are contemplated having half-life of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours. Another particularly convenient measure of chemical stability is the resistance of a noggin or chimeric noggin molecule to protease digestion, such as trypsin or chymotrypsin digestion. 5 The binding affinity of a noggin or chimeric noggin molecule to its ligand or receptor may be measured as the equilibrium dissociation constant (Kd) or functionally equivalent measure. 10 The solubility of a protein may be measured as the amount of protein that is soluble in a given solvent and/or the rate at which the protein dissolves. Furthermore, the rate and or level of solubility of a noggin or chimeric noggin molecule in solvents of differing properties such as polarity, pH, temperature and the like may also provide measurable physiochemical characteristics of the noggin or chimeric noggin molecule. 15 Any "measurable physiochemical parameters" may be determined, measured, quantified or qualified using any methods known to one of skill in the art. Described below is a range of methodologies which may be useful in determining, measuring, quantifying or qualifying one or more measurable physiochemical parameters of an isolated noggin or chimeric 20 noggin molecule. However, it should be understood that the present invention is in no way limited to the particular methods described, or to the measurable physiochemical parameters that are measurable using these methods. Glycoproteins can be said to have two basic components that interact with each other to 25 create the molecule as a whole- the amino acid sequence and the carbohydrate or sugar side chains. The carbohydrate component of the molecule exists in the form of monosaccharide or oligosaccharide side chains attached to the amine side chain of Asn or the hydroxyl side chain of Ser/Thr residues of the amino acid backbone by N- or 0 linkages, respectively. A monosaccharide is the term given to the smallest unit of a 30 carbohydrate that is regarded as a sugar, having the basic formula of (CH 2 0), and most often forming a ring structure of 5 or 6 atoms (pentoses and hexoses respectively). Oligosaccharides are combinations of monosaccharides forming structures of varying WO 2007/028212 PCT/AU2006/001325 - 128 complexities that may be either linear or branched but which generally do not have long chains of tandem repeating units (such as is the case for polysaccharides). The level of branching that the oligosaccharide contains as well as the terminal and branching substitutions dramatically affect the properties of the glycoprotein as a whole, and play an 5 important role in the biological function of the molecule. Oligosaccharides are manufactured and attached to the amino acid backbone in the endoplasmic reticulum (ER) and Golgi apparatus of the cell. Different organisms and cell types have different ratios of glycotransferases and endoglycosidases and exoglycosidases and therefore produce different oligosaccharide structures. One of the fundamental defence mechanisms of the 10 body is the detection and destruction of aberrant isoforms and as such it is important to have correct glycosylation of a biological therapeutic not only to increase effectiveness but also to decrease detection by neutralizing antibodies. Glycan chains are often expressed in a branched fashion, and even when they are linear, 15 such chains are often subject to various modifications. Thus, the complete sequencing of oligosaccharides is difficult to accomplish by a single method and therefore requires iterative combinations of physical and chemical approaches that eventually yield the details of the structure under study. 20 Determination of the glycosylation pattern of a protein can be performed using a number of different systems, for example using SDS-PAGE. This technique relies on the fact that glycosylated proteins often migrate as diffuse bands by SDS-PAGE. Differentiation between different isoforms are performed by treating a protein with a series of agents. For example, a marked decrease in band width and change in migration position after digestion 25 with peptide-N4-(N-acetyl-p-D-glucosaminyl) asparagine amidase (PNGase) is considered diagnostic of N-linked glycosylation. To determine the composition of N-linked glycosylation, N-linked oligosaccharides are removed from the protein with PNGase cloned from Flavobacterium meningosepticum and 30 expressed in E coli. The removed N-linked oligosaccharides may be recovered from Alltech Carbograph SPE Carbon columns (Deerfield, Illinois, USA) as described by Packer et al. Glycoconj J 5(8):737-47, 1998. The sample can then be taken for WO 2007/028212 PCT/AU2006/001325 -129 monosaccharide analysis, sialic acid analysis or sulfate analysis on a Dionex system with a GP50 pump ED50 pulsed Amperometric or conductivity detector and a variety of pH anion exchange columns. 5 The extent of O-linked glycosylation may be determined by first removing O-linked oligosaccharides from the target protein by p-elimination. The removed 0-linked oligosaccharides may be recovered from Alltech Carbograph SPE Carbon columns (Deerfield, Illinois, USA) as described by Packer et al. 1998, supra. The sample can then be taken for monosaccharide analysis, sialic acid analysis or sulfate analysis on a Dionex 10 system with a GP50 pump ED50 pulsed Amperometric or conductivity detector and a variety of pH anion exchange columns. Monosaccharide subunits of an oligosaccharide have variable sensitivities to acid and thus can be released from the target protein by mild trifluoro-acetic acid (TFA) conditions, 15 moderate TFA conditions, and strong hydrochloric acid (HCl) conditions. The monosaccharide mixtures are then separated by high pH anion exchange chromatography (HPAEC) using a variety of column media, and detected using pulsed amperometric electrochemical detection (PAD). 20 High-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC PAD) has been extensively used to determine monosaccharide composition. Fluorophore based labeling methods have been introduced and many are available in kit form. A distinct advantage of fluorescent methods is an increase in sensitivity (about 50-fold). One potential disadvantage is that different monosaccharides may demonstrate different 25 selectivity for the fluorophore during the coupling reaction, either in the hydrolyzate or in the external standard mixture. However, the increase in sensitivity and the ability to identify which monosaccharides are present from a small portion of the total amount of available glycoprotein, as well as the potential for greater sensitivity using laser-induced fluorescence, makes this approach attractive. In addition a conductivity detector may be 30 used to determine the sulfate and phosphate composition. By using standards, the peak areas can be calculated to total amounts of each monosaccharide present. These data can indicate the level of N- and 0-linked glycosylation, the extent of sialylation, and in WO 2007/028212 PCT/AU2006/001325 -130 combination with amino acid composition, percent by weight glycosylation, percent by weight acidic glycoproteins. Monosaccharide composition analysis of small amounts of protein is best performed with 5 PVDF (PSQ) membranes, after electroblotting, or, if smaller aliquots are to be analyzed, on dot blots. PVDF is an ideal matrix for carbohydrate analysis because neither monosaccharides nor oligosaccharides bind to the membrane, once released by acid or enzymatic hydrolysis. 10 Determination of the oligosaccharide content of the target molecule is performed by a number of techniques. The sugars are first removed from the amino acid backbone by enzymatic (such as digestion with PNGase)) or chemical (such as beta elimination with hydroxide) means. The sugars may be stabilised by reduction or labeled with a fluorophore for case of detection. The resultant free oligosaccharides are then separated either by high 15 pH anion exchange chromatography with pulsed amperometric electrochemical detection (HPAEC-PAD), which can be used with known standards to determine the ratios of the various structures and levels of sialylation, or by fluorophore assisted carbohydrate electrophoresis (FACE) a process similar to SDS-PAGE separation of proteins. In this process the oligosaccharides are labeled with a fluorophore that imparts electrophoretic 20 mobility. They are separated on high percentage polyacrylamide gels and the resultant band pattern provides a profile of the oligosaccharide content of the target molecule. By using standards it is possible to gain some information on the actual structures present or the bands can be cut and analysed using mass spectrometry to determine each of their structures. 25 Fluorophore assisted carbohydrate electrophoresis (FACE) is a polyacrylamide gel electrophoresis system designed to separate individual oligosaccharides that have been released from a glycoconjugate. Oligosaccharides are removed from the sample protein by either chemical or enzymatic means in such a way as to retain the reducing terminus. 30 Oligosaccharides are then either digested into monosaccharides or left intact and labeled with a fluorophore (either charged or non charged). High percentage polyacrylamide gels and various buffer systems are used to migrate the oligosaccharides/monosaccharides WO 2007/028212 PCT/AU2006/001325 -131 which migrate relative to their size/composition in much the same way as proteins. Sugars are visualised by densitometry and relative amounts of sugars can be determined by fluorophore detection. This process is compatible with MALDI-TOF MS, hence the method can be used to elucidate actual structures. 5 Quartz crystal microbalance and surface plasmon resonance (QCM and SPR, respectively) are two methods of obtaining biological information through the physiochemical properties of a molecule. Both measure protein-protein interactions indirectly through the change that the interaction causes in the physical characteristics of a prefabricated chip. In QCM a ,10 single crystal quartz wafer is treated with a receptor/antibody etc which interacts with the ligand of interest. This chip is oscillated by the microbalance and the frequency of the chip recorded. The protein of interest is allowed to pass over the chip and the interaction with the bound molecule causes the frequency of the wafer to change. By changing the conditions by which the ligand interacts with the chip, it is possible to determine the 15 binding characteristics of the target molecule. Apparent molecular weight is also a physiochemical property which can be used to determine the similarities between the noggin or chimeric noggin molecule of the present invention and those produced using alternative means. 20 As used herein, the term "molecular weight" is defined as the sum of atomic weights of the constituent atoms in a molecule, sometimes also referred to as "molecular mass" (Mr). Molecular weight can be determined theoretically by summing the atomic masses of the constituent atoms in a molecule. The term "apparent molecular weight" is defined as the 25 molecular weight determined by one or more analytical techniques such as SDS page or ultra centrifugation and depends on the relationship between the molecule and the detection system. The apparent molecular weight of a noggin or chimeric noggin molecule can be determined using any one of a range of experimental methods. Analytical methods for determining the molecular weight of a protein include, without being limited to, size 30 exclusion chromatography (SEC), gel electrophoresis, Rayleigh light scattering, analytical ultracentrifugation, and, to some extent, time-of-flight mass spectrometry.
WO 2007/028212 PCT/AU2006/001325 -132 Gel electrophoresis is a process of determining some of the physiochemical properties (specifically apparent molecular weight and pI) of a protein and in the case of 2 dimensional electrophoresis to separate the molecule into isoforms, thereby providing information on the post-translational modifications of the protein product. Specifically, 5 electrophoresis is the process of forcing a charged molecule (such as protein or DNA) to migrate through a gel matrix (most commonly polyacrylamide or agarose) by applying an electric potential through its body. The most common forms of electrophoresis used on proteins are isoelectric focussing, native, and SDS polyacrylamide gel electrophoresis. In isoelectric focussing a protein is placed into a polyacrylamide gel that has a pH gradient 10 across its length. The protein will migrate to the point in the gel where it has a net charge of zero thereby giving its isoelectric point. Glyco mass fingerprinting (GMF) is the process by which the oligosaccharide profile of a protein or one of its isoforms is identified by electrophoresis followed by specific mass 15 spectrometric techniques. Sample protein is purified either by 1D SDS-PAGE for total profile determination or 2D gel electrophoresis for specific isoform characterization. The protein band/spot is excised from the gel and de-stained to remove contaminants. The sugars are released by chemical or enzymatic means and desalted/separated using a nanoflow LC system and a graphitised carbon column. The LC flow can be directly 20 injected into an electrospray mass spectrometer that is used to determine the mass and subsequently the identity of the oligosaccharides present on the sample. This provides a profile or fingerprint of each isoform which can be combined with quantitative techniques such as Dionex analysis to determine the total composition of the molecule being tested. 25 Primary structure can be evaluated in determining the physiochemical properties of the noggin or chimeric noggin molecule of the present invention. The primary structure of a noggin or chimeric noggin molecule can be assayed using one or more of the following systems. 30 WO 2007/028212 PCT/AU2006/001325 - 133 Information on the primary structure of a noggin or chimeric noggin molecule can be determined using a combination of mass spectrometry (MS), DNA sequencing, amino acid composition, protein sequencing and peptide mass fingerprinting. 5 To determine the sequence of the amino acid backbone either N-terminal chemical sequencing, tandem mass spectrometry sequencing, or a combination of both is used. N terminal chemical sequencing utilises Edman chemistry (Edman P. "Sequence determination" Mol Biol Biochem Biophys 8:211-55, 1970), which states that the peptide bond between the N-terminal amino acid and the amino acid in position 2 of the protein is 10 weaker than all other peptide bonds in the sequence. By using moderate acidic conditions the N-terminal amino acid is removed derivatised with a fluorophore (FTIC) and the retention time on a reversed-phase HPLC column determined, and compared to a standard to identify what the amino acid is. This method will determine the actual primary structure of the molecule but is not quantitative. Alternatively tandem mass spectrometry in 15 conjunction with nanoflow liquid chromatography may be used (LC-MS/MS). In this process the protein is digested into peptides using specific endoproteases and the molecular weight of the peptides determined. High energy collision gases such as nitrogen or argon are then used to break the peptide bonds and the masses of the resultant peptides measured. By calculating the change in mass of the peptides it is possible to determine the sequence 20 of each of the peptides (each amino acid has a unique mass). By using different proteases the peptides may then be overlapped to determine their order and thus the entire sequence of the protein. Clearly, the combination of enzymatic digestion, chemical derivatization, liquid 25 chromatography (LC)/MS and tandem MS provides an extremely powerful tool for AA sequence analysis. For example, the detailed structure of recombinant soluble CD4 receptor was characterized by a combination of methods, which confirmed over 95% of the primary sequence of this 369 AA glycoprotein and showed the whole nature of both N-and C-termini, the positions of attachment of the glycans, the structures of the glycans and the 30 correct assignment of the disulfide bridges (Carr et al. JBiol Chem 264(35):21286-21295, 1989).
WO 2007/028212 PCT/AU2006/001325 - 134 Mass spectrometry (MS) is the process of measuring the mass of a molecule through extrapolation of its behavior in a charged environment under a vacuum. MS is very useful in stability studies and quality control. The method first requires digestion of samples by proteolytic enzymes (trypsin, V8 protease, chymotrypsin, subtilisin, and clostripain) 5 (Franks et al. Characterization ofproteins, Humana Press, Clifton, NJ, 1988; Hearn et al. Methods in Enzymol 104:190-212, 1984) and then separation of digested samples by reverse phase chromatography (RPC). With tryptic digestion in conjunction with LC-MS, the peptide map can be used to monitor the genetic stability, the homogeneity of production lots, and protein stability during fermentation, purification, dosage form 10 manufacture and storage. Before a mass analysis, several ways are used to interface a HPLC to a mass spectrometer: 1) direct liquid injection; 2) supercritical fluid; 3) moving belt system; 4) thermospray. The HPLC/MS interface used in Caprioli's work used a fused silica capillary column to 15 transport the eluate from the column to the tip of the sample probe in the ionization chamber of the mass spectrometer. The probe tip is continuously bombarded with energetic Xe atoms, causing sputtering of the sample solution as it emerges from the tip of the capillary. The mass is then analyzed by the instrument (Caprioli et al. Biochem Biophys Res Commun 146:291-299, 1987). 20 MS/MS and LC/MS interfaces expand the potential applications of MS. MS/MS allows direct identification of partial to full sequence for peptides up to 25 AAs, sites of deamidation and isomerization (Carr et al. Anal Chem 63:2802-2824, 1991). Coupled with RPC or capillary electrophoresis (CE), MS can perform highly sensitive analysis of 25 proteins (Figeys and Aebersold, Electrophoresis 19:885-892, 1998; Nguyen et al. J Chromatogr A 705:21-45, 1995). LC/MS allows LC methodology to separate peptides before entering the MS, such as the continuous flow FAB interfaced with microbore HPLC (Caprioli et al. 1987, supra). The latter "interface" allows the sequencing of individual peptides from complex mixtures: Fragmentation of the peptides selected by the first MS is 30 followed by passing through a cloud of ions in a collision cell: CID (collision induced dissociation). The collision generates characteristic set of fragments, from which the sequence may be deduced, without knowing other information, such as the cDNA WO 2007/028212 PCT/AU2006/001325 - 135 sequence. In a single MS experiment, an unfractionated mixture of peptides (e.g. from an enzyme digest) is injected and the masses of the major ions are compared with those predicted from the cDNA sequence. The sequence of the recombinant human interleukin-2 was verified by fast atom bombardment (FAB)-MS analysis of CNBr and proteolytic 5 digests (Fukuhara et al. JBiol Chen 260:10487-10494, 1985). Electrospray ionization MS (ESI-MS) uses an aerosol of solution protein to introduce into a needle under a high voltage, generating a series of charged peaks of the same molecules with various charges. Because each peak generated from the differently charged species 10 produces an estimation of the molecular weights, these estimations can be combined to increase the overall precision of the molecular weight estimation. Matrix Assisted Laser Desorption Ionization MS (MALDI-MS) uses a high concentration of a chromophore. A higher intensity laser pulse will be absorbed by the matrix and the energy absorbed evaporates part of the matrix and carries the protein sample with it into the vapor phase 15 almost entirely. The resulting ions are then analyzed in a time of flight MS. The mild ionization may enhance the capacity of the method to provide quaternary structure information. MALDI-MS can be run rapidly in less than 15 minutes. It does not need to fragment the molecules and the result is easy to interpret as a densitometric scan of an SD S-PAGE gel, with a mass range up to over 1 00kDa. 20 Amino acid sequence can be predicted by sequencing DNA that encodes a noggin or chimeric noggin molecule. However, occasionally the actual protein sequence may be different. Traditionally, DNA sequencing reactions are just like the PCR reactions for replicating DNA (DNA denaturation, replication). By DNA cloning technology, the gene 25 is cloned, and the nucleotide sequence determined. The amino acid sequence of a noggin or chimeric noggin molecule can be assayed using one or more of the following systems. 30 Full sequence description of the noggin or chimeric noggin molecule is usually required to describe the product. Amino acid sequencing includes: in gel tryptic digestion, fractionation of the digested peptides by RPC-HPLC, screening the peptide peaks that have WO 2007/028212 PCT/AU2006/001325 -136 the most symmetrical absorbance profile by MALDI-TOF MS, and the first peptide (N terminal) by Edman degradation. Edman chemically derived primary sequence data is the classical method to identify proteins at the molecular level. MALDI-TOF MS can be used for N-terminal sequence analysis. However, all enzymatic digests for HPLC and peptide 5 sequencing are recommended to first be subjected to MS or MS/MS protein identification to decrease the time and cost. The internal amino acid sequences from SDS-PAGE separated proteins are obtained by elution of the peptides with HPLC separation after an in situ tryptic or lysyl endopeptidase digestion in the gel matrix. 10 Internal sequencing of the standard peptide is recommended to be run with the analyzed samples to maintain the instruments at the peak performance. More than 80% of higher eukaryotic proteins are reported to have blocked amino-termini that prevent direct amino acid sequencing. When a blocked eukaryotic protein is encountered, the presence of the sequence of the internal standard assures that the instrument is operating properly. 15 Edman degradation can be used for direct N-terminal sequencing with a chemical procedure, which derivatizes the N-terminal amino acids to release the amino acids and expose the amino terminal of the next AAs. The Edman sequencing includes: 1). By microbore HPLC, N-terminal sequence analysis is repeated by Edman chemistry cycles. 20 Every cycle of the Edman chemistry can identify one amino acid. 2). After in-gel or PVDF bound protein digestions followed by HPLC separation of the resulting peptides, internal protein sequence analysis is conducted by Edman degradation chemistry. Microbore HPLC and capillary HPLC are used for analysis and purification of peptide 25 mixtures using RPC-HPLC. In-gel samples and PVDF samples are purified using different columns. MALDI-TOF MS analysis can be used for N-terminal analysis after HPLC fractionation. The selection criteria are: 1) The apparent purity of the HPLC fraction. 2) The mass and thus the estimated length of the peptide. The peptide mass information is useful for confirming the Edman sequencing amino acid assignments, and also in the 30 possible detection of co- or post-translational modifications.
WO 2007/028212 PCT/AU2006/001325 - 137 In-gel digests are suitable for purification on the higher sensitivity HPLC system. The internal protein sequence analysis is first enzymatically digested by SDS-PAGE. Proteins in an SDS-PAGE mini-gel can be reliably digested in-gel only with trypsin. The peptide fragments are purified by RPC-HPLC and then analyzed by MALDI-TOF MS, screening 5 for peptides suitable for Edman sequence analysis. Proteins in a gel can only be analyzed by internal sequencing analysis, but very accurate peptides masses can be obtained, which provides additional information useful in both amino acid assignment and database searching. 10 PVDF-bound proteins are suitable for both N-terminal and internal Edman sequencing analysis. PVDF-bound proteins are digested with the proper enzyme (trypsin, endoproteinase Lys-C, endoproteinase Glu-C, clostripain, endoproteinase Asp-N, thermolysin) and a non-ionic detergent such as hydrogenated Triton X-100. In PVDF bound proteins, the detergents used for releasing digested peptides from the membrane can 15 interfere with MALDI-TOF MS analysis. Before the enzyme is added, Cys is reduced with DTT and alkylated with iodoacetamide to generate carboxyamidomethyl Cys, which can be identified during N-terminal sequence analysis. To determine the amino acid composition of a noggin or chimeric noggin molecule, the 20 sample is hydrolyzed using phenol catalyzed strong hydrochloric acid (HCl) acidic conditions in the gaseous phase. Once the hydrolysis is performed the liberated amino acids are derivatised with a fluorophore compound that imparts a specific reversed phase characteristic on the combined molecule. The derivatized amino acids are separated using reversed phase high performance liquid chromatography (RP-HPLC) and detected with a 25 fluorescence detector. By using external and internal standards it is possible to calculate the amount of each amino acid present in the sample from the observed peak area. This information may be used to determine sample identity and to quantify the amount of protein present in the sample. For instance, discrepancies between theoretical and actual results can be used to initially identify the possibility of a de-amidation site. In 30 combination with monosaccharide analysis it may determine the composition % by weight glycosylation and percent by weight acidic glycoproteins. This method is limited in the information that it can provide on the actual sequence of the backbone however as there is WO 2007/028212 PCT/AU2006/001325 - 138 inherent variability due to environmental contaminants and occasional destruction of amino acids. For example, it is not possible for this method to detect point mutations in the sequence. 5 Peptide mass fingerprinting (PMF) is another method by which the identity of a noggin or chimeric noggin molecule may be determined. The procedure involves an initial separation of the sample by electophoretic means (either 1 or 2 dimensional), excision of the spot/band from the gel and digestion with a specific endoprotease (typically porcine trypsin). Peptides are eluted from the gel fragment and analysed by mass spectrometry to 10 determine the peptide masses present. The resultant peptide masses are then compared to a database of theoretical mass fragments for all reported proteins (or in the case of constructs for the theoretical peptide masses of the designed sequence). The technique relies on the fact that the "fingerprint" of a protein (i.e. its combination of peptide masses) is unique. Identity can be confidently determined (greater than 90% accuracy) with as little as 4 15 peptides and 30% sequence coverage. Modifications such as lipid moieties and de amidation can be identified during the PMF stage of analysis. Peaks that do not correspond to those of the identified protein are further analysed by tandem mass spectrometry (MS MS), a technique that uses the energy created by the impact of a collision gas to break the weaker bond of the PTM. The newly freed molecule and the original peptide are then re 20 analysed for mass to identify the post-translational modification and the peptide fragment to which it was attached. HPLC is classified into different modes depending on the size, charge, hydrophobicity, function or specific content of the target biomolecules. Generally, two or more 25 chromatographic methods are used to purify a protein. It is of paramount importance to consider both the characteristics of the protein and the sample solvent when the chromatographic modes are selected. Secondary structures of a noggin or chimeric noggin molecule of the present invention can 30 also be evaluated in characterising their properties.
WO 2007/028212 PCT/AU2006/001325 -139 The secondary structure of a noggin or chimeric noggin molecule can be assayed using one or more of the following systems. To study the secondary structures of proteins, most commonly several spectroscopic 5 methods should be applied and compared. Electromagnetic energy can be defined as a continuous waveform of radiation, depending on the size and shape of the wave. Different spectroscopic methods use different electromagnetic energy. The wavelength, is the extent of a single wave of radiation (the distance between two 10 successive maxima of the waves). When the radiant energy increases, the wavelength becomes shorter. The relationship between frequency and wavenumber is: Wavenumber (cm"') = Frequency (s") / The speed of light (cm/s). 15 The absorption of electromagnetic radiation by molecules includes vibrational and rotational transitions, and electronic transitions. Infrared (IR) and Raman spectroscopy are most commonly used to measure the vibrational energies of molecules in order to determine secondary structure. However, they are different in their approach to determine molecular absorbance. 20 The energy of the scattered radiation is less than the incident radiation for the Stokes line. The energy of the scattered radiation is more than the incident radiation for the anti-Stokes line. The energy increase or decrease from the excitation is related to the vibrational energy spacing in the ground electronic state of the molecule. Therefore, the wavenumber 25 of the Stokes and anti-Stokes lines are a direct measure of the vibrational energies of the molecule. Only the Stokes shift is observed in a Raman spectrum. The Stokes lines are at smaller wavenumbers (or higher wavelengths) than the exciting light. A high power excitation 30 source, such as a laser, should be used to enhance the efficiency of Raman scattering. The excitation source should be monochromatic because we are interested in the energy (wavenumber) difference between the excitation and the Stokes lines.
WO 2007/028212 PCT/AU2006/001325 -140 For a vibrational motion to be IR active, the dipole moment of the molecule must change. Therefore, the symmetric stretch in carbon dioxide is not IR active because there is no change in the dipole moment. The asymmetric stretch is IR active due to a change in dipole -5 moment. For a vibration to be Raman active, the polarizability of the molecule must change with the vibrational motion. The symmetric stretch in carbon dioxide is Raman active because the polarizability of the molecule change. Thus, Raman spectroscopy complements IR spectroscopy (Herzberg et al. Infrared and Raman Spectra of Polyatomic Molecules, Van Nostrand Reinhold, New York, NY, 1945). For example, IR is not able to 10 detect a homonuclear diatomic molecule due to the lack of dipole moments, but Raman spectroscopy can detect it because the molecular polarizability is changed by stretching and contraction of the bond, further, the interactions between electrons and nuclei are changed. 15 For highly symmetric polyatomic molecules with a center of inversion (such as benzene), it is more likely that bands active in the IR spectrum are not active in the Raman spectrum or vice-versa. In molecules with little or no symmetry, modes are likely to be active in both infrared and Raman spectroscopy. 20 IR spectroscopy measures the wavelength and intensity of the absorption of infrared light by a sample. Infrared light is so energetic that it can excite the molecular vibrations to higher energy levels. Both infrared and RAMAN spectroscopy measure the vibrations of bond lengths and angles. 25 IR characterizes vibrations in molecules by measuring the absorption of light of certain energies corresponding to the vibrational excitation of the molecule from v = 0 to v 1 (or higher) states. There are selection rules that govern the ability of a molecule to be detected by infrared spectroscopy - Not all of the normal modes of vibration can be excited by infrared radiation (Herzberg et al. 1945, supra). 30 IR spectra can provide qualitative and quantitative information of the secondary structures of proteins, such as a helix, p sheet, P turn and disordered structure. The most informative WO 2007/028212 PCT/AU2006/001325 - 141 IR bands for protein analysis are amide 1 (1620-1700 cm), amide 11 (1520-1580 cm') and amide III (1220-1350 cm). Amide I is the most intense absorption band in proteins. It consists of stretching vibration of the C=O (70-85% and C-N groups (10-20%). The exact band position is dictated by the backbone conformation and the hydrogen bonding pattern. 5 Amide II is more complex than Amide I. Amide II is governed by in-plane N-H bending (40-60%), C-N (18-40%) and C-C (10%) stretching vibrations. Amide III bands are not very useful (Krimm and Bandekar, Adv Protein Chem 38:181-364, 1986). Most of the p sheet structures of FTIR amide I band usually are located at about 1629 cm" with a minimum of 1615 cm 1 and a maximum of 1637 cm 1 ; the minor component may show 10 peaks around 1696 cm' (lowest value 1685 cm 1 ). a-helix is mainly found at 1652 cm'. An absorption near 1680 cm 1 is now assigned to p turns. The principle of Raman scattering is different from that of infrared absorption. Raman spectroscopy measures the wavelength and intensity of inelastically scattered light from 15 molecules. The Raman scattered light occurs at wavelengths that are shifted from the incident light by the energies of molecular vibrations. To be Raman active, for the vibration to be inelastically scattered, a change in polarizability during the vibration is essential. In the symmetric stretch, the strength of 20 electron binding is different between the minimum and maximum internuclear distances. Therefore the polarizability changes during the vibration, and this vibrational mode scatters Raman light, the vibration is Raman active. In the asymmetric stretch the electrons are more easily polarized in the bond that expands but are less easily polarized in the bond that compresses. There is no overall change in polarizability and the asymmetric stretch is 25 Raman inactive (Herzberg et al. 1945, supra). Circular dichroism can be used to detect any asymmetrical structures, such as proteins. Optically active chromophores absorb different amount of right and left polarized light, this absorbance difference results in either a positive or negative absorption spectrum 30 (Usually, the right polarized spectrum is subtracted from the left polarized spectrum). Commonly, the far UV or amide region (190-250nm) is mainly contributed from peptide bonds, providing information on the environment of the carbonyl group of the amide bond WO 2007/028212 PCT/AU2006/001325 -142 and consequently the secondary structure of the protein. a helix usually displays two negative peaks at 208, 222 nm (Holzwarth et al. JAm Chem Soc 178:350, 1965), 0 sheet displays one negative peak at 218 nm, random coils has a negative peak at 196 nm. Near UV region peaks are (250-350 nm) contributed from the environment of the aromatic 5 chromophores (Phe, Tyr, Trp). Disulfide bonds give rise to minor CD bands around 250 nm. Intense dichroism is commonly associated with the side-chain structures being held tightly in a highly folded, three-dimensional structure. Denaturation of the protein mostly releases 10 the steric hindrance, a weaker CD spectrum is obtained along with an increasing degree of denaturation. For example, the side chain CD spectrum of hGH is quite sensitive to the partial denaturation by adding denaturants. Some reversible chemical alterations of the molecules, such as reduction of the disulfide bonds, or alkaline titrations will change the side-chain CD spectrum. For hGH, these spectral difference can be caused by entirely the 15 removal of a chromophores, or by affecting changes in the particular chromophore's CD response, but not by the gross denaturation or conformational changes (Aloj et al. J Biol Chem 247:1146-1151, 1971). UV absorption spectroscopy is one of the most significant methods to determine protein 20 properties. It can provide information about protein concentrations and the immediate environments of chromophoric groups. Proteins functional groups, such as amino, alcoholic (or phenolic) hydroxyl, carbonyl, carboxyl, or thiol can be transformed into strong chromophores. Visible and near UV spectroscopy are used to monitor two types of chromophores: metalloproteins (more than 400 nm) and proteins that contains Phe, Trp, 25 Tyr residues (260-280nm). The change in UV or fluorescence signal can be negative or positive, depending on the protein sequence and solution properties. Fluorescence measures the emission energy after the molecule has been irradiated into an excited state. Many proteins emitted fluorescence in the range of 300 to 400 nm when 30 excited at 250 to 300 nm from their aromatic amino acids. Only proteins with Phe, Trp, Tyr residues can be measured with the order of intensity Trp>> Tyr>> Phe. Fluorescence spectra can reflect the microenvironments information that are affected by the folding of WO 2007/028212 PCT/AU2006/001325 - 143 the proteins. For example, a buried Trp is usually in a hydrophobic environment and will fluoresce at maximum 325 to 330 nm range, but an exposed residue or free amino acids fluoresces at around 350 to 355 nm. An often used agent to probe protein unfolding is Bis ANS. The fluorescence of Bis-ANS is pH-independent. Even though its signal is weak in 5 water, it can be increased significantly by binding to unfolding-exposed hydrophobic sites in proteins (James and Bottomley Arch Biochem Biophy 356:296-300, 1998). Effective quenching of Tyr and Trp in the folded proteins causes significant signal increase upon unfolding. A simple solute may cause the change also. To maximize detection 10 sensitivity, a signal ratio can be used. For example, In the study of rFXIII unfolding, a ratio of fluorescence intensity at 350nm to that at 330nm was used (Kurochkin et al. JMol Biol 248:414-430, 1995). Conformational changes may be studied by means of excitation energy transfer between a fluorescent donor and an absorbing acceptor, because the efficiency of transfer depends on the distance between the two chromophores (Honroe et 15 al. Biochem J 258:199-204, 1989). Fluorescence was used to probe a-Antitrypsin conformation (Kwon and Yu, Biophim Biophys Acta 1335:265-272, 1997), to determine Tm of HSA (Farruggia et al. Int J Biol Macronol 20:43-51, 1997), and to detect MerP unfolding interactions (Aronsson et al. FEBS Lett 411:359-364, 1997). 20 At neutral pH, the intensity of the fluorescence emission spectrum is in the order of Trp> Tyr. At acidic pH, due to the conformational changes which disrupts the energy transfer, the fluorescence from Tyr dominates over Trp. Fluorescence studies also confirm the presence of intermediates in the guanidine-induced unfolding transition of the proteins. 25 Tertiary and quaternary structures of the physiochemical forms of a noggin or chimeric noggin molecule of the present invention are also important in ascertaining their function. The tertiary and quaternary structures of a noggin or chimeric noggin molecule can be assayed using one or more of the following systems. 30 NMR and X-ray crystallography are the most often used techniques to study the 3D structure of proteins. Other less detailed methods to probe protein tertiary structure include WO 2007/028212 PCT/AU2006/001325 -144 CD in near UV region, second-derivative of UV spectroscopy (Ackland et al. J Chromatogr 540:187-198, 1991) and fluorescence. NMR is one of the main experimental methods for molecular structure and intermolecular 5 interactions in structural biology. In addition to studying protein structures, NMR can also be utilised to study the carbohydrate structures of a noggin or chimeric noggin molecule of the present invention. NMR spectroscopy is routinely used by chemists to study chemical structure using simple one-dimensional techniques. The structure of more complicated molecules can also be determined by two-dimensional techniques. Time domain NMR are 10 used to probe molecular dynamics in solutions. Solid state NMR is used to determine the molecular structure of solids. NMR can be used to study structural and dynamic properties of proteins, nucleic acids, a variety of low molecular weight compounds of biological, pharmacological and medical interests. However, not all nuclei possess the correct property in order to be read by NMR, i.e., not all nuclei posses spin, which is required for 15 NMR. The spin causes the nucleus to produce an NMR signal, functioning as a small magnetic field. The crystal structure of a noggin or chimeric noggin molecule can be assayed using one or more of the following systems. 20 X-ray crystallography is an experimental technique that applies the fact that X-rays are diffracted by crystals. X-rays have the appropriate wavelength (in the Angstrom range, -10-8 cm) to be scattered by the electron cloud of an atom of comparable size. The electron density can be reconstructed based on the diffraction pattern obtained from X-ray 25 scattering off the periodic assembly of molecules or atoms in the crystal. Additional phase information either from the diffraction data or from supplementing diffraction experiments should be obtained to complete the reconstruction. A model is then progressively built into the experimental electron density, refined against the data and the result is a very accurate molecular structure. 30 WO 2007/028212 PCT/AU2006/001325 - 145 X ray diffraction has been developed to study the structure of all states of matter with any beam, e.g., ions, electrons, neutrons, and protons, with a wavelength similar to the distance between the atomic or molecular structures of interest. 5 Light scattering spectroscopy is based on the simple principle that larger particles scatter light more than the smaller particles. A slope base line in the 310-400nm region originates from light scattering when large particles, such as aggregates, present in the solution (Schmid et al. Protein structure, a practical approach, Creighton Ed., IRI Press, Oxford, England, 1989) 10 Light scattering spectroscopy can be used to estimate the molecular weight of a protein and is a simple tool to monitor protein quaternary structure or protein aggregation. The degree of protein aggregation can be indicated by simple turbidity measurement. Final product pharmaceutical solutions are subjected to inspection of clarity because most aggregated 15 proteins are present as haze and opalescence. Quasielastic light scattering spectroscopy (QELSS), sometimes called photon correlation spectroscopy (PCS), or dynamic light scattering (DLS), is a noninvasive probe of diffusion in complex fluids for macromolecules (proteins, polysaccharides, synthetic polymers, micelles, colloidal particles and aggregations). In most cases, light scattering spectroscopy yields directly the mutual 20 diffusion coefficient of the scattering species. When applied to dilute monodisperse solutions, the diffusion coefficient obtained by QELSS can estimate the size. With polydisperse system, it estimates the width of molecular weight distribution. For accurate measurement, 200-500 mW laser power is mandatory, conventional Ar+/Kr+ gas lasers are widely used (Phillies Anal Chem 62:1049A-1057A, 1990). Protein aggregation was 25 detected by human relaxin (Li et al. Biochemistry 34:5762-5772, 1995). Stability of a noggin or chimeric noggin molecule is also an important determinant of function. Methods for analysing such characteristics include DSC, TGA and freeze-dry cryostage microscopy, analysis of freeze-thaw resistance, and protease resistance. 30 A noggin or chimeric noggin molecule of the present invention may be more stable for lyophilization (freeze drying). Lyophilization is used to enhance the stability and/or shelf WO 2007/028212 PCT/AU2006/001325 -146 life of the product as it is stored in powder rather than liquid form. The process involves an initial freezing of the sample, then removal of the liquid by evaporation under vacuum. The end result is a dessicated "cake" of protein and excipients (other substances used in the formulation). The consistency of the resulting cake is critical for successful reconstitution. 5 The lyophilization process can result in changes to the protein, especially aggregate formation though crosslinking, but also deamidation and other modifications. These can reduce efficacy by either losses, reduced activity or by inducing immune reactions against aggregates. In order to test lyophilization stability, the protein can be formulated for lyophilization using standard stabilizers (e.g. mannitol, trehalose, Tween 80, human serum 10 albumin and the like). After lyophilization, the amount of protein recovered can be assayed by ELISA, while its activity can be assayed by a suitable bioassay. Aggregates of the protein can be detected by HPLC or Western blot analysis. Prior to lyophilization, the Tg or Te (define Tg or Te) of the formulation should be 15 determined to set the maximum allowable temperature of the product during primary drying. Also, information about the crystallinity or amorphousity of the formulation helps to design the lyophilization cycle in a more rationale manner. Product information on these thermal parameters can be obtained by using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) or freeze-dry cryostage microscope. 20 Differential Scanning Calorimetry (DSC) is a physical thermo-analytical method to measure, characterize and analyze thermal properties of materials and determine the heat capacities, melting enthalpies and transition points accordingly. DSC scans through a temperature range at a linear rate. Individual heaters within the instrument provide heat to 25 sample and reference pans separately, based on the "power compensated null balance" principle. During a physical transition, the absorption or evolution of the energy causes an imbalance in the amount of energy supplied to that of the sample holder. Depending on the varying thermal behavior of the sample, the energy will be taken or diffused from the sample, and the temperature difference will be sensed as an electrical signal to the 30 computer. As a result, an automatic adjustment of the heaters makes the temperature of the sample holder identical to the reference holder. The electrical power needed for the compensation is equivalent to the calorimetric effect.
WO 2007/028212 PCT/AU2006/001325 -147 The purity of an organic substance can be estimated by DSC based on the shape and temperature of the DSC melting endotherm. The power-compensated DSC provides very high resolution compared to a heat flux DSC under the identical conditions. More well 5 defined and more accurate partial areas of melting can be generated from power compensated DSC because the partial areas of melting are not "smeared" over a narrow temperature interval, as for the lesser-resolved heat flux DSC. The power-compensated DSC produces inherently better partial melting areas and therefore better purity analysis. By the help of StepScan DSC, the power-compensated DSC can provide a direct heat 10 capacity measurement using the traditional and time-proven means without the need for deconvolution or the extraction of sine wave amplitudes. Thermogravimetric Analysis (TGA) measures sample mass loss and the rate of weight loss as a function of temperature or time. 15 As DSC, freeze-dry cryostage can reach a wide temperature range rapidly. Currently, as an preformulation and formulation study tool, simulating the lyophilization cycle in a freeze dry cryostage provides the best platform to study thermal parameters of the protein formulations on a miniature scale. Freeze dry microscope can predict the influence of 20 formulations and process factors on freezing and drying. Only a 2-3mL sample is required for a cryostage study, which makes this technique a valuable tool to study scarce, difficult to-obtain drugs. It is a good tool to study the effect of freezing, rate, drying rate, thawing rate on the lyophilization cycle. Annealing research may be advanced by the studies from freeze-dry cryostage microscope. Because of extensive applications of lyophilization 25 technology, and larger demand to stabilize the extremely expensive drugs (such as proteins and gene therapy drugs), it is expected that an in-process microscopic monitor should be realized in the pharmaceutical industries soon. The freeze-thaw resistance of a noggin or chimeric noggin molecule can be assayed using 30 one or more of the following systems.
WO 2007/028212 PCT/AU2006/001325 - 148 Co- or post translational modification such as glycosylation may protect proteins from repeated freeze/thaw cycles. To determine this, a noggin or chimeric noggin molecule of the present invention can be compared to carrier-free E. coli-produced counterparts. A noggin or chimeric noggin molecule are diluted into suitable medium (e.g. cell growth 5 medium, PBS or the like) then frozen by various methods, for instance, snap frozen in liquid nitrogen, slowly frozen by being placed at -70 degrees or rapidly frozen on dry ice. The samples are then thawed either rapidly at room temperature or slowly at 4 degrees. Some samples are then refrozen and the process are repeated for a number of cycles. The amount of protein present can be measured by ELISA, and the activity measured in a 10 suitable bioassay chosen by a skilled artisan. The amount of activity/protein remaining is compared to the starting material to detennine the resistance over many the freeze/thaw cycles. A noggin or chimeric noggin molecule of the present invention may have altered thermal 15 stability in solution. The thermal stability of the present invention may be determined in vitro as follows. A noggin or chimeric noggin molecule of the present invention can be mixed into buffer e.g. phosphate buffered saline containing carrier protein e.g. human serum albumin and 20 incubated at a particular temperature for a particular time (e.g. 37 degrees for 7 days). The amount of noggin or chimeric noggin molecule remaining after this treatment can be determined by ELISA and compared to material stored at -70 degrees. The biological activity of the remaining noggin or chimeric noggin molecule is determined by performing a suitable bioassay chosen by a person skilled in the relevant art. 25 The protease resistance of a noggin or chimeric noggin molecule can be assayed using one or more of the following systems. To compare protease resistance, solution containing a noggin or chimeric noggin molecule 30 of the present invention and solution containing E. coli expressed counterparts can be incubated with a protease of choice (e.g. unpurified serum proteases, purified proteases, recombinant proteases) for different time periods. The amount of protein remaining is measured by an appropriate ELISA (e.g. one in which the epitopes recognized by the WO 2007/028212 PCT/AU2006/001325 - 149 capture and detection antibodies are separated by the protease cleavage site), and the activity of the remaining noggin or chimeric noggin molecule is determined by a suitable bioassay chosen by a skilled artisan. 5 The bioavailability of a noggin or chimeric noggin molecule can be assayed using one or more of the following systems. Bioavailability is the degree to which a drug or other substance becomes available to the target tissue after administration. Bioavailability may depend on half life of the drug or its 10 ability to reach the target tissue. Compositions comprising a noggin or chimeric noggin molecule of the present invention is injected subcutaneously or intramuscularly. The levels of noggin can then be measured in the blood by ELISA or radioactive counts. Alternatively, the blood samples can be assayed 15 for activity of the noggin by a suitable bioassay chosen by a skilled artisan, for instance, stimulation of proliferation of a particular target cell population. As the sample will be from plasma or serum, there may be a number of other molecules that could be responsible for the output activity. This can be controlled by using a neutralizing antibody to the protein being tested. Hence, any remaining bioactivity is due to the other serum 20 components. The stability or half-life of a noggin or chimeric noggin molecule can be assayed using one or more of the following systems. 25 A noggin or chimeric noggin molecule of the present invention may have altered half-life in serum or plasma. The half-life of the present invention may be determined in vitro as follows. Composition containing the noggin or chimeric noggin molecule of the present invention can be mixed into human serum/plasma and incubated at a particular temperature for a particular time (e.g. 37 degrees for 4 hours, 12 hours etc). The amount of noggin or 30 chimeric noggin molecule remaining after this treatment can be determined by ELISA. The biological activity of the remaining noggin or chimeric noggin molecule is determined by performing a suitable bioassay chosen by a person skilled in the relevant art. The serum chosen may be from a variety of human blood groups (e.g. A, B, AB, 0 etc.) WO 2007/028212 PCT/AU2006/001325 - 150 The half-life of a noggin or chimeric noggin molecule can also be determined in vivo. Composition containing a noggin or chimeric noggin molecule, which may be labeled by a radioactive tracer or other means, can be injected intravenously, subcutaneously, retro 5 orbitally, tail vein, intramuscularly or intraperitoneally) into the species of choice for the study, for instance, mouse, rat, pig, primate, human. Blood samples are taken at time points after injection and assayed for the presence of the noggin or chimeric noggin molecule (either by ELISA or by TCA-precipitable radioactive counts). A comparison composition consisting of E. coli or CHO-produced noggin or chimeric noggin molecule can be run as a 10 control. To determine the half-life of noggin or chimeric noggin molecule of the present invention, in vivo, male Wag/Rij rats, or other suitable animals can be injected intravenously with a noggin or chimeric noggin molecule. 15 Just before the administration of the substrate, 200pl of EDTA blood is sampled as negative control. At various time points after the injection, 200pl EDTA blood can be taken from the animals using the same technique. After the last blood sampling, the animals are sacrificed. The specimen is centrifuged for 15 min at RT within 30 min of 20 collection. The plasma samples are tested in a specific ELISA to determine the concentration of noggin or chimeric noggin molecule of the present invention in each sample. A noggin or chimeric noggin molecule of the present invention may cross the blood brain 25 barrier. An in vitro assay to determine if noggin or chimeric noggin molecule of the present invention binds human brain endothelial cells can be tested using the following assays. Radiolabeled noggin or chimeric noggin molecule of the present invention can be tested 30 for its ability to bind to human brain capillary endothelial cells. An isolated noggin or chimeric noggin molecule of the present invention can be custom conjugated with WO 2007/028212 PCT/AU2006/001325 - 151 radiolabel to a specific activity using a method known in the art, for instance, with 125I by the chloramine T method, or with 3H Primary cultures of human brain endothelial cells can be grown in flat-bottom 96-well plates until five days post-confluency then lightly fixed using acetone. Cells are lysed, 5 transferred to glass fibre membranes. Radiolabeled noggin or chimeric noggin molecule of the present invention can be detected using a liquid scintillation counter. In vivo assays for the determination of noggin or chimeric noggin molecule of the present invention binding to human brain endothelial cells can be tested using the following 10 assays. A human-specific noggin or chimeric noggin molecule of the present invention are tested for binding to human brain capillaries using sections of human brain tissue that are fresh frozen (without fixation), sectioned on a cryostat, placed on glass slides and fixed in 15 acetone. Binding of [3 H]-noggin or chimeric noggin molecule of the present invention are examined on brain sections using quantitative autoradiography. In vivo assay can be used to measure tissue distribution and blood clearance of human specific noggin or chimeric noggin molecule of the present invention in a primate system. 20 A noggin or chimeric noggin molecule of the present invention is used to determine the tissue distribution and blood clearance of [14 C]-labeled noggin or chimeric noggin molecule of the present invention in 2 male cynomolgus monkeys or other suitable primates. noggin or chimeric noggin molecule of the present invention is administered 25 concurrently with a [3 H]-labeled control protein to the animals with an intravenous catheter. During the course of the study, blood samples are collected to determine the clearance of the proteins from the circulation. At 24 hours post-injection, the animals are euthanized and selected organs and representative tissues collected for the determination of isotope distribution and clearance by combustion. In addition, capillary depletion 30 experiments are performed to samples from different regions of the brain in accordance with Triguero, et al. J of Neurochemistry 54:1882-1888 (1990). This method removes WO 2007/028212 PCT/AU2006/001325 - 152 greater than 90% of the vasculature from the brain homogenate (Triguero et al. cited supra). The time-dependent redistribution of the radiolabeled noggin or chimeric noggin molecule 5 of the present invention from the capillary fraction to the parenchyma fraction is consistent with the time dependent migration of a noggin or chimeric noggin molecule of the present invention across the blood-brain barrier. Noggin or chimeric noggin molecule of the present invention may have a distinct 10 immunoreactivity profile determined by immunoassay techniques, which involve the interaction of the molecule with one or more antibodies directed against the molecule. Examples of immunoassay techniques include enzyme-linked immunoabsorbant assays (ELISA), dot blots and immunochromatographic assays such as lateral flow tests or strip tests. 15 The level of the noggin or chimeric noggin molecule may be measured using an immunoassay procedure, for example, a commerically purchased ELISA kit. Noggin or chimeric noggin molecule of the present invention may have a different immunoreactivity profile to non-human cell expressed noggin or chimeric noggin molecules due to the 20 specificity of the antibodies provided in an immunoassay kit. For instance, the capture and/or detection antibodies of the immunoassay may be antibodies specifically directed against non-human cell expressed human noggin. In addition, incorrect folding of the non-human cell expressed human noggin may result in 25 the exposure of antigenic epitopes which are not exposed on the correctly folded human cell expressed human noggin. Incorrect folding may arise through, for instance, overproduction of heterologous proteins in the cytoplasm of non-human cells, for example, E. coli (Baneyx Current Opinion in Biotechnology 10:411-421, 1999). Further, non-human cell expressed human noggin may have a different pattern of post-translational 30 modifications to that of the noggin or chimermic noggin molecule of the present invention. For example, the non-human cell expressed human noggin may exhibit abnormal quantities and/or types of carbohydrate structures, phosphate, sulfate, lipid or other WO 2007/028212 PCT/AU2006/001325 - 153 residues. This may result in the exposure of antigenic epitopes which are not exposed on the noggin or chimeric noggin of the present invention. Conversely, an altered pattern of post-translational modifications may result in an absence of antigenic epitopes on the noggin or chimeric noggin of the present invention which are exposed on the non-human 5 cell expressed human noggin. Any one of, or combination of, the above-mentioned factors may lead to inaccurate measurements of: 10 (a) naturally occurring human noggin in laboratory samples or human tissues, or (b) human cell expressed recombinant human noggin or chimeric noggin molecule in laboratory samples, human tissues or in human embryonic stem cell (hES) culture media. 15 The immunoreactivity profile of a human cell expressed human noggin or chimeric noggin molecule, as determined by the use of a suitable immunoassay, may provide an indication of the protein's immunogenicity in the human, as described hereinafter. The immunogenicity of noggin or chimeric noggin molecule can be assayed using one or 20 more of the following systems. Most biologic products elicit a certain level of antibody response against them. The antibody response can, in some cases, lead to potentially serious side effects and/or loss of efficacy. For instance, some patients treated with recombinant noggin or chimeric noggin 25 molecule expressed from non-human cells may generate neutralizing antibodies particularly during long-term therapeutic use reducing noggin efficacy and or contributing to side effects. Noggin expressed from human cells is unlikely to generate neutralizing antibodies therefore increasing its therapeutic efficacy compared with non-human cell expressed noggin. 30 Immunogenicity is the property of being able to evoke an immune response within an organism. Immunogenicity depends partly upon the size of the substance in question and WO 2007/028212 PCT/AU2006/001325 - 154 partly upon how unlike host molecules it is. A noggin or chimeric noggin molecule may have altered immunogenicity due to its novel physiochemical characteristics. For instance, the glycosylation structure of a noggin or chimeric noggin molecule may shield or obscure the epitope(s) recognized by the antibody and therefore preventing or reducing antibody 5 binding to the noggin or chimeric noggin molecule. Alternatively, some antibodies may recognize a glycopeptide epitope not present in the non-glycosylated version of the protein. The ability of patient samples to recognize a noggin or chimeric noggin molecule with a distinctive physiochemical form can be determined by various immunoassays. A properly 10 designed immunoassay involves considerations directing to appropriate detection, quantitation and characterization of antibody responses. A number of recommendations for the design and optimization of immunoassays are outlined in Mire-Sluis et al. JImmunol Methods 289(1-2):1-16, 2004, which is incorporated by reference. 15 The use of noggin or chimeric noggin molecule on therapeutic implants can be assayed using one or more of the following systems. The present invention extends to the use of a noggin or chimeric noggin molecule to manipulate stem cells, such as achieving higher proliferation, enhanced differentiation, 20 maintenance in a desired state of differentiation and/or greater lineage specificity of differentiation. In particular, the present invention extends to the use of a noggin or chimeric molecule to maintain hES in a desired state of differentiation. Methods known in the art (as described in Hoffman et al. Nat Biotechnol 23(6):699-708, 2005) can be used to assess whether the hES have been maintained in a particular desired state, indicators to 25 assess the phenomenon include: expression of markers (e.g. SSEA-4, TRA-1-60, TRA-1 81), expression of telomerase, ability to differentiate and maintenance of a nonnal karyotype. In addition, the subject invention also provides a human derived noggin or chimeric noggin 30 molecule for use in culturing hES, reducing the potential risks of transferring animal derived infectious agents to hES, thus allowing hES to be applicable for therapeutic applications.
WO 2007/028212 PCT/AU2006/001325 - 155 A major therapeutic use of stem cells is in regeneration of tissue, cartilage or bone. In one embodiment, the cells are likely to be introduced to the body in a biocompatible three dimensional matrix, The implant will consist of a mixture of cells, the scaffold, growth 5 factors and accessory components such as biodegradable polymers, proteoglycans and the like. Incorporation of a noggin or chimeric noggin molecule into these matrices during their construction is proposed to regulate the behavior of the cells. Such implants may be used for the formation of bone, the growth of neurons from progenitor cells, chondrocyte implantation for cartilage replacement and other applications. 10 A noggin or chimeric noggin molecule of the present invention may interact differently with the matrix used for the formation of the implant, as well as regulating the cells incorporated within the implant. It is anticipated that the combination of a noggin or chimeric noggin molecule of the present invention with the implant components will result 15 in one or more of the following pharmacological traits, such as higher proliferation, enhanced differentiation, maintenance in a desired state of differentiation, greater lineage specificity of differentiation, enhanced secretion of matrix components, better 3 dimensional structure formation, enhanced signaling, better structural performance, reduced toxicity, reduced side effects, reduced inflammation, reduced immune cell 20 infiltrate, reduced rejection, longer duration of the implant, longer function of the implant, better stimulation of the cells surrounding the implant, better tissue regeneration, better organ function, or better tissue remodeling. The effects of noggin or chimeric noggin molecule on differential gene expression can be 25 assayed using one or more of the following systems. The differences in gene expression can be analyzed in cells exposed to a noggin or chimeric noggin molecule. 30 Microarray technology enables the simultaneous determination of the mRNA expression of almost all genes in an organism's genome. This method uses gene "chips" in which oligonucleotides corresponding to the sequences of different genes are attached to a solid WO 2007/028212 PCT/AU2006/001325 - 156 support. Labeled cDNA derived from mRNA isolated from the cell or tissue of interest is incubated with the chips to allow hybridisation between cDNA and the attached complementary sequence. A control is also used, and following hybridisation and washing the signal from both is compared. Specialised software is used to determine which genes 5 are up or down regulated or which have unchanged expression. Many thousands of genes can be analysed on each chip. For example using Affymetrix technology, the Human Genome U133 (HG-U133) Set, consisting of two GeneChip (registered trade mark) arrays, contains almost 45,000 probe sets representing more than 39,000 transcripts derived from approximately 33,000 well-substantiated human genes. The GeneChip (registered trade 10 mark) Mouse Genome 430 2.0 contains over 39,000 transcripts on a single array. This type of analysis reveals changes in the global mRNA expression pattern and therefore differences in the expression of genes not known to be controlled by a particular stimulus may be uncovered. This technology is hence suitable to analyze the induced gene 15 expression associated with noggin or chimeric noggin molecule of the present invention. The definition of known and novel genes regulated by the particular stimulus will assist in the identification of the biochemical pathways that are important in the biological activity of the particular noggin or chimeric noggin molecule of the present invention. This 20 information will be useful in the identification of novel therapeutic targets. The system could also be used to look at differences in gene expression induced by a noggin or chimeric noggin molecule of the present invention as compared to commercially available products. 25 The effects of noggin or chimeric noggin molecule on binding ability can be assayed using one or more of the following systems. The binding ability of a noggin or chimeric noggin molecule of the present invention to 30 various substances, including extracellular matrix, artificial materials, heparin sulfates, carriers or co-factors can be investigated.
WO 2007/028212 PCT/AU2006/001325 - 157 The effects of a noggin or chimeric noggin molecule on the ability of a particular protein to bind an extracellular matrix can be determined using the following assays. A surface is coated with extracellular matrix proteins, including but not limited to collagen, 5 vitronectin, fibronectin, laminin, in an appropriate buffer. The unbound sites can be blocked by methods known in the art, for instance, by incubation with BSA solution. The surface is washed, for instance, with PBS solutions, then a solution containing the protein to be tested, for instance a noggin or chimeric noggin molecule of the present invention, is added to the surface. After coating, the surface is washed and incubated with an antibody 10 that recognizes a noggin or chimeric noggin molecule. Bound antibody is then detected, for instance, by an enzyme-linked secondary antibody that recognizes the primary antibody. The bound antibodies are visualized by incubating with the appropriate substrate and observing a colour change reaction. Glycosylated proteins may adhere more strongly to the extracellular matrix proteins than unglycosylated proteins. 15 Alternatively, an equivalent amount (specified by ELISA concentration or bioassay activity units) of a noggin or chimeric noggin molecule of the present invention, or a counterpart noggin or chimeric noggin molecule expressed by non-human cells, are incubated with matrix coated wells, then following washing of the wells the amount bound 20 is determined by ELISA. The amount bound can be indirectly measured by a drop in ELISA reactivity following incubation of the sample with the coated surface. The ability of noggin or chimeric noggin molecule to bind artificial materials can be assayed using one or more of the following systems. 25 In order to determine the binding ability of a noggin or chimeric noggin molecule to artificial materials, a surface is coated with artificial material, including but not limited to metals, scaffolds, in an appropriate buffer. The surface is washed, for instance, with PBS solutions, then a solution containing the protein to be tested, for instance a noggin or 30 chimeric noggin molecule of the present invention, is added to the surface. After coating, the surface is washed and incubated with an antibody that recognizes a noggin or chimeric noggin molecule. Bound antibody is then detected, for instance, by a enzyme-linked secondary antibody that recognizes the primary antibody. The bound antibodies are WO 2007/028212 PCT/AU2006/001325 - 158 visualized by incubating with the appropriate substrate and observing a color change reaction. Alternatively, an equivalent amount (specified by ELISA concentration or bioassay 5 activity units) of a noggin or chimeric noggin molecule of the present invention, and a counterpart noggin or chimeric noggin molecule expressed by non-human cells, are incubated with wells coated by artificial materials, the wells are then washed and the amount bound is determined by ELISA. The amount bound can be indirectly measured by a drop in ELISA reactivity following incubation of the sample with the coated surface. 10 Ability to bind to artificial surfaces may have biological consequences, for instance, in stent coating. Alternatively, a scaffold coated with a noggin or chimeric noggin molecule of the present invention is used to seed cells on. The cell growth and differentiation is then monitored and compared to uncoated or differentially coated scaffolds. 15 The ability of noggin or chimeric noggin molecule to bind to heparin sulfates can be assayed using one or more of the following systems. A noggin or chimeric noggin molecule of the present invention is expected to interact 20 differentially with heparin sulfates due to their physiochemical form. These differences are expected to be evident in experimental models of cell proliferation, differentiation, migration and the like. The combination of a noggin or chimeric noggin molecule with heparin sulfates is expected to have distinctive pharmacological traits for a given treatment. This may be an increase in serum half-life, bioavailability, reduced immune 25 related clearance, greater efficacy, reduced dosage fewer side effects and related advantages. The ability of noggin or chimeric noggin molecule to bind to carriers or co-factors can be assayed using one or more of the following systems. 30 WO 2007/028212 PCT/AU2006/001325 -159 Noggin or chimeric noggin molecule will be bound to other molecules when they are present in plasma. These molecules may be termed "carriers" or "co-factors" and will influence such factors as bioavailability or serum half life. 5 Incubating purified versions of the proteins in plasma and analyzing the resulting solution by size exclusion chromatography can determine the interaction of a noggin or chimeric noggin molecule of the present invention with their binding partners. If the noggin or chimeric noggin molecule binds a co-factor, the resulting complex will have a larger molecular weight, resulting in an altered elution time. The complex can be compared for 10 biological activity, in vitro or in vivo half-life and bioavailability. The effects of noggin or chimeric noggin molecule on bioassays can be assayed using one or more of the following systems. 15 Various bioassays can be performed to test the activity of a noggin or chimeric noggin molecule of the present invention, including assays on cell proliferation, cell differentiation, cell apoptosis, cell size, cytokine/cytokine receptor adhesion, cell adhesion, cell spreading, cell motility, migration and invasion, chemotaxis, ligand-receptor binding, receptor activation, signal transduction, and alteration of subgroup ratios. 20 The effects of noggin or chimeric noggin molecule on cell proliferation can be assayed using one or more of the following systems. Cells, in a particular embodiment, exponentially growing cells, are incubated in a growth 25 medium in the presence of a noggin or chimeric noggin molecule of the present invention. This can be performed in flasks or 96 well plates. The cells are grown for a period of time and then the number of cells is determined by either a direct (e.g. cell counting) or an indirect (MTT, MTS, tritiated thymidine) method. The increase or decrease in proliferation is determined by comparison with a medium only control assay. Different concentrations 30 of noggin or chimeric noggin molecule can be used in parallel series of experiments to get a dose response profile. This can be used to determine the ED50 and ED100 (the dose required to generate the half maximal and maximal response effectively).
WO 2007/028212 PCT/AU2006/001325 -160 The effects of noggin or chimeric noggin molecule on cell differentiation or maintenance of cells in an undifferentiated state can be assayed using one or more of the following systems. 5 Cells are incubated in a growth medium in the presence of a noggin or chimeric noggin molecule of the present invention. After a suitable period of time, the cells are assayed for indicators of differentiation. This may be the expression of particular markers on the cell surface, cytoplasmic markers, an alteration in the cell dimensions, shape or cytoplasmic 10 characteristics. The markers may include proteins, sugar structures (e.g. glycosaminocglycans such as heparin sulfates, chondroitin sulfates etc.) lipids (glycosphingolipids or lipid bilayer components). These changes can be assayed by a number of techniques including microscopy, western blot, FACS staining or forward/side scatter profiles. 15 The effects of noggin or chimeric noggin molecule on cell apoptosis can be assayed using one or more of the following systems. Apoptosis is defined as programmed cell death, and is distinct from other methods of cell 20 death such as necrosis. It is characterized by defined changes in the cells, such as activation of signaling pathways (e.g. Fas, TNFR) resulting in the activation of a subset of proteases know as caspases. Initiator caspase activation leads to the activation of the executioner caspases which cleave a variety of cellular proteins resulting in nuclear fragmentation, cleavage of nuclear lamins, blebbing of the cytoplasm and destruction of 25 the cell. Apoptosis can be induced by protein ligands such as FasL, TNFa and lymphotoxin or by signals such as UV light and substances causing DNA damage. Cells are incubated in a growth medium in the presence of noggin or chimeric noggin molecule and or other agents as suitable for the assay. For instance, the presence of agents 30 able to block transcription (actinomycin D) or translation (cycloheximide) may be required. Following incubation for an appropriate period, the number of cells is determined by a suitable method. A decrease in cell number may indicate apoptosis. Other indications of apoptosis may be obtained by staining of the cells, for instance, for annexins or WO 2007/028212 PCT/AU2006/001325 - 161 observing characteristic laddering patterns of DNA. Further evidence for the confirmation of apoptosis may be achieved by preventing the expression of apoptotic markers by incubating with cell permeable caspases inhibitors (e.g. z-VAD FMK), then assaying for apoptotic markers. 5 A noggin or chimeric noggin molecule of the present invention may prevent apoptosis by providing a survival signal through cellular survival pathways such as the Bcl2 or Akt pathways. Activation of these pathways can be confirmed by western blotting for an increase in cellular Bcl2 expression, or for an increase in the activated (phosphorylated) 10 form of Akt using a phospho-specific antibody directed against Akt. For this assay, cells are incubated in the presence or absence of the survival factor (e.g. IL 3 and certain immune cells). A proportion of cells incubated in the absence of the survival factor will die by apoptosis upon extended culture, whereas cells incubated in sufficient 15 quantities of survival factor will survive or proliferate. Activation of the cellular pathways responsible for these effects can be determined by western blotting, immunocytochemistry and FACS analysis. The effects of noggin or chimeric noggin molecule on the inhibition of apoptosis can be 20 assayed using one or more of the following systems. A noggin or chimeric noggin molecule of the present invention is tested for it's in vitro activity to protect rat-, mouse-and human cortical neural cells from cell death under hypoxic conditions and with glucose deprivation. For this, neural cell cultures are prepared 25 from rat embryos. To evaluate the effects of noggin or chimeric noggin molecule of the present invention, the cells are maintained in modular incubator chambers in a water jacketed incubator for up to 48 hours at 37C*, in serum-free medium with 30 mM glucose and humidified 95% air/ 5%CO 2 (normoxia) or in serum-free medium without glucose and humidified 95% N 2 / 5% CO 2 (hypoxia and glucose deprivation), in the absence or 30 presence of noggin or chimeric noggin molecule of the present invention. The cell cultures are exposed to hypoxia and glucose deprivation for less than 24 hours and thereafter returned to normoxic conditions for the remainder of 24 hours. The cytotoxicity is WO 2007/028212 PCT/AU2006/001325 -162 analyzed by the fluorescence of Alamar blue, which reports cell viability as a function of metabolic activity. In another method, the neural cell cultures are exposed for 24 hours to 1 mM L-glutamate 5 or a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) under normoxic conditions, in the absence or presence of various concentrations of noggin or chimeric noggin molecule of the present invention. The cytotoxicity is analyzed by the fluorescence of Alamar blue, which reports cell-viability as a function of metabolic activity. 10 Noggin or chimeric noggin molecule may affect the growth, apoptosis, development, or differentiation of a variety of cells. These changes can be reflected by, among other measurable parameters, changes in the cell size and changes in cytoplasmic complexity, which are due to intracellular organelle development. For instance, keratinocytes induced to differentiate by suspension culture exhibit downregulation of surface markers such as PI 15 integrins, an increase in cell size and cytoplasmic complexity. The effects of noggin or chimeric noggin molecule on cell size, or cytoplasmic complexity can be assayed using one or more of the following systems. FACS measures the amount of light scattered off by a cell when a beam of laser is incident 20 on it. An argon laser providing light with a wavelength of 488nm is frequently used. The larger the size of the cell, the greater the disruption of the beam of light in the forward direction, hence the level of forward scatter corresponds to the size of the cell. In order to measure changes in cell size, cells treated with a noggin or chimeric noggin molecule of the present invention are diluted in sheath fluid and injected into the flow cytometer 25 (FACSVantage SE, Becton Dickinson). Untreated cells act as a control. The cells pass through a beam of light and the amount of forward scattering of the light corresponds to the size of the cells. Changes in intracellular organelle growth and development (cytoplasmic complexity) can 30 also be measured by FACS. The intracellular organelles of the cell scatter light sideways. Hence, change in cytoplasmic complexity can be measured by the amount of side scattering of light by the cells by the above method, and the level of complexity of WO 2007/028212 PCT/AU2006/001325 - 163 intracellular organelles and the level of granularity of the cell can be estimated by measuring the level of side scatter of light given off by the cells. The effect of a noggin or chimeric noggin molecule on cell size or cytoplasmic complexity 5 can be assessed by using FACS to compare the profiles given off by, for instance, 20,000 treated cells with the signals emitted by identical number of untreated cells. By comparing the signals from the different treated populations of cells, the relative changes in cell size and cytoplasmic complexity can be determined. 10 The effects of noggin or chimeric noggin molecule on cell growth, apoptosis, development, or differentiation can be assayed using one or more of the following systems. Noggin or chimeric noggin molecule induced apoptosis and changes in cell growth or cycles can be assessed by labeling the DNA of treated cells with dyes such as propidium 15 iodine which has an excitation wavelength in the range of 488 nm and emission at 620 m. Cells undergoing apoptosis has condensed DNA as well as different size and granularity. These factors give specific forward and size scatter profiles as well as fluorescence signal, and hence the population of cells undergoing apoptosis can be differentiated from normal cells. The amount of DNA in a cell also reflects which state of the cell cycle the cell is in. 20 For instance, a cell in G2 stage will have twice the amount of DNA as a cell in Go state. This will be reflected by a doubling of the fluorescence signal given off by a cell in G 2 phase. The effect of a noggin or chimeric noggin molecule can be assessed by using FACS to compare the fluorescence signals given off by for instance, 20,000 treated cells with the signals emitted by identical number of untreated cells. 25 Noggin or chimeric noggin molecule may also alter the expression of various proteins. The effects of noggin or chimeric noggin molecule on protein expression by cells can be assayed using one or more of the following systems. 30 To assess the increase and decrease in expression of a protein in an entire cell, the cells can be fixed and permeabilised, then incubated with fluorescence conjugated antibody targeting the epitope of the protein of interest. A large variety of fluorescent labels can be WO 2007/028212 PCT/AU2006/001325 -164 used with an Argon laser system. Fluorescent molecules such as FITC, Alexa Fluor 488, Cyanine 2, Cyanine 3 are commonly used for this experiment. This method can also be used to estimate the changes in expression of surface markers and proteins by labeling non-permeabilised cells where only the epitope exposed on the cell surface can be labeled 5 with antibodies. The effect of a noggin or chimeric noggin molecule can be assessed by using FACS to compare the fluorescence signals given off by, for instance, 20,000 treated cells with the signals emitted by identical number of untreated cells. The effects of noggin or chimeric noggin molecule on ligand/receptor adhesion can be 10 assayed using one or more of the following systems. A noggin or chimeric noggin molecule of the present may be more or less adhesive to substrates compared to those of a previously known physiochemical form. The interaction may be with protein receptors for sugar structures (e.g. L-selectin), with extracellular 15 matrix components such as fibronectin, collagens, vitronectins, and laminins, or with non protein components such as sugar molecules (heparin sulfates, other glycosaminoglycans). A noggin or chimeric noggin molecule may also interact differently with non-biological origin materials such as tissue culture plastics, medical device components (e.g. stents or 20 other implants) or dental materials. In the case of medical devices this may alter the engraftment rates, the interaction of the implant with particular classes of cell type or the type of linkage formed with the body. Any suitable assays for protein adhesion can be employed. For instance, a solution 25 containing a noggin or chimeric noggin molecule of the present invention is incubated with a binding partner, in a particular embodiment, on an immobilised surface. Following incubation, the amount of noggin or chimeric noggin molecule present in the solution is assayed by ELISA and the difference between the amount remaining and the starting material is what has bound to the binding partner. For instance, the interaction between 30 noggin or chimeric noggin molecule and an extracellular matrix protein could be determined by first coating wells of a 96 well plate with the ECM protein (e.g. fibronectin). Non-specific binding is then blocked by incubation with a BSA solution. Following washing, a known concentration of a noggin or chimeric noggin molecule WO 2007/028212 PCT/AU2006/001325 - 165 solution is added for a defined period. The 'solution is then removed and assayed for the amount of noggin or chimeric noggin molecule remaining in solution. The amount bound to the ECM protein can be determined by incubating the wells with an antibody to a noggin or chimeric noggin molecule, then detecting with an appropriate system (either a 5 labeled secondary antibody or by biotin-avidin enzyme complexes such as those used for ELISA). Methods for determining the amount bound to other surfaces may involve hydrolyzing a noggin or chimeric noggin molecule from the inert implant surface, then measuring the 10 amino acids present in the solution. The effects of noggin or chimeric noggin molecule on cell adhesion can be assayed using one or more of the following systems. 15 Cell adhesion to matrix (e.g. extracellular matrix components such as fibronectin, vitronectin, collagen, laminin etc.) is mediated at least in part by the integrin molecules. Integrin molecules consist of alpha and beta subunits, and the particular combinations of alpha and beta subunit give rise to the binding specificity to a particular ligand (e.g. a2bl integrin binds collagen, a5bl binds fibronectin etc). The integrins subunits have large 20 extracellular domains responsible for binding ligand, and shorter cytoplasmic domains responsible for interaction with the cytoskeleton. In the presence of ligand, the cytoplasmic domains are responsible for the induction of signal transduction events ("outside in signaling"). The affinity of integrins for their ligands can be modulated by extracellular signaling events that in turn lead to changes in the cytoplasmic tails of the integrins 25 ("inside out signaling"). Incubation with noggin or chimeric noggin molecule of the present invention can potentially alter cell adhesion in a number of ways. First, it can alter qualitatively the expression of particular integrin subsets, leading to changes in binding ability. Secondly, 30 the amount of a particular integrin expressed may alter, leading to altered cell binding to its target matrix. Thirdly, the affinity of a particular integrin may be altered without changing its surface expression (inside-out signaling). All these changes may alter the binding of cells to either a spectrum of ligands, or alter the binding to a particular ligand.
WO 2007/028212 PCT/AU2006/001325 -166 A noggin or chimeric noggin molecule of the present invention can be tested in Cell-ECM adhesion assays which are generally performed in 96 well plate. Wells are coated with matrix, then unbound sites within the wells are blocked with BSA. A defined number of 5 cells are incubated with the coated wells, then unbound cells are washed away and the bound cells incubated in the presence or absence of noggin or chimeric noggin molecule. The number of cells is determined by an indirect method such as MTT/MTS. Alternatively, the cells are labeled with a radioactive label (e.g. 51 Cr) and a known amount of radioactivity (i.e. cells) is added to each well. The amount of bound radioactivity is 10 determined and calculated as a percentage of the amount loaded. Cells also adhere to other cells, for instance, adhesion of one population of cells to a monolayer of another type of cells. To assay for this, the suspension cells added to the monolayer cells would be labeled with radioactivity. The cells are then incubated in the 15 presence or absence of noggin or chimeric noggin molecule. The unbound cells would be washed away and the remaining mixed population of cells can be lysed and assayed for the amount of radioactivity present. The effects of noggin or chimeric noggin molecule on cell spreading can be assayed using 20 one or more of the following systems. A noggin or chimeric noggin molecule of the present invention may have altered effects on cell spreading. Initiation of cell spreading is a key step in cell motility and invasive behavior. Cells spreading can be initiated in vitro in a number of ways. Plating a 25 suspension of cells onto ECM components will result in attachment and ligand binding by integrin receptors. This initiates signal transduction events resulting in the activation of a family of the Cdc42, Rac and Rho small GTPases. Activation of these proteins results in actin polymerization and an extension of a lamellipodium, resulting in gradual flattening of the cells and contact of more integrins with their receptors. Eventually the cells have 30 flattened totally and formed focal adhesions (large structures containing integrins and signaling proteins). Cell spreading can also be initiated by stimulation of adherent cells with growth factors, again resulting in activation of the Cdc42/Rac/Rho proteins and lamellipodium formation.
WO 2007/028212 PCT/AU2006/001325 -167 Cell spreading can be quantitated by examining a large number of cells at different time points following stimulation with noggin or chimeric noggin molecule. The area of each cell can be determined using image analysis programs and the percentage of cells spread as 5 well as the degree of cell spreading can be compared with time. More rapid spreading may be initiated by a higher activation of the Cdc42/Rac/Rho pathways, alternatively, temporal, qualitative and quantitative differences in their activation may be observed with a noggin or chimeric noggin molecule of the present invention. This in turn may reflect differences in the signaling events induced by the noggin or chimeric noggin moleculeof the present 10 invention. The effects of noggin or chimeric noggin molecule on cell motility, migration and invasion can be assayed using one or more of the following systems. 15 Cells adherent to a tissue culture dish do not remain statically anchored to one spot, but rather constantly extend and retract portions of their cell body. When viewed under time lapse photography, the cells can be observed to move around the dish, either as isolated single cells or as a cell colony. This motion may be either "random walk" (i.e. not directed in a particular direction), or directional. Both types of motion can be increased by the 20 addition of growth factors. Time-lapse photography can be used to quantitate the overall distance covered by the cells in a given time period, as well as the overall directionality. In the case of directional migration, cells will move towards a source of chemoattractant by sensing the chemical gradient and orienting their migration machinery towards it. In many 25 instances, the chemoattractant is a growth factor. Directional migration can be quantitated by providing a source of chemoattractant (e.g. via a thin pipette) then imaging the cells migrating towards it with timelapse photography. An alternative system for determining directed migration is the Boyden chamber assay. In 30 this assay, cells are placed in an upper chamber that is connected to a lower chamber via small holes in the partitioning membrane. Growth medium is put in both chambers, but chemoattractant is added only to the lower chamber, resulting in a diffusion gradient WO 2007/028212 PCT/AU2006/001325 -168 between the two chambers. The cells are attracted to the growth factor source and migrate through the holes in the separation membrane and on to the lower side of the membrane. After a number of hours, the membrane is removed and the number of cells that has migrated onto the bottom of the membrane is determined. 5 The process of cellular invasion utilises many of the same components as migration. Cell invasion can be modeled using layers of extracellular matrix through which the cells invade. For instance, Matrigel is a mixture of basement membrane components (ECM components, growth factors etc.) that is liquid at 4 degrees but rapidly sets at 37 degrees to 10 form a gel. This can be used to coat the upper surface of a Boyden chamber, and the chemoattractant added to the lower layer. For cells to pass onto the lower surface of the membrane, they must degrade the matrigel using enzymes such as collagenases and matrix metalloproteinases (MMPs) as well as migrating directionally towards the chemoattractant. This assay mimics the various processes required for cellular invasion. 15 The effects of noggin or chimeric noggin molecule on chemotaxis can be assayed using one or more of the following systems. The migration of cells toward the chemoattractant can be measured in vitro in a Boyden 20 chamber. A noggin or chimeric noggin molecule of the present in invention is placed in the lower chamber and an appropriate target cell population is placed in the upper chamber. To mimic the in vitro process of immune cells migrating from the blood to sites of inflammation, migration through a layer of cells may be measured. Coating the upper surface of the well of the Boyden chamber with a confluent sheet of cells, for instance, 25 epithelial, endothelial or fibroblastic cells, will prevent direct migration of immune cells through the holes in the well. Instead, the cells will need to adhere to the monolayer and migrate through it towards the protein to be tested. The presence of cells on the under surface of the Boyden chamber or in the medium in the lower well in only those wells treated with noggin or chimeric noggin molecule is indicative of the chemotactic ability of 30 the noggin or chimeric noggin molecule. To show that the effect is specific to a noggin or chimeric noggin molecule, a neutralising antibody can be incubated with the protein in the lower chamber.
WO 2007/028212 PCT/AU2006/001325 -169 Alternatively, to test the ability of a substance (chemical, protein, sugar) to prevent chemotaxis, the substance is included in the lower chamber of the Boyden chamber along with a solution containing known chemotactic ability (this may be a specific chemokine, 5 conditioned medium from a cell source or cells secreting a range of chemokines). A susceptible target cell population is then added to the upper chamber and the assay performed as described above. The effects of noggin or chimeric noggin molecule on ligand-receptor binding can be 10 assayed using one or more of the following systems. A noggin or chimeric noggin molecule of the present invention may have different ligand receptor binding abilities. Ligand-receptor binding can be measured by various parameters, for instance, the dissociation constant (Kd), dissociation rate constant (off rate) (k~), 15 association rate constant (on rate) (k+). Differences in ligand-receptor binding may correlate with different timing and activation of signaling, leading to different biological outcomes. Ligand-receptor binding can be measured and analysed by either Scatchard plot or by other 20 means such as Biacore. For Scatchard analysis, noggin or chimeric noggin molecule, labeled with, for instance, radioactively labeled (eg, 1251), is incubated in the presence of differing amounts of cold competitor of a noggin or chimeric noggin molecule, with cells, or extracts thereof, 25 expressing the corresponding ligand or receptor. The amount of specifically bound labeled noggin or chimeric noggin molecule is determined and the binding parameters calculated. For the Biacore, the corresponding recombinant ligand or receptor of noggin or chimeric noggin molecule is coupled to the detection unit. Solutions containing a noggin or chimeric 30 noggin molecule of choice are then passed over the detection cell and binding is determined by a change in the properties of the detection unit. On rates can be determined by passing solutions containing the noggin or chimeric noggin molecule over the detection WO 2007/028212 PCT/AU2006/001325 - 170 cell until a fixed reading is recorded (when the available sites are all occupied). A solution not containing the noggin or chimeric noggin molecule is then passed over the cell and the protein dissociates from the corresponding ligand or receptor, giving the off rate. 5 The effects of noggin or chimeric noggin molecule on receptor activation can be assayed using one or more of the following systems. Interaction with noggin or chimeric noggin molecule and its corresponding ligand or receptor may be paralleled by differences in the signaling events induced from the cell's 10 endogenous noggin. The timing of interaction may be characteristic of a noggin or chimeric noggin molecule as definitely on/off rates or dissociation constants. Activated receptors are often internalized by the cells. The receptor/ligand complex can then be dissociated (e.g., be lowering the pH within cellular vesicles, resulting in 15 detachment of the ligand) and the receptor recycled to the cell surface. Alternatively, the complex may be targeted for destruction. In this case the receptors are effectively down regulated and unable to generate more signal, whereas when they are recycled they are able to repeat the signaling process. Differential receptor binding or activation may result in the receptor being switched from a destruction to a recycling pathway, resulting in a stronger 20 biological response. The effects of noggin or chimeric noggin molecule on signal transduction can be assayed using one or more of the following systems. 25 Binding of ligands or receptors to noggin or chimeric noggin molecule initiates signaling, and may include reverse signaling, through a variety of cytoplasmic proteins. Reverse signaling occurs when a membrane-bound form of a ligand transduces a signal following binding by a soluble or membrane bound version of its receptor. Reverse signaling can also occur after binding of the membrane bound ligand by an antibody. These signaling events 30 (including reverse signaling events) lead to changes in gene and protein expression. Hence, a noggin or chimeric noggin molecule of the present invention can induce or inhibit different signal transductions in various pathways or other signal transduction events, such WO 2007/028212 PCT/AU2006/001325 - 171 as the activation of JAK/STAT pathway, Ras-erk pathway, AKT pathway, the activation of PKC, PKA, Src, Fas, TNFR, NFkB, p38MAPK, c-Fos, recruitment of proteins to receptors, receptor phosphorylation, receptor internalization, receptor cross-talk or secretion. 5 The ligands or receptors recruited to the noggin or chimeric noggin molecule may be unique to the noggin or chimeric noggin molecule of the present invention, due to different conformations of the ligand or receptors being induced. One way of assaying for these differences is to immunoprecipitate the ligand or receptor using an antibody crosslinked to 10 sepahrose beads. Following immunoprecipitation and washing, the proteins are loaded on a 2D gel and the comparative spot patterns are analysed. Different spots can be cut out and identified by mass spectrometry. The effects of noggin or chimeric noggin molecule on up regulation and down regulation 15 of surface markers can be assayed using one or more of the following systems. Cells have a variety of responses to noggin or chimeric noggin molecule. There are a range of proteins on cell surfaces responsible for communication between the cells and the extracellular environment. Through regulated processes of endocytosis and exocytosis, 20 various proteins are transported to and from the cell surface. Typical proteins found on the cells surface includes receptors, binding proteins, regulatory proteins and signaling molecules. Changes in expression and degradation rate of the proteins also changes the level of the proteins on the cell surface. Some proteins are also stored in intracellular reservoirs where specific signals can induce trafficking of proteins between this storage 25 and the cellular membrane. Cells are incubated for an appropriate amount of time in medium containing a noggin or chimeric noggin molecule of the present invention and their responses can be compared with cells exposed to the same medium without noggin or chimeric noggin molecule of the 30 present invention. The proteins on the cell membrane can be solubilised and separated from the cells by centrifugation. The level of expression of a specific protein can be measured by Western blotting. Cells can also be labeled with fluorescence conjugated antibodies, and visualized under confocal microscopy system or counted by fluorescence WO 2007/028212 PCT/AU2006/001325 -172 activated cell sorting (FACS). This will detect any changes in expression and distribution of proteins on the cells. By using multiple antibodies, changes in protein interaction can also be studied by confocal microscopy and immuno-precipitation. Similarly, these experiments can be extended to in vivo animal models. Cells from specific part of animals 5 treated with different noggins or chimeric noggin molecules can be extracted and examined with identical methodologies. Cells induced to differentiate in vitro or in vivo by the addition of noggin or chimeric noggin molecule of the present invention will express differentiation markers that 10 distinguish them from the untreated cells. Some cells, for instance, progenitor or stem cells, can differentiate into many subpopulations, distinguishable by their surface markers. A noggin or chimeric noggin molecule of the present invention may stimulate the progenitor cells to differentiate into subgroups in a particular ratio. 15 Noggin or chimeric noggin molecule may have effects upon cell repulsion. The effects of noggin or chimeric noggin molecule on the modulation of the growth and guidance of cells and neurons is a convenient assay for cell repulsion. 20 Disrupting the interactions between a noggin or chimeric noggin molecule subunits and other components leads to a way to inhibit the biological effects of noggin or chimeric noggin molecule. Compounds inhibiting such biological effects are identified by a number of ways. 25 High throughput screening programs use a library of small chemical entities (chemicals or peptides) to generate lead compounds for clinical development. A number of assays can be used to screen a library compounds for their ability to affect a biologically relevant endpoint. Each potential compound in a library is tested with a particular assay in a single well, and the ability of the compound to affect the assay determined. Some examples of 30 the assays are provided below: WO 2007/028212 PCT/AU2006/001325 - 173 For this assay, cells are plated into a microtitre plate (96 plate, 384 plate or the like). The cells will have a readout mechanism for activation of a noggin or chimeric noggin molecule. This may involve assaying for cell growth, assaying for stimulation of a particular pathway (e.g., FRET based techniques), assaying for induction of a reporter gene 5 (e.g., CAT, beta-galactosidase, fluorescent proteins), assaying for apoptosis and assaying for differentiation. Cells are then exposed to the noggin or chimeric noggin molecule of the present invention in the presence or absence of a particular small molecule. The drug can be added before, after or during the addition of the noggin or chimeric noggin molecule. After an appropriate period of time, the individual wells are read using an 10 appropriate method (eg, Fluorescence for FRET or induction of fluorescent proteins, cell number by MTT, beta-galactosidase activity etc). Control wells without addition of any drug or cytokine serve as comparisons. Any molecule able to inhibit the receptor/cytokine complex will give a different readout to the control wells. Further experiments will be required to show specificity of the inhibition. Alternatively, the drug could affect the 15 detection method by a non-cytokine, non-receptor mechanism (a false positive). A ligand or receptor of noggin or chimeric noggin molecule is immobilised on a solid surface. A noggin or chimeric noggin molecule and the compound to be tested are then added. This can be performed by adding a noggin or chimeric noggin molecule first, then 20 the compound; the compound first, then a noggin or chimeric noggin molecule; or the compound and the noggin or chimeric noggin molecule can be added together. Bound noggin or chimeric noggin molecule is then detected by an appropriate detection antibody. The detection antibody can be labeled with an enzyme (e.g., alkaline phosphatase or Horse-radish peroxidase for colorimetric detection) or a fluorescent tag for fluorescence 25 detection. Alternatively, a noggin or chimeric noggin molecule can be labeled (e.g., Biotin, radioactive labeling) and be detected with an appropriate technique (e.g., for Biotin labeling, streptavidin linked to a colorimetric detection system, for radiolabeling the complex is solubilised and counted). Inhibition of noggin or chimeric noggin molecule binding is measured by a drop in the reading compared to the control wells. 30 Soluble ligands or receptors of noggin or chimeric noggin molecule are bound to beads. This binding reaction can be either an adsorption process or involve chemically linking WO 2007/028212 PCT/AU2006/001325 -174 them to the plate. The beads are incubated with noggin or chimeric noggin molecule and a candidate compound in an appropriate well. This can be performed as noggin or chimeric noggin molecule first, then compound; compound first then noggin or chimeric noggin molecule; or compound and noggin or chimeric noggin molecule together. A fluorescently 5 labeled detection antibody that recognizes a noggin or chimeric noggin molecule is then added. The unbound antibody is removed and the beads are passed through a FACS. The amount of fluorescence detected will decrease if a compound inhibits the interaction of a noggin or chimeric noggin molecule with its receptor. 10 To enable screening of multiple interactions between protein and its corresponding ligand/receptor against one inhibitory compound, the ability of the FACS machine to analyse scatter profiles is used. A bead with a larger diameter will have a different scatter profile to that of a smaller bead, and this can be separated out for analysis ("gating"). 15 A number of different proteins, one of which is noggin or chimeric noggin molecule, are each linked to beads of a particular diameter. A mixture of ligands/receptors to the above mentioned proteins are then added to the bead mixture in the presence of one candidate compound. The bound ligands/receptors are then detected using a specific secondary antibodies that is fluorescently labeled. The antibodies can be all labeled with the same 20 detection fluorophore. The ability of the compound to prevent binding of a protein to its ligand/receptor is then determined by running the sample though a FACS machine and gating for each known bead size. The individual binding results are then analysed separately. The major benefit of this method of analysis is that the screening each compound can be tested in parallel with a number of proteins to decrease the time taken for 25 screening proportionally. A noggin or chimeric noggin molecule may also be characterised by its crystal structure. The physiochemical form of a noggin or chimeric noggin molecule may provide a unique 3D crystal structure. In addition, the crystal structure of the protein-ligand/receptor 30 complex may also be generated using a noggin or chimeric noggin molecule of the present invention. Since the present invention provides noggin or chimeric noggin molecule which is substantially similar to a human naturally occurring form, the complex is likely to be a WO 2007/028212 PCT/AU2006/001325 - 175 more reflective representation of the in vivo structure of the naturally occurring protein ligand/receptor complex. Once a crystal structure has been obtained, interactions between a noggin or chimeric noggin molecule and potential compounds inhibiting such interactions can be identified. 5 Once potential compounds are identified by high throughput screening or from the crystal structure of the protein-ligand/receptor complex, a process of rational drug design can begin. 10 There are several steps commonly taken in the design of a mimetic from a compound having a given desired property. First, the particular parts of the compound that are critical and/or important in determining the desired property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. Alanine scans of peptides are commonly used to 15 refine such peptide motifs. These parts or residues constituting the active region of the compound are known as its "pharmacophore". Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of 20 sources, e.g. spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process. 25 In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modeled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic. Modeling can be used to generate inhibitors which interact with the linear sequence or a three-dimensional configuration. 30 A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted WO 2007/028212 PCT/AU2006/001325 - 176 onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. Alternatively, where the mimetic is peptide-based, further stability can be achieved by cyclizing the peptide, increasing its rigidity. The mimetic or 5 mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing. The goal of rational drug design is to produce structural analogs of biologically active 10 polypeptides of interest or of small molecules with which they interact (e.g. agonists, antagonists, inhibitors or enhancers) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g. enhance or interfere with the function of a polypeptide in vivo. See, e.g. Hodgson (Bio/Technology 9:19-21, 1991). In one approach, one first determines the three-dimensional structure of a protein of interest 15 by x-ray crystallography, by computer modeling or most typically, by a combination of approaches. Useful information regarding the structure of a polypeptide may also be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al. Science 249:527-533, 1990). In addition, target molecules may be analyzed by an alanine scan 20 (Wells, Methods Enzymol 202:2699-2705, 1991). In this technique, an amino acid residue is replaced by Ala and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide. 25 It is also possible to isolate a target-specific antibody, selected by a functional assay and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site 30 of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.
WO 2007/028212 PCT/AU2006/001325 -177 In one aspect, the noggin or chimeric noggin molecule of the present invention is used as an immunogen to generate antibodies. The physiochemical form of a noggin or chimeric noggin molecule of the present invention may raise antibodies to a noggin or chimeric 5 noggin molecule of the present invention; glycopeptides specific to a noggin or chimeric noggin molecule of the present invention; or antibodies directed to another co- or post translationally modified peptide within a noggin or chimeric noggin molecule. Noggin or chimeric noggin molecule may present epitopes not normally accessible (but 10 possibly present) in vivo. For instance, there may be regions within a receptor domain that are normally in contact with another component of a heteromeric receptor. These epitopes may be used to generate monoclonal antibodies that cross react with the endogenous receptor. Such antibodies may block interaction of one receptor component with another and therefore prevent signal transduction. This may be therapeutically useful in the case of 15 overexpression of a cytokine or receptor. The antibodies may also be therapeutically useful in diseases where the receptor is overexpressed and signals without needing the ligand. The antibodies are also useful to detect the levels of the noggin or chimeric noggin molecule during the treatment of the disease (e.g., serum levels for half-life determination). 20 In addition, the antibodies are useful as diagnostic for determining the presence of a noggin or chimeric noggin molecule of the present invention in a particular sample. Reference to an "antibody" or "antibodies" includes reference to all the various forms of 25 antibodies, including but not limited to: full antibodies (e.g. having an intact Fc region), including, for example, monoclonal antibodies; antigen-binding antibody fragments, including, for example, Fv, Fab, Fab' and F(ab') 2 fragments; humanized antibodies; human antibodies (e.g., produced in transgenic animals or through phage display); and immunoglobulin-derived polypeptides produced through genetic engineering techniques. 30 Unless otherwise specified, the terms "antibody" or "antibodies" and as used herein encompasses both full antibodies and antigen-binding fragments thereof. Unless stated otherwise, specificity in respect of an antibody of the present invention is WO 2007/028212 PCT/AU2006/001325 - 178 intended to mean that the antibody binds substantially only to its target antigen with no appreciable binding to unrelated proteins. However, it is possible that an antibody will be designed or selected to bind to two or more related proteins. A related protein includes different splice variants or fragments of the same protein or homologous proteins from 5 different species. Such antibodies are still considered to have specificity for those proteins and are encompassed by the present invention. The term "substantially" means in this context that there is no detectable binding to a non-target antigen above basal, i.e. non specific, levels. 10 The antibodies of the present invention may be prepared by well-known procedures. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). 15 One method for producing an antibody of the present invention comprises immunizing a non-human animal, such as a mouse or a transgenic mouse, with a noggin or chimeric noggin molecule of the present invention, or immunogenic parts thereof, such as, for example, a peptide containing the receptor binding domain, whereby antibodies directed 20 against the polypeptide of a noggin or chimeric noggin molecule, or immunogenic parts thereof, are generated in the animal. Various means of increasing the antigenicity of a particular noggin or chimeric noggin molecule, such as administering adjuvants or conjugated antigens, comprising the antigen against which an antibody response is desired and another component, are well known to those in the art and may be utilized. 25 Immunizations typically involve an initial immunization followed by a series of booster immunizations. Animals may be bled and the serum assayed for antibody titer. Animals may be boosted until the titer plateaus. Conjugates may be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response. 30 Both polyclonal and monoclonal antibodies can be produced by this method. The methods for obtaining both types of antibodies are well known in the art. Polyclonal antibodies are WO 2007/028212 PCT/AU2006/001325 -179 less preferred but are relatively easily prepared by injection of a suitable animal with an effective amount of a noggin or chimeric noggin molecule of the present invention, or immunogenic parts thereof, collecting serum from the animal and isolating specific antibodies to a noggin or chimeric noggin molecule by any of the known immunoadsorbent 5 techniques. Antibodies produced by this technique are generally less favoured, because of the potential for heterogeneity of the product. The use of monoclonal antibodies is particularly favored because of the ability to produce them in large quantities and the homogeneity of the product. Monoclonal antibodies may 10 be produced by conventional procedures. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations 15 that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being 20 obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. Nature 256:495, 1975, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The 25 "monoclonal antibodies" may also be isolated from phage antibody libraries using for example, the techniques described in Clackson et al. Nature 352:624-628, 1991 and Marks et al. JMol Biol 222:581-597, 1991. The present invention contemplates a method for producing a hybridoma cell line which 30 comprises immunizing a non-human animal, such as a mouse or a transgenic mouse, with a noggin or chimeric noggin molecule of the present invention; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line to generate WO 2007/028212 PCT/AU2006/001325 - 180 hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds a noggin or chimeric noggin molecule. Such hybridoma cell lines and the monoclonal antibodies produced by them are 5 encompassed by the present invention. Monoclonal antibodies secreted by the hybridoma cell lines are purified by conventional techniques. Hybridomas or the monoclonal antibodies produced by them may be screened further to identify monoclonal antibodies with particularly desirable properties, such as the ability to inhibit cytokine-signaling through its receptor. 10 A noggin or chimeric noggin molecule or immunogenic part thereof that may be used to immunize animals in the initial stages of the production of the antibodies of the present invention should be from a human-expressed source. 15 Antigen-binding fragments of antibodies of the present invention may be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab, Fab', F(ab') 2 and Fv fragments, including single chain Fv fragments (termed sFv or scFv). Antibody fragments and derivatives produced by genetic engineering techniques, such as disulfide stabilized Fv fragments (dsFv), single chain variable region domain (Abs) 20 molecules, minibodies and diabodies are also contemplated for use in accordance with the present invention. Such fragments and derivatives of monoclonal antibodies directed against a noggin or chimeric noggin molecule may be prepared and screened for desired properties, by known 25 techniques, including the assays herein described. The assays provide the means to identify fragments and derivatives of the antibodies of the present invention that bind to a noggin or chimeric noggin molecule, as well as identify those fragments and derivatives that also retain the activity of inhibiting signaling by a noggin or chimeric noggin molecule. Certain of the techniques involve isolating DNA encoding a polypeptide chain 30 (or a portion thereof) of a mAb of interest, and manipulating the DNA through recombinant DNA technology. The DNA may be fused to another DNA of interest, or WO 2007/028212 PCT/AU2006/001325 - 181 altered (e.g. by mutagenesis or other conventional techniques) to add, delete, or substitute one or more amino acid residues. DNA encoding antibody polypeptides (e.g. heavy or light chain, variable region only or 5 full length) may be isolated from B-cells of mice that have been immunized with a noggin or chimeric noggin molecule of the present invention. The DNA may be isolated using conventional procedures. Phage display is another example of a known technique whereby derivatives of antibodies may be prepared. In one approach, polypeptides that are components of an antibody of interest are expressed in any suitable recombinant 10 expression system, and the expressed polypeptides are allowed to assemble to form antibody molecules. Single chain antibodies may be formed by linking heavy and light chain variable region (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single 15 polypeptide chain. Such single-chain Fvs (scFvs) have been prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable region polypeptides (VL and VH). The resulting antibody fragments can form dimers or trimers, depending on the length of a flexible linker between the two variable domains (Kortt et al. Protein Engineering 10:423, 1997). Techniques developed for the production of single chain 20 antibodies include those described in U.S. Patent No. 4,946,778; Bird (Science 242:423, 1988), Huston et al. (Proc Nati Acad Sci USA 85:5879, 1988) and Ward et al. (Nature 334:544, 1989). Single chain antibodies derived from antibodies provided herein are encompassed by the present invention. 25 In one embodiment, the present invention provides antibody fragments or chimeric, recombinant or synthetic forms of the antibodies of the present invention that bind to noggin or chimeric noggin molecule and inhibit signaling by noggin or chimeric noggin molecule. 30 Techniques are known for deriving an antibody of a different subclass or isotype from an antibody of interest, i.e., subclass switching. Thus, IgG1 or IgG4 monoclonal antibodies may be derived from an IgM monoclonal antibody, for example, and vice versa. Such WO 2007/028212 PCT/AU2006/001325 - 182 techniques allow the preparation of new antibodies that possess the antigen-binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques may be employed. Cloned DNA encoding particular 5 antibody polypeptides may be employed in such procedures, e.g. DNA encoding the constant region of an antibody of the desired isotype. The monoclonal production process described above may be used in animals, for example mice, to produce monoclonal antibodies. Conventional antibodies derived from such 10 animals, for example murine antibodies, .are known to be generally unsuitable for administration to humans as they may cause an immune response. Therefore, such antibodies may need to be modified in order to provide antibodies suitable for administration to humans. Processes for preparing chimeric and/or humanized antibodies are well known in the art and are described in further detail below. 15 The monoclonal antibodies herein specifically include "chimeric" antibodies in which the variable domain of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a non-human species (e.g., murine), while the remainder of the chain(s) is identical with or homologous to corresponding 20 sequences in antibodies derived from humans, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al. Proc NatlAcad Sci USA 81:6851-6855, 1984). "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies which 25 contain minimal sequence derived from the non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which the complementarity determining regions (CDRs) of the recipient are replaced by the corresponding CDRs from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired properties, for example specificity, and 30 affinity. In some instances, framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody.
WO 2007/028212 PCT/AU2006/001325 - 183 These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non-human immunoglobulin and all or substantially all of 5 the framework region residues are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin. For further details, see Jones et al. Nature 321:522-525, 1986; Reichmann et al. Nature 332:323-329, 1988; Presta, Curr Op Struct Biol 2:593-596, 1992; Liu et al. Proc Natl Acad Sci USA 84:3439, 10 1987; Larrick et al. Bio/Technology 7:934, 1989; and Winter and Harris, TIPS 14:139, 1993. In a further embodiment, the present invention provides an immunoassay kit with the ability to assay the level of human noggin or chimeric noggin molecule expressed from 15 human cells present in a biological preparation, including a biological preparation comprising naturally occurring human noggin. A biological preparation which can be assayed using the immunoassay kit of the present invention includes but is not limited to laboratory samples, cells, tissues, blood, serum, 20 plasma, urine, stool, saliva and sputum. The immunoassay kit of the present invention comprises a solid phase support matrix, not limited to but including a membrane, dipstick, bead, gel, tube or a multi-well, flat bottomed, round-bottomed or v-bottomed microplate, for example, a 96-well microplate; a 25 preparation of antibody directed against human noggin or chimeric noggin molecule (the capture antibody); a preparation of blocking solution (for example, BSA or casein); a preparation of secondary antibody (the detection antibody), also directed against human noggin or chimeric noggin molecule and conjugated to a suitable detection molecule (for example, alkaline phosphatase); a solution of chromagenic substrate (for example, nitro 30 blue tetrazolium); a solution of additional substrate (for example, 5-bromo-4-chloro-3 indolyl phosphate); a stock solution of substrate buffer (for example, 0.lM Tris-HCL (pH 7.5) and 0.lM NaCl, 50mM MgCl 2 ); a preparation of the noggin or chimeric noggin WO 2007/028212 PCT/AU2006/001325 - 184 molecule of the present invention with known concentration (the standard); and instructions for use. A suitable detection molecule may be chosen from the list consisting an enzyme, a dye, a 5 fluorescent molecule, a chemiluminescent, an isotope or such agents as colloidal gold conjugated to molecules including, but not limited to, such molecules as staphylococcal protein A or streptococcal protein G. In a particular embodiment, the capture and detection antibodies are monoclonal 10 antibodies, the production of which comprises immunizing a non-human animal, such as a mouse or a transgenic mouse, with a noggin or chimeric noggin molecule of the present invention, followed by standard methods, as hereinbefore described. Monoclonal antibodies may alternatively be produced by recombinant methods, as hereinbefore described and may comprise human or chimeric antibody portions or domains. 15 In another embodiment, the capture and detection antibodies are polyclonal antibodies, the production of which comprises immunizing a non-human animal, such as a mouse, rabbit, goat or horse, with a noggin or chimeric noggin molecule of the present invention, followed by standard methods, as hereinbefore described. 20 The components of the immunoassay kit are provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide for concentrations in solution of the reagents that substantially maximize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including 25 excipients, which on dissolution provide for each reagent solution having the appropriate concentration for combining with the biological preparation to be tested. The instructions for use may detail the method for using the immunoassay kit of the present invention. For example, the instructions for use may describe the method for 30 coating the solid phase support matrix with a prepared solution of capture antibody under suitable conditions, for example, overnight at 4'C. The instructions for use may further detail blocking non-specific protein binding sites with the prepared blocking solution; WO 2007/028212 PCT/AU2006/001325 - 185 adding and incubating a serially diluted sample containing noggin or chimeric noggin molecule under suitable conditions, for example, 1 hour at 37'C or 2 hours at room temperature, followed by a series of washes using a suitable buffer known in the art, for example, a solution of 0.05% Tween 20 in 0.1M PBS (pH 7.2). In addition, the instructions 5 may provide that a preparation of detection antibody is applied followed by incubation under suitable conditions, for example, 1 hour at 37"C or 2 hours at room temperature, followed by a further series of washes. A working solution of detection buffer is prepared from the supplied detection substrate(s) and substrate buffer, then added to each well under a suitable conditions ranging from 5 minutes at room temperature to 1 hour at 37*C. The 10 chromatogenic reaction may be halted with the addition of 1N NaOH or 2N H 2 SO4. In an alternative embodiment, the instructions for use may provide the simultaneous addition of any combination of any or all of the above components to be added in predetermined ratios, with the relative amounts of the various reagents suitably varied to 15 provide for concentrations in solution of the reagents that substantially maximize the formation of a measurable signal from formation of a complex. The level of colored product, or fluorescent or chemiluminescent or radioactive or other signal generated by the bound, conjugated detection reagents can be measured using an 20 ELISA-plate reader or spectrophotometer, at an appropriate optical density (OD), or as emitted light, using a spectrophotometer, fluorometer or flow cytometer, at an appropriate wavelength, or using a radioactivity counter, at an appropriate energy spectrum, or by a densitometer, or visually by comparison to a chart or guide. A serially diluted solution of the standard preparation is assayed in parallel with the above sample. A standard curve or 25 chart is generated and the level of the noggin or chimeric noggin molecule present within the sample can be interpolated from the standard curve or chart. The subject invention also provides a human derived noggin or chimeric noggin molecule for use as a standard protein in an immunoassay. The present invention further extends to a 30 method for determining the level of human cell-expressed human noggin in a biological preparation comprising a suitable assay for measuring the human noggin wherein the assay comprises (a) combining the biological preparation with one or more antibodies directed WO 2007/028212 PCT/AU2006/001325 - 186 against the human noggin; (b) determining the level of binding of the or each antibody to the human noggin in the biological preparation; (c) combining a standard human noggin sample with one or more antibodies directed against the human noggin; (d) determining the level of binding of the or each antibody to the standard human noggin sample; (e) 5 comparing the level of the or each antibody bound to the human noggin in the biological preparation to the level of the or each antibody bound to the standard human noggin sample. In particular, the standard human noggin sample is a preparation comprising the noggin or 10 chimeric noggin molecule of the present invention. The biological preparation includes but is not limited to laboratory samples, cells, tissues, blood, serum, plasma, urine, stool, saliva and sputum. The biological preparation is bound to one or more capture antibody as described hereinbefore or by methods known in the art. 15 For instance, the solid phase support matrix is first coated with a prepared solution of capture antibody under suitable conditions (for example, overnight at 4"C); followed by blocking non-specific protein binding sites with the prepared blocking solution; then adding and incubating serially diluted sample containing noggin or chimeric noggin molecule under suitable conditions (for example, I hour at 37'C or 2 hours at room 20 temperature), followed by a series of washes using a suitable buffer known in the art (for example, a solution of 0.05% Tween 20 in 0.lM PBS (pH 7.2)). The biological preparation is then combined with one or more detection antibodies conjugated to a suitable detection molecule as described herein. For instance, applying a 25 preparation of detection antibody followed by incubation under suitable conditions (for example, 1 hour at 37'C or 2 hours at room temperature), followed by a further series of washes. Determination of the level of binding may be carried out as described hereinbefore or by 30 methods known in the art. For instance, a working solution of detection buffer is prepared from the detection substrate(s) and substrate buffer, then adding to each well under a WO 2007/028212 PCT/AU2006/001325 - 187 suitable conditions ranging from 5 minutes at room temperature to 1 hour at 37 0 C. The chromatogenic reaction may be halted with the addition of 1N NaOH or 2N H 2 S0 4 . In a particular embodiment, the present invention contemplates an isolated noggin or 5 chimeric noggin molecule as hereinbefore described. In an embodiment, a noggin of the present invention is characterized by a profile of one or more of the following physiochemical parameters (Px) and pharmacological traits (Ty), comprising: 10 - an apparent molecular weight (P 1 ) of I to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 15 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 kDa and in one embodiment 23 to 35 kDa; - a pI (P 2 ) of noggin molecule is 2 to 14 such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and in one embodiment 8 to 14; - about 2 to 50 isoforms, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 isoforms and in one embodiment 2 to 16 isoforms; - a percentage by weight carbohydrate (P 5 ) of about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 25 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% and in one embodiment, 0 to 35%; - an observed molecular weight of the molecule after the N-linked oligosaccharides 30 are removed (P 6 ) of about 23 to 29 kDa; - an observed molecular weight of the molecule after the N-linked and 0-linked oligosaccharides are removed (P7) of about 23 to 27 kDa; WO 2007/028212 PCT/AU2006/001325 - 188 - one or more N-glycan structures as listed in Table 9 in the N-linked fraction (P 19 ); - one or more 0-glycan structures as listed in Table 10 in the 0-linked fraction (P 2 0 ); - a site of N-glycosylation (P 21 ) which includes N-62 (numbering from the start of the signal sequence); 5 - an immunoreactivity profile (T 1 3 ) distinct from that of a mammalian noggin expressed in a non-human cell system, and in one embodiment, the protein concentration of the noggin of the present invention is underestimated when assayed using a quantitative immunoassay in comparison to a mammalian noggin expressed in a non-human cell system; 10 - a biological activity that is distinct from that of a human noggin expressed in a non human cell system, and in one embodiment, a greater ability of noggin of the present invention to inhibit BMP-4 mediated alkaline phosphatise production (T 3 ) of MC3T3 pre-osteoblast cells than that of a human noggin expressed in E. coli cells; - an effect on stem cell expansion (T 73 ) that is distinct from that of a human noggin 15 expressed in a non-human cell system, and in one embodiment, a greater ability of noggin of the present invention to inhibit BMP-4 mediated expansion of CD34* human hematopoietic progenitor cells than that of a human noggin expressed in K coli. 20 In one embodiment, the noggin or chimeric noggin molecule of the present invention contains at least one of the structures in the N-linked fraction as described herein. In one embodiment, the noggin or chimeric noggin molecule of the present invention contains at least one of the structures in the 0-linked fraction as described herein. 25 In one embodiment, a noggin or chimeric noggin molecule is produced using a human cell line transformed with either a-2,3 or a-2,6 sialytransferase, or both a-2,3 sialytransferase and a-2,6 sialytransferase. 30 In particular, a noggin or chimeric noggin molecule produced using a human cell line transformed with either a-2,3 or a-2,6 sialytransferase, or both cc-2,3 sialytransferase and a-2,6 sialytransferase is characterized by a profile of physiochemical parameters (P,) WO 2007/028212 PCT/AU2006/001325 - 189 comprising monosaccharide (P 9 ) and sialic acid contents (Pio) of, when normalized to GalNAc, I to 0.1-100 NeuNAc; and when normalized to 3 times of mannose 3 to 0.1-100 NeuNAc. 5 Neutral percentage of N-linked oligosaccharides (P 13 ) is 0 to 99% such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. Acidic 10 percentage of N-linked oligosaccharides (P 1 4 ) is 1 to 100% such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. 15 Neutral percentage of O-linked oligosaccharides (P 1 5 ) is 0 to 99% such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 20 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. Acidic percentage of O-linked oligosaccharides (P 16 ) is 1 to 100% such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 25 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. The in vivo half-life (Tiu) of a noggin or chimeric noggin molecule produced using a human cell line transformed with either a-2,3 or a-2,6 sialytransferase, or both a-2,3 sialytransferase and a-2,6 sialytransferase is increased in comparison to the half-life of a 30 noggin or chimeric noggin molecule of the present invention.
WO 2007/028212 PCT/AU2006/001325 -190 In one embodiment, the noggin or chimeric noggin molecule of the present invention produced using a human cell line transformed with either a-2,3 or a-2,6 sialytransferase, or both a-2,3 sialytransferase and a-2,6 sialytransferase contains at least one of the structural formulae described herein or at least one of the structural formulae described 5 herein where one or more NeuNAc linkage is a a 2,6 linkage in the N-linked fraction. In one embodiment, the noggin or chimeric noggin molecule of the present invention produced using a human cell line transformed with either a-2,3 or a-2,6 sialytransferase, or both a-2,3 sialytransferase and a-2,6 sialytransferase contains at least one of the 10 structural formulae described herein or at least one of the structural formulae described herein where one or more NeuNAc linkage is a a 2,6 linkage in the O-linked fraction. Using forward primer (SEQ ID NO: 21) and reverse primer (SEQ ID NO: 22), the cDNA of noggin was amplified from the EST by Polymerase Chain Reaction (PCR) by methods 15 known in the art, for example, according to the method of Invitrogen's PCR Super Mix High Fidelity (Cat. No.:10790-020). The amplicon is digested and ligated into the corresponding restriction enzyme sites of an appropriate vector, for instance, pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA3.1/GS, pCEP4, pIRESpuro3, pIRESpuro4. The ligated vector is transformed into an appropriate E. coli host cell, for 20 instance, XLGold, ultracompetant cell (Strategene), XL-Blue, DH5a, DH1 GB or the like. For the production of chimeric molecules, the DNA sequence for the Fc domain of an immunoglobulin, such as IgG1, IgG2, IgG3, IgG4, IgGA1, IgGA2, IgGM, IgGE, IgGD is amplified from the EST using the appropriate forward and reverse primers by PCR. The 25 amplicon is cloned into the corresponding restriction enzyme sites of an appropriate vector, for instance, pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA3.1/GS, pCEP4, pIRESpuro3, pIRESpuro4. The DNA sequence of noggin is amplified and cloned into the corresponding restriction enzyme sites of the respective Fc-vector in frame with the Fe. 30 In a particular embodiment, the Fc receptor binding region or the complement activating region of the Fe region may be modified recombinantly, comprising one or more amino WO 2007/028212 PCT/AU2006/001325 - 191 acid insertions, deletions or substitutions relative to the amino acid sequence of the Fc region. In addition, the receptor binding region or the complement activating region of the Fc region may be modified chemically by changes to its glycosylation pattern, the addition or removal of carbohydrate moieties, the addition of polyunsaturated fatty acid moieties or 5 other lipid based moieties to the amino acid backbone or to any associated co- or post translational entities. The Fc region may also be in a truncated form, resulting from the cleavage by an enzyme including papain, pepsin or any other site-specific proteases. The Fc region may promote the spontaneous formation by the chimeric protein of a dimer, trimer or higher order multimer that is better capable of binding to its corresponding ligand 10 or receptor. Diagnostic digests using the appropriate restriction enzymes are performed to identify/isolate bacterial colonies containing the vector bearing the correct gene. Positive colonies are isolated and stored as Glycerol stocks at -70 0 C. The clone is then expanded to 15 750ml of sterile LB broth containing ampicillin (100pig/ml) at 37'C with shaking for 16 hours. The plasmid is prepared in accordance with methods known in the art, in a particular embodiment, in accordance with a Qiagen Endofree Plasmid Mega Kit (Qiagen Mega Prep Kit #12381). 20 Human host cells suitable for the introduction of the cloned DNA sequence comprising a noggin or chimeric noggin molecule include but are not limited to HEK 293 and any derivatives thereof, HEK 293 ci 8, HEK 293-T, HEK 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD- 293 (Stratagene), 293A (Invitrogen), Hela cells and any derivatives thereof, HepG2, PA-1 Jurkat, THP-1, HL-60, H9, HuT 78, Hep-2, Hep G2, MRC-5, 25 PER.C6, SKO-007, U266, Y2 (Apollo), WI-38, WI-L2. The physiochemical form of noggin or chimeric noggin molecule of the present invention may be achieved by modifying the host cell by a variety of ways known in the art, including but not limited to the introduction of a transgene into the host cell that encodes 30 an enzyme or enzymes that will produce the desired physiochemical form. Such transgenes include but are not limited to various types of sialyltransferases, such as 2, 3 sialyltransferase, 2, 6 sialyltransferase, ST3Gall, ST3Gal3, ST6Gall; WO 2007/028212 PCT/AU2006/001325 - 192 galactosyltransferases, such as GalT1, GalT2, antenna-cleaving enzymes, lactosamine repeats, sulfotransferases; fucosyltransferases such as FTI, FTII, FTIII, FTIV, FTV, FTVI, FTVII, and GlcNAc transferases such as GNTI, GNTII, GNTIII, GNTIV, GNTV. 5 For instance, inefficient terminal sialylation of N-glycan structures that results in reduced serum half-life of an expressed protein such as recombinant human AchE can be ameliorated by the addition of a rat beta-galactoside alpha-2,6-sialyltransferase transgene to HEK 293 cells (JBiochem 336:647-658, 1998; JBiochem 363:619-631, 2002). 10 Similarly, the introduction of specific DNA sequences can be used to optimize the integration of the cloned DNA sequence into the host cell genome, the various types of integration including but not limited to site-specific, targeted, direct or enzyme-mediated integration. 15 The DNA of noggin or chimeric noggin molecule can be introduced into suitable host cells by various transfection methods known in the art, for instance, using chemical reagents such as DEAE-dextran, calcium phosphate, artificial liposomes, or by direct microinjection, electroporation, biolistic particle delivery or infection or transfection with viral constructs as described below. 20 DEAE-dextran is a cationic polymer that associates with negatively charged nucleic acids. An excess of positive charge, contributed by the polymer in the DNA/polymer complex allows the complex to come into closer association with the negatively charged cell membrane. Uptake of the complex is presumably by endocytosis. Other synthetic cationic 25 polymers including polybrene, polyethyleneimine and dendrimers have also been used for transfection. Calcium phosphate co-precipitation can be used for transient and stable transfection of a variety of cell types. The DNA is mixed with calcium chloride in a controlled manner and 30 added to a buffered saline/phosphate solution and the mixture is incubated at room temperature. A precipitate is generated and is taken up by the cells via endocytosis or phagocytosis.
WO 2007/028212 PCT/AU2006/001325 - 193 The most commonly used synthetic lipid component of liposomes for liposome-mediated gene delivery is one which has overall net positive charge at physiological pH. Often the cationic lipid is mixed with a neutral lipid such as L-dioleoyl phosphatidylethanolamine 5 (DOPE). The cationic portion of the lipid molecule associates with the negatively charged nucleic acids, resulting in compaction of the nucleic acid in a liposome/nucleic acid complex. Uptake of the complex is by endocytosis. Direct microinjection of DNA into cultured cells or nuclei is an effective, although 10 laborious technique, which is not appropriate if a large number of transfected cells are required. Electroporation utilizes an electric pulse, which generates pores that allow the passage of nucleic acids into the cells. This technique requires fine-tuning and optimization for 15 duration and strength of the pulse for each type of cell used. Commercially available electroporation device includes Amaxa Biosystems' Nucleofector Kits (Amaxa Biosystems, Germany). This method relies upon high velocity delivery of nucleic acids on microprojectiles to 20 recipient cells. Infection or transfection with viral or retroviral constructs include the use of retrovirus, such as lentivirus, or DNA viruses, such as adenovirus. The process involves using a viral or retroviral vector to transfer a foreign gene to the host's cells. 25 In some embodiments, noggin or chimeric noggin molecule is produced by either transient methods or from stably transfected cell lines. Transient transfection is performed using either adherent or suspension cell lines. For adherent cell lines, the cells are grown in serum containing medium (between 2-10% serum) and in medium such as DMEM, 30 DMIEM/F12 (JRH). Serum used can be fetal calf serum (FCS), donor calf serum (DCS), new born calf serum (NBCS) or the like. Plasmid vectors are introduced into the cells by standard methods known in the art. In a particular embodiment, the DNA of noggin or WO 2007/028212 PCT/AU2006/001325 -194 chimeric noggin molecule is transfected using DEAE dextran or calcium phosphate precipitation. Following transfection, the cells are switched to an appropriate collection medium (e.g. serum free DMEM/F 12) for collection of the expressed noggin or chimeric noggin molecule. 5 Transient expression of the noggin or chimeric noggin molecule from suspension cells can be performed by introducing the plasmid vector using the methods outlined above. The suspension cells can be grown in either serum containing medium, or in serum free medium (e.g. Freestyle medium (Invitrogen), CD293 medium (Invitrogen), Excell medium 10 (JRH) or the like). The transfection can be performed in the absence of serum by transfecting in an appropriate media using a suitable transfection method, for instance, lipofectamine in OptiMEM medium. Transient expression usually results in a peak of expression 2-3 days after transfection. 15 Episomal vectors are replicated within the cell and give sustained expression. Therefore, to obtain large amounts of product, episomal expression vectors are transfected into cells and the cells are expanded. A noggin or chimeric noggin molecule is expressed into the medium, which is collected as the cells are expanded over a period of weeks. The expression medium can be serum containing or serum free and the cells can be either 20 adherent or suspension adapted. Stable clones are obtained by transfection of the expression vector into the cells, then selecting with an appropriate agent, for instance, phleomycin, hygromycin, puromycin, neomycin G418, methotrexate or the like. Stable clones will survive selection as the 25 plasmid contains a resistance gene in addition to the gene encoding the noggin or chimeric noggin molecule. One to two days after introduction of the gene, selection is begun on either the whole population of cells (stable pools) or on cells plated at clonal density. A non-transfected population of cells is also selected to determine the efficacy of cell killing by the selective agent. For adherent cells, the cells are allowed to grow on a tissue culture 30 plate until visible separate clones are obtained. They are then removed from the plate by trypsinization, or physical removal and placed into tissue culture wells (eg, one clone per well of a 96 well plate). For suspension cells, limiting dilution cloning is performed WO 2007/028212 PCT/AU2006/001325 - 195 subsequent to selection. The clones are then expanded, then either characterized and/or subjected to a further round of limiting dilution analysis. Stable clones growing in serum containing medium can be adapted by gradual reduction of 5 serum levels followed by detachment and growth under low serum in suspension. The serum levels are then reduced further until serum free status is achieved. Some growth media allow more rapid adaptation (e.g. a straight swap from serum containing adherent conditions to serum free suspension growth), an example of which is Invitrogen's CD293 media. 10 Following growth in serum free media, the clones can begin media optimization. The clones are tested for production characteristics in many different growth media until an optimum formulation or formulations are obtained. This may depend on the method of production of the product. For instance, the cells may be expanded in one medium, then 15 additives that enhance expression added prior to product collection. The over-expressed noggin or chimeric noggin molecule may accumulate within host cells. Recovery of intracellular protein involves treatment of the host cells with lysis buffers including but not limited to buffers containing: NP40, Triton X-100, Triton X-1 14, sodium 20 dodecyl sulfate (SDS), sodium cholate, sodium deoxycholate, CHAPS, CHAPSO, Brij-35, Brij-58, Tween-20, Tween-80, Octylglucoside and Octylthioglucoside. Alternative methods of host cell lysis may include sonication, homogenization, french press treatment and repeated cycles of freeze thawing and treatment of the cells with hypotonic solutions. 25 The final product can be produced in many different sorts of bioreactors, by way of non limiting examples, including stirred tank, airlift, packed bed perfusion, microcarriers, hollow fibre, bag technologies, cell factories. The methods may be continuous culture, batch, fed batch or induction. 30 Noggin or chimeric noggin molecule is purified using a purification strategy specifically tailored for noggin or chimeric noggin molecule of the present invention. Purification methods include but are not limited to: tangential flow filtration (TFF); ammonium sulfate WO 2007/028212 PCT/AU2006/001325 -196 precipitation; size exclusion chromatography (SEC); gel filtration chromatography (GFC); affinity chromatography (AFC); Protein A Affinity Purification; Receptor mediated Ligand Chromatography (RMLC); dye ligand chromatography (DLC); ion exchange chromotogaphy (IEC), including anion or cation exchange chromatography (AEC or 5 CEC); reversed-phase chromatography (RPC); hydrophobic interaction chromatography (HIC); metal chelating chromatography (MCC). TFF is a rapid and efficient method for biomolecule separation and is used for concentrating, desalting, or fractionating samples. TFF can concentrate samples as large as 10 hundreds of litres down to as little as 10 ml. In conjunction with a suitable molecular weight cut off membrane, TFF can separate and isolate biomolecules of differing size and molecular weight (nominal molecular weight cutoff (NMWC) 5 KDa, 10 KDa, 30 KDa, 100 KDa). The process of diafiltration involving dilution of the sample followed by re concentration can be used to desalt or exchange the sample buffer. 15 Salting out or ammonium sulfate precipitation is useful for concentrating dilute solutions of proteins. It is also useful for fractionating a mixture of proteins. Increases in the ionic strength of a solution containing protein causes a reduction in the repulsive effect of like charges between protein molecules. It also reduces the forces holding the solvation shell 20 around the protein molecules. When these forces are sufficiently reduced, the protein will precipitate; hydrophobic proteins precipitating at lower salt concentrations than hydrophilic proteins. Fractionation of protein mixtures by the stepwise increase in the ionic strength followed by centrifugation can be a very effective way of partly purifying proteins. 25 SEC separates proteins by size, based on the flow of the sample through a porous matrix. SEC has the same principle as GFC when it is used to separate molecules in aqueous systems. In SEC, molecules larger than pores of the packing elute with the solvent front first and are completely excluded. Intermediate sizes of molecules, between the 30 completely excluded and the retained, pass through the pores of the matrix according to their sizes. Small molecules which freely pass in and out of the pores are retained. Therefore, different sizes of proteins have different elution volume and retention times.
WO 2007/028212 PCT/AU2006/001325 - 197 For structurally similar molecules, the larger the molecular sizes, the earlier they elute out. Before running any samples, a standard curve should be established to determine the working limits and reference retention time. 5 When the protein shapes are the same, molecular weight can be screened in the elutes from the column rapidly by UV absorption, fluorescence or light scattering, according to the packing materials of various pore sizes on the column. Photon correlation spectroscopy (PCS) has been usually performed on static samples and for liquid chromatographic detection. Low angle laser light scattering has also been coupled to chromatographic 10 detection to detect the molecular weights directly, independent of the shapes of the proteins (Carr et al. Anal Biochem 175:492-499, 1988). SEC-HPLC was used to detect hGH degradation and aggregation (Pikal et al. Pharm Res 8:427-436, 1991). It was also used for estimation of contamination in studying p-galactosidase (Yoshioka et al. Pharm Res 10:103-108, 1993). 15 AFC purifies biological molecules according to specific interactions between their chemical structures and the suitable affinity ligands. The target molecule is adsorbed by a complementary immobilized ligand specifically and reversibly. The ligand can be an inhibitor, substrate, analog or cofactor, or an antibody which can recognize the target 20 molecules specifically. Subsequently, the adsorbed molecules are either eluted by competitive displacement, or by the conformation change through a pH or ionic strength shift. Protein A Affinity Purification is an example of affinity purification utilising the affinity of 25 certain bacterial proteins that bind generally to antibodies, regardless of the antibody's specificity to antigen. Protein A, Protein G and Protein L are three that have well characterised antibody-binding properties. These proteins have been produced recombinantly and used routinely for affinity purification of key antibody types from a variety of species. A genetically engineered recombinant form of Protein A and G, called 30 Protein A/G, is also available. These antibody-binding proteins can be immobilized to support matrixes. This method has been modified to purify recombinant proteins that have had the Protein A binding region of an antibody (Fe region) linked to the target protein.
WO 2007/028212 PCT/AU2006/001325 - 198 Binding to the immobilised Protein A molecule is performed under physiological conditions and eluted by change in pH or ionic strength. RMLC is a special kind of AFC utilising the inherent affinity of a receptor for its cognate 5 target molecule. The receptor molecule is immobilised on a suitable chromatography support matrix via reactive amines, reactive hydrogens, carbonyl, carboxyl or sulfhydryl groups. In one example of RMLC, the receptor-Fc chimera molecule is immobilised on Protein A sepharose beads via affinity of the Fc portion of the receptor to the Protein A. This method has the advantage of immobilising the receptor in an orientation that exposes 10 its ligand-binding site to its cognate cytokine. Adsorption of the target molecule to the receptor is performed under physiological conditions and elution is achieved by change in pH or ionic strength. DLC is a kind of ALC utilizing the ability of reactive dyes to bind proteins in a selective 15 and reversible manner. The dyes are generally monochlorotriazine compounds. The reactive chloro group allows easy immobilization of the triazine dye to a support matrix, such as Sepharose or agarose, and, more recently, to nylon membranes. The initial discovery of the ability of these dyes to bind proteins came from the observation 20 that blue dextran (a conjugate of cibacron blue FG-3A), used as a void volume marker on gel filtration columns, could retard the elution of certain proteins. A number of studies have been carried out on the specificity of the dyes for particular proteins, mostly using the prototype cibacron blue dye. The dyes appear to be most effective at binding proteins and enzymes that utilize nucleotide cofactors, such as kinases and dehydrogenases, although 25 other proteins such as serum albumin also bind tightly. It has been proposed that the aromatic triazine dye structure resembles the nucleotide structure of nicotinamide adenine dinucleotide (NAD) and that the dye interacts with the dinucleotide fold in these proteins. In many cases, bound proteins can be eluted from the columns by a substrate or nucleotide cofactor in a competitive fashion, and dyes have been shown to compete for substrate 30 binding sites in free solution. It seems likely that these dyes can bind proteins by electrostatic and hydrophobic interactions and by more specific "pseudoaffinity" interactions with ligand-binding sites. Enhancing the specificity of dye ligands by WO 2007/028212 PCT/AU2006/001325 -199 modification to further resemble ligands (biomimetic dyes) has been successful in the purification of a number of dehydrogenases and proteases (McGettrick et al. Methods Mol Biol 244:151-7, 2004). 5 Ion Exchange Chromatography (IEC) purifies proteins using protein retention on columns resulting from the electrostatic interactions between the ion exchange column matrix and the proteins. When the pH of the mobile phase is above the pI of the target protein will be negatively charged and will interact with an anion exchange column (AEC). When the pH of the mobile phase is below the pI of the target protein the protein will be positively 10 charged and a cation exchange column (CEC) should be used. The target proteins are eluted by increasing the concentrations of a counter ion with the same charge as the target molecule. RPC separates biological molecules according to the hydrophobic interactions between the 15 molecule and a chromatographic support matrix. Ionizable compounds are best analyzed in their neutral form by controlling the pH of the separation. Mobile phase additives, such as trifluoroacetic acid, increase protein hydrophobicity by forming ion pairs which strongly adsorb to the stationary phase. By changing the polarity of the mobile phase, the biological molecules are eluted from the chromatographic support. 20 HIC is similar to RPC, but with a larger nominal pore size. In HIC, the elution solvent uses an aqueous salt solution, instead of the aqueous or organic mobile phases used in RPC. Also, the order of sample elution is reversed from that obtained from RPC. The surfaces of proteins consist of hydrophilic residues and hydrophobic "patches", which are 25 usually located in the interior of the folded proteins to stabilize the proteins. When the hydrophobic patches become exposed to the aqueous environment, they will disrupt the normal solvation properties of the protein, which is thermodynamically unfavorable. In the aqueous mobile phase, the higher the concentrations of inorganic salts (e.g. ammonium sulfate), the higher surface tension, thereby increasing the strength of hydrophobic 30 interactions between the hydrophobic groups of the HIC resin and the proteins, which are adsorbed. However, while descending the salt concentration gradient, the surface tension WO 2007/028212 PCT/AU2006/001325 - 200 of the aqueous mobile phase is decreased, thus reducing the hydrophobic interaction, resulting in the proteins desorbing from the hydrophobic groups of the column. MCC is a technique in which proteins are separated on the basis of their affinity for 5 chelated metal ions. Various metal ions including but not limited to Cu 2 +, Co 2 +, Zn 2 +, Mn2+, Mg 2 + or Ni 2 + are immobilized on the stationary phase of a chromatographic support via a covalently bound chelating ligand (e.g. iminodiacetic acid ). Free coordination sites of the metal ions are used to bind different proteins and peptides. Elution can occur by displacement of the protein with a competitive molecule or by changing the pH. For 10 instance, a lowering of the pH in the buffer results in a reduced binding affinity of the protein-metal ion complex and desorption of the protein. Alternatively, bound proteins can be eluted from the column using a descending pH gradient, in the form of a step gradient or as linear gradient. 15 In a particular embodiment, the process of ion exchange chromatography (IEC) is used as the primary step in the purification of noggin or chimeric noggin molecule. The physiochemical form of the noggin or chimeric noggin molecule of the present invention may be achieved by chemical and/or enzymatic modification to the expressed 20 molecule in a variety of ways known in the art. The present invention contemplates chemical or enzymatic coupling of carbohydrates to the peptide chain of noggin or chimeric noggin molecule at a time after a noggin or chimeric noggin molecule is expressed and purified. Chemical and/or enzymatic coupling 25 procedures may be used to modify, increase or decrease the number or profile of carbohydrate substituents. Depending on the coupling mode used, the sugar(s) may be attached to (a) amide group of arginine, (b) free carboxyl groups, (c) sulfhydroxyl groups such as those of cysteine, (d) hydroxyl groups such as those of serine, threonine, hydroxylysine or hydroxyproline, (e) aromatic residues such as those of phenylalanine, 30 tyrosine, or tryptophan, (f) the amide group of glutamine, or (g) the amino groups such as those of histidine, arginine or lysine. Additions can be carried out chemically or enzymatically. For example serial addition of sugar units to noggin or chimeric noggin WO 2007/028212 PCT/AU2006/001325 -201 molecule can be performed using appropriate recombinant glycosyltransferases. Glycosyltransferases can also be used to add sugars that have covalently attached substituents. For example, sialic acid with covalently attached polyethylene glycol (PEG) can be transferred by a sialyltransferase to a terminal galactosyl residue to increase 5 molecular size and serum half-life. The carbohydrate side chain of a noggin or chimeric noggin molecule can also be modified chemically or enzymatically to incorporate a variety of functionalities, including phosphate, sulfate, hydroxyl, carboxylate, O-sulfate and N-acetyl groups. 10 Carbohydrates present on a noggin or chimeric noggin molecule may also be removed chemically or enzymatically. Trifluoromethanesulfonic acid or an equivalent compound can be used for chemical deglycosylation. This treatment can result in the cleavage of most or all sugars, except the linking sugar, while leaving the polypeptide intact. Individual 15 sugars or the entire chain can also be removed from a noggin or chimeric noggin molecule by a variety of endoglycosidases and exoglycosidases. The glycan component of noggin or chimeric noggin molecule may be modified synthetically by treatment with sialidases, or mild acid treatment to remove any residual 20 sialic acids; treatment with exo- or endo- glycosidases to trim down the antennae of N linked oligosaccharides or shorten O-linked oligosaccharides. It may also be treated with fucosidases or sulfatases to remove side groups such as fucose and sulfate. Pseudo glycan structures such as polyethylene glycol or dextrans may be chemically added to the amino acid backbone, or a glycotransferase cocktail can be used with sugar-dUDP precursors to 25 synthetically add sugar subunits to the glycan. An isolated noggin or chimeric noggin molecule described herein may be delivered to the subject by any means that produces contact of the isolated noggin or chimeric noggin molecule with the target receptor or ligand in the subject. In a particular embodiment, a 30 noggin or chimeric noggin molecule is delivered to the subject as a "pharmaceutical composition".
WO 2007/028212 PCT/AU2006/001325 -202 In another aspect, the present invention contemplates a pharmaceutical composition comprising one or more isolated noggins or chimeric noggin molecules as hereinbefore described together with a pharmaceutically acceptable carrier or diluent. 5 Composition forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dilution medium comprising, for example, water, ethanol, 10 polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of surfactants. The preventions of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it 15 will be favorable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. 20 Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with the active ingredient and optionally other active ingredients as required, followed by filtered sterilization or other appropriate means of sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, suitable methods of preparation include vacuum drying and the freeze-drying 25 technique which yield a powder of active ingredient plus any additionally desired ingredient. When the active agent is suitably protected, it may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or 30 soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet or administered via breast milk. For oral therapeutic administration, the active ingredient may be incorporated with excipients and used in the WO 2007/028212 PCT/AU2006/001325 -203 form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. Such compositions and preparations should contain at least 1% by weight of active agent. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of 5 the unit. The amount of active agent in such therapeutically useful compositions is such that a suitable dosage will be obtained. In a particular embodiment, compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 [ig and 200 mg of modulator. Alternative dosage amounts include from about 1 pig to about 1000 mg and from about 10 pig to about 500 mg. These 10 dosages may be per individual or per kg body weight. Administration may be per hour, day, week, month or year. The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter. A binder such as gum, acacia, corn starch or gelatin; excipients such as 15 dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be 20 present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically 25 pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations. The present invention also contemplates topical formulations. In a topical composition, the active agent may be suspended within a cream or lotion or wax or other liquid solution 30 such that topical application of the cream or lotion or wax or liquid solution results in the introduction of the active agent to a biological surface in the subject. The term "biological surface" as used herein, contemplates any surface on or within the organism. Examples of WO 2007/028212 PCT/AU2006/001325 - 204 "biological surfaces" to which the topical compositions of the present invention may be applied include any epithelial surface such as the skin, respiratory tract, gastrointestinal tract and genitourinary tract. 5 In addition to traditional cream, emulsion, patch or spray formulations, the agents of the present invention may also be delivered topically and/or transdermally using a range of iontophoric or poration based methodologies. "Iontophoresis" is predicated on the ability of an electric current to cause charged particles 10 to move. A pair of adjacent electrodes placed on the skin set up an electrical potential between the skin and the capillaries below. At the positive electrode, positively charged drug molecules are driven away from the skin's surface toward the capillaries. Conversely, negatively charged drug molecules would be forced through the skin at the negative electrode. Because the current can be literally switched on and off and modified, 15 iontophoretic delivery enables rapid onset and offset, and drug delivery is highly controllable and programmable. Poration technologies, use high-frequency pulses of energy, in a variety of forms (such as radio frequency radiation, laser, heat or sound) to temporarily disrupt the stratum corneum, 20 the layer of skin that stops many drug molecules crossing into the bloodstream. It is important to note that unlike iontophoresis, the energy used in poration technologies is not used to transport the drug across the skin, but facilitates its movement. Poration provides a "window" through which drug substances can pass much more readily and rapidly than they would normally. 25 Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and except insofar as any conventional media or agent 30 is incompatible with the modulator; their use in the pharmaceutical compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
WO 2007/028212 PCT/AU2006/001325 -205 In one embodiment, the pharmaceutical composition of the present invention can be used either alone or in conjunction with other drugs or therapies in the same manner as noggin or chimeric noggin molecule expressed by non-human cell line, such as, but not limited to 5 noggin-Fc expressed by E. coli, yeast, or CHO, including treatment alone or in conjunction with another drug for A-Beta-Lipoproteinemia, A-V, A Beta-2-Microglobulin Amyloidosis, A-T, AlAD, AlAT, Aagenaes, Aarskog syndrome, Aarskog-Scott Syndrome, Aase-smith syndrome, Aase Syndrome, AAT, Abderhalden-Kaufmann-Lignac Syndrome, Abdominal Muscle Deficiency Syndrome, Abdominal Wall Defect, Abdominal 10 Epilepsy, Abdominal Migraine, Abductor Spasmodic Dysphonia, Abductor Spastic Dysphonia, Abercrombie Syndrome, blepharon-Macrostomia Syndrome, ABS, Absence of IPRT, Absence of Corpus Callosum Schinzel Typ, Absence Defect of Limbs Scalp and Skull, Absence of Menstruation Primar, Absence of HGPRT, Absorptive Hyperoxaluriaor Enteric, Abt-Letterer-Siwe Disease, ACADL, ACADM Deficiency, ACADM, ACADS, 15 Acanthocytosis-Neurologic Disorder, Acanthocytosis, Acantholysis Bullosa, Acanthosis Nigricans, Acanthosis Bullosa, Acanthosis Nigricans With Insulin Resistance Type A, Acanthosis Nigricans With Insulin Resistance Type B, Acanthotic Nevus, Acatalasemia, Acatalasia, ACC, Accessory Atrioventricular Pathways, Accessory Atrioventricular Pathways, Acephaly, ACF with Cardiac Defects, Achalasia, Achard-Thiers Syndrome, 20 ACHARD (Marfan variant), Achard's syndrome, Acholuric Jaundice, Achondrogenesis, Achondrogenesis Type IV, Achondrogenesis Type III, Achondroplasia, Achondroplasia Tarda, Achondroplastic Dwarfism, Achoo Syndrome, Achromat, Achromatope, Achromatopic, Achromatopsia, Achromic Nevi, Acid Ceramidase Deficiency, Acid Maltase Deficiency, Acid Beta-glucosidase Deficiency, Acidemia Methylmalonic, 25 Acidemia Propionic, Acidemia with Episodic Ataxia and Weakness, Acidosis, Aclasis Tarsoepiphyseal, ACM, Acoustic Neurilemoma, Acoustic Neuroma, ACPS with Leg Hypoplasia, ACPS II, ACPS IV, ACPS III, Acquired Aphasia with Convulsive Disorder, Acquired Brown Syndrome, Acquired Epileptic Aphasia, Acquired Factor XIII Deficiency, Acquired Form of ACC (caused by infection while still in womb), Acquired 30 Hyperoxaluria, Acquired Hypogammaglobulinemia, Acquired Immunodeficiency Syndrome (AIDS), Acquired Iron Overload, Acquired Lipodystrophy, Acquired Partial Lipodystrophy, Acquired Wandering Spleen, ACR, Acral Dysostosis with Facial and WO 2007/028212 PCT/AU2006/001325 -206 Genital Abnormalities, Acro Renal, Acrocallosal Syndrome Schinzel Type, Acrocephalosyndactyly, Acrocephalosyndactyly Type I, Acrocephalosyndactyly Type I Subtype I, Acrocephalopolysyndactyly Type II, Acrocephalopolysyndactyly Type III, Acrocephalopolysyndactyly Type IV, Acrocephalosyndactyly V (ACS5 or ACS V) 5 Subtype I, Acrocephaly Skull Asymmetry and Mild Syndactyly, Acrocephaly, Acrochondrohyperplasia, Acrodermatitis Enteropathica, Acrodysostosis, Acrodystrophic Neuropathy, Acrofacial Dysostosis Nager Type, Acrofacial Dysostosis Postaxial Type, Acrofacial Dysostosis Type Genee-Wiedep, Acrogeria Familial, Acromegaly, Acromelalgia Hereditary, Acromesomelic Dysplasia, Acromesomelic Dwarfism, 10 Acromicric Skeletal Dysplasia, Acromicric Dysplasia, Acroosteolysis with Osteoporosis and Changes in Skull and Mandible, Acroosteolysis, Acroparesthesia, ACS I, ACS Type II, ACS Type III, ACS, ACS3, ACTH Deficiency, Action Myoclonus, Acute Brachial Neuritis Syndrome, Acute Brachial Radiculitis Syndrome, Acute Cerebral Gaucher Disease, Acute Cholangitis, Acute Disseminated Encephalomyeloradiculopathy, Acute 15 Disseminated Histiocytosis-X, Acute Hemorrhagic Polioencephalitis, Acute Idiopathic Polyneuritis, Acute Immune-Mediation Polyneuritis, Acute Infantile Pelizaeus-Merzbacher Brain Sclerosis, Acute Intermittant Porphyria, Acute Porphyrias, Acute Sarcoidosis, Acute Shoulder Neuritis, Acute Toxic Epidermolysis, Acyl-CoA Dehydrogenase Deficiency Long-Chain, Acyl-CoA Dehydrogenase Deficiency Short-Chain, Acyl-CoA 20 Dihydroxyacetone Acyltransferase, Acyl-coenzyme A Oxidase Deficiency, ADA, ADA Deficiency, Adam Complex, Adamantiades-Behcet's Syndrome, Adamantinoma, Adams Oliver Syndrome, Adaptive Colitis, ADD combined type, ADD, Addison Disease with Cerebral Sclerosis, Addison's Anemia, Addison's Disease, Addison-Biermer Anemia, Addison-Schilder Disease, Addisonian Pernicious Anemia, Adducted Thumbs-Mental 25 Retardation, Adductor Spasmodic Dysphonia, Adductor Spastic Dysphonia, Adenoma Associated Virilism of Older Women, Adenomatosis of the Colon and Rectum, Adenomatous polyposis of the Colon, Adenomatous Polyposis Familial, Adenosine Deaminase Deficiency, Adenylosuccinase deficiency, ADHD predominantly hyperactive impulsive type, ADHD predominantly inattentive type, ADHD, Adhesive Arachnoiditis, 30 Adie Syndrome, Adie's Syndrome, Adie's Tonic Pupil, Adie's Pupil, Adipogenital Retinitis Pigmentosa Polydactyly, Adipogenital-Retinitis Pigmentosa Syndrome, Adiposa Dolorosa, Adiposis Dolorosa, Adiposogenital Dystrophy, Adolescent Cystinosis, ADPKD, WO 2007/028212 PCT/AU2006/001325 -207 Adrenal Cortex Adenoma, Adrenal Disease, Adrenal Hyperfunction resulting from Pituitary ACTH Excess, Adrenal Hypoplasia, Adrenal Insufficiency, Adrenal Neoplasm, Adrenal Virilism, Adreno-Retinitis Pigmentosa-Polydactyly Syndrome, Adrenocortical Insufficiency, Adrenocortical Hypofunction, Adrenocorticotropic Hormone Deficiency 5 Isolated, Adrenogenital Syndrome, Adrenoleukodystrophy, Adrenomyeloneuropathy, Adreno-Retinitis Pigmentosa-Polydactyly Syndrome, Adult Cystinosis, Adult Dermatomyositis, Adult Hypophosphatasia, Adult Macula Lutea Retinae Degeneration, Adult Onset ALD, Adult-Onset Ceroidosis, Adult Onset Medullary Cystic Disease, Adult Onset Pernicious Anemia, Adult Onset Schindler Disease, Adult-Onset Subacute 10 Necrotizing Encephalomyelopathy, Adult Polycystic Kidney Disease, Adult Onset Medullary Cystic Disease, Adynlosuccinate Lyase Deficiency, AE, AEC Syndrome, AFD, Afibrinogenemia, African Siderosis, AGA, Aganglionic Megacolon, Age Related Macular Degeneration, Agenesis of Commissura Magna Cerebri, Agenesis of Corpus Callosum, Agenesis of Corpus Callosum-Infantile Spasms-Ocular Anomalies, Agenesis of Corpus 15 Callosum and Chorioretinal Abnonnality, Agenesis of Corpus Callosum-Chorioretinitis Abnornality, Aggressive mastocytosis, Agnosis Primary, AGR Triad, AGU, Agyria, Agyria-pachygria-band spectrum, AHC, AHD, AHDS, AHF Deficiency, AHG Deficiency, AHO, Ahumada Del Castillo, Aicardi Syndrome, AIED, AIMP, AIP, AIS, Akinetic Seizure, ALA-D Porphyria, Alactasia, Alagille Syndrome, Aland Island Eye Disease (X 20 Linked), Alaninuria, Albers-Schonberg Disease, Albinism, Albinismus, Albinoidism, Albright Hereditary Osteodystrophy, Alcaptonuria, Alcohol-Related Birth Defects, Alcoholic Embryopathy, Alcoholic Liver Cirrohsis, Ald, ALD, ALD, Aldosterone, Aldosteronism With Normal Blood Pressure, Aldrich Syndrome, Alexander's Disease, Alexanders Disease, Algodystrophy, Algoneurodystrophy, Alkaptonuria, Alkaptonuric 25 Ochronosis, Alkyl DHAP synthase deficiency, Allan-Herndon-Dudley Syndrome, Allan Herndon Syndrome, Allan-Herndon-Dudley Mental Retardation, Allergic Granulomatous Antitis, Allergic Granulomatous Angiitis of Cronkhite-Canada, Alobar Holoprosencephaly, Alopecia Areata, Alopecia Celsi, Alopecia Cicatrisata, Alopecia Circumscripta, Alopecia-Poliosis-Uveitis-Vitiligo-Deafness-Cutaneous-Uveo-O, Alopecia 30 Seminuniversalis, Alopecia Totalis, Alopecia Universalis, Alpers Disease, Alpers Diffuse Degeneration of Cerebral Gray Matter with Hepatic Cirrhosis, Alpers Progressive Infantile Poliodystrophy, Alpha-1-Antitrypsin Deficiency, Alpha-1 4 Glucosidase Deficiency, WO 2007/028212 PCT/AU2006/001325 -208 Alpha-Galactosidase A Deficiency, Alpha-Galactosidase B Deficiency, Alpha High Density Lipoprotein Deficieny, Alpha-L-Fucosidase Deficiency Fucosidosis Type 3, Alpha-GalNAc Deficiency Schindler Type, Alphalipoproteinemia, Alpha Mannosidosis, Alpha-N-Acetylgalactosaminidase Deficiency Schindler Type, Alpha-NAGA Deficiency 5 Schindler Type, Alpha-Neuraminidase Deficiency, Alpha-Thalassemia/mental retardation syndrome non-deletion type, Alphalipoproteinemia, Alport Syndrome, ALS, Alstroem's Syndrome, Alstroem, Alstrom Syndrome, Alternating Hemiplegia Syndrome, Alternating Hemiplegia of Childhood, Alzheimer's Disease, Amaurotic Familial Idiocy, Amaurotic Familial Idiocy Adult, Amaurotic Familial Infantile Idiocy, Ambiguous Genitalia, AMC, 10 AMD, Ameloblastoma, Amelogenesis Imperfecta, Amenorrhea-Galactorrhea Nonpuerperal, Amenorrhea-Galactorrhea-FSH Decrease Syndrome, Amenorrhea, Amino Acid Disorders, Aminoaciduria-Osteomalacia-Hyperphosphaturia Syndrome, AMN, Amniocentesis, Amniotic Bands, Amniotic Band Syndrome, Amniotic Band Disruption Complex, Amniotic Band Sequence, Amniotic Rupture Sequence, Amputation Congenital, 15 AMS, Amsterdam Dwarf Syndrome de Lange, Amylo-1 6-Glucosidase Deficiency, Amyloid Arthropathy of Chronic Hemodialysis, Amyloid Corneal Dystrophy, Amyloid Polyneuropathy, Amyloidosis, Amyloidosis of Familial Mediterranean Fever, Amylopectinosis, Amyoplasia Congenita, Amyotrophic Lateral Sclerosis, Amyotrophic Lateral Sclerosis, Amyotrophic Lateral Sclerosis-Polyglucosan Bodies, AN, AN 1, AN 2, 20 Anal Atresia, Anal Membrane, Anal Rectal Malformations, Anal Stenosis, Analine 60 Amyloidosis, Analphalipoproteinemia, Analrectal, Analrectal, Anaplastic Astrocytoma, Andersen Disease, Anderson-Fabry Disease, Andersen Glycogenosis, Anderson-Warburg Syndrome, Andre Syndrome, Andre Syndrome Type II, Androgen Insensitivity, Androgen Insensitivity Syndrome Partial, Androgen Insensitivity Syndrome Partial, Androgenic 25 Steroids, Anemia Autoimmune Hemolytic, Anemia Blackfan Diamond, Anemia, Congenital, Triphalangeal Thumb Syndrome, Anemia Hemolytic Cold Antibody, Anemia Hemolytic with PGK Deficiency, Anemia Pernicious, Anencephaly, Angelman Syndrome, Angio-Osteohypertrophy Syndrome, Angiofollicular Lymph Node Hyperplasia, Angiohemophilia, Angiokeratoma Corporis, Angiokeratoma Corporis Diffusum, 30 Angiokeratoma Diffuse, Angiomatosis Retina, Angiomatous Lymphoid, Angioneurotic Edema Hereditary, Anhidrotic Ectodermal Dysplasia, Anhidrotic X-Linked Ectodermal Dysplasias, Aniridia, Aniridia-Ambiguous Genitalia-Mental Retardation, Aniridia WO 2007/028212 PCT/AU2006/001325 -209 Associated with Mental Retardation, Aniridia-Cerebellar Ataxia-Mental Deficiency, Aniridia Partial-Cerebellar Ataxia-Mental Retardation, Aniridia Partial-Cerebellar Ataxia Oligophrenia, Aniridia Type I, Aniridia Type II, Aniridia-Wilms' Tumor Association, Aniridia-Wilms' Tumor-Gonadoblastoma, Ankyloblepharon-Ectodermal Defects-Cleft 5 Lip/Palate, Ankylosing Spondylitis, Annular groves, Anodontia, Anodontia Vera, Anomalous Trichromasy, Anomalous Dysplasia of Dentin,Coronal Dentin Dysplasia, Anomic Aphasia, Anophthalmia, Anorectal, Anorectal Malformations, Anosmia, Anterior Bowing of the Legs with Dwarfism, Anterior Membrane Corneal Dystrophy, Anti Convulsant Syndrome, Anti-Epstein-Barr Virus Nuclear Antigen (EBNA) Antibody 10 Deficiency, Antibody Deficiency, Antibody Deficiency with near normal Immunoglobulins, Antihemophilic Factor Deficiency, Antihemophilic Globulin Deficiency, Antiphospholipid Syndrome, Antiphospholipid Antibody Syndrome, Antithrombin III Deficiency, Antithrombin III Deficiency Classical (Type 1), Antitrypsin Deficiency, Antley-Bixler Syndrome, Antoni's Palsy, Anxietas Tibialis, Aorta Arch 15 Syndrome, Aortic and Mitral Atresia with Hypoplasic Left Heart Syndrome, Aortic Stenosis, Aparoschisis, APC, APECED Syndrome, Apert Syndrome, Aperts, Aphasia, Aplasia Axialis Extracorticales Congenital, Aplasia Cutis Congenita, Aplasia Cutis Congenita with Terminal Transverse Limb Defects, Aplastic Anemia, Aplastic Anemia with Congenital Anomalies, APLS, Apnea, Appalachian Type Amyloidosis, Apple Peel 20 Syndrome, Apraxia, Apraxia Buccofacial, Apraxia Constructional, Apraxia Ideational, Apraxia Ideokinetic, Apraxia Ideomotor, Apraxia Motor, Apraxia Oculomotor, APS, Arachnitis, Arachnodactyly Contractural Beals Type, Arachnodactyly, Arachnoid Cysts, Arachnoiditis Ossificans, Arachnoiditis, Aran-Duchenne, Aran-Duchenne Muscular Atrophy, Aregenerative Anemia, Arginase Deficiency, Argininemia, Arginino Succinase 25 Deficiency, Argininosuccinase Deficiency, Argininosuccinate Lyase Deficiency, Argininosuccinic Acid Lyase-ASL, Argininosuccinic Acid Synthetase Deficiency, Argininosuccinic Aciduria, Argonz-Del Castillo Syndrome, Arhinencephaly, Armenian Syndrome, Arnold-Chiari Malformation, Arnold-Chiari Syndrome, ARPKD, Arrhythmic Myoclonus, Arrhythmogenic Right Ventricular Dysplasia, Arteriohepatic Dysplasia, 30 Arteriovenous Malformation, Arteriovenous Malformation of the Brain, Arteritis Giant Cell, Arthritis, Arthritis Urethritica, Arthro-Dento-Osteodysplasia, Arthro Ophthalmopathy, Arthrochalasis Multiplex Congenita, Arthrogryposis Multiplex WO 2007/028212 PCT/AU2006/001325 -210 Congenita, Arthrogryposis Multiplex Congenita, Distal, Type IIA, ARVD, Arylsulfatase-B Deficiency, AS, ASA Deficiency, Ascending Paralysis, ASD,Atrioseptal Defects, ASH, Ashermans Syndrome, Ashkenazi Type Amyloidosis, ASL Deficiency, Aspartylglucosaminuria, Aspartylglycosaminuria, Asperger's Syndrome, Asperger's Type 5 Autism, Asphyxiating Thoracic Dysplasia, Asplenia Syndrome, ASS Deficiency, Asthma, Astrocytoma Grade I (Benign), Astrocytoma Grade II (Benign), Asymmetric Crying Facies with Cardiac Defects, Asymmetrical septal hypertrophy, Asymptomatic Callosal Agenesis, AT, AT III Deficiency, AT III Variant IA, AT III Variant Ib, AT 3, Ataxia, Ataxia Telangiectasia, Ataxia with Lactic Acidosis Type II, Ataxia Cerebral Palsy, 10 Ataxiadynamia, Ataxiophemia, ATD, Athetoid Cerebral Palsy, Atopic Eczema, Atresia of Esophagus with or without Tracheoesophageal Fistula, Atrial Septal Defects, Atrial Septal Defect Primum, Atrial and Septal and Small Ventricular Septal Defect, Atrial Flutter, Atrial Fibrillation, Atriodigital Dysplasia, Atrioseptal Defects, Atrioventricular Block, Atrioventricular Canal Defect, Atrioventricular Septal Defect, Atrophia Bulborum 15 Hereditaria, Atrophic Beriberi, Atrophy Olivopontocerebellar, Attention Deficit Disorder, Attention Deficit Hyperactivity Disorder, Attentuated Adenomatous Polyposis Coli, Atypical Amyloidosis, Atypical Hyperphenylalaninemia, Auditory Canal Atresia, Auriculotemporal Syndrome, Autism, Autism Asperger's Type, Autism Dementia Ataxia and Loss of Purposeful Hand Use, Autism Infantile Autism, Autoimmune Addison's 20 Disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune Polyendocrinopathy-Candidias, Autoimmune Polyglandular Disease Type I, Autosomal Dominant Albinism, Autosomal Dominant Compelling Helioophthalmic Outburst Syndrome, Autosomal Dominant Desmin Distal myopathy with Late Onset, Autosomal Dominant EDS, Autosomal Dominant Emery-Dreifuss Muscular Dystrophy, Autosomal 25 Dominant Keratoconus, Autosomal Dominant Pelizaeus-Merzbacher Brain Sclerosis, Autosomal Dominant Polycystic Kidney Disease, Autosomal Dominant Spinocerebellar Degeneration, Autosomal Recessive Agammaglobulinemia, Autosomal Recessive Centronuclear myopathy, Autosomal Recessive Conradi-Hunermann Syndrome, Autosomal Recessive EDS, Autosomal Recessive Emery-Dreifuss Muscular Dystrophy, 30 Autosomal Recessive Forms of Ocular Albinism, Autosomal Recessive Inheritance Agenesis of Corpus Callosum, Autosomal Recessive Keratoconus, Autosomal Recessive Polycystic Kidney Disease, Autosomal Recessive Severe Combined Immunodeficiency, WO 2007/028212 PCT/AU2006/001325 -211 AV, AVM, AVSD, AWTA, Axilla Abscess, Axonal Neuropathy Giant, Azorean Neurologic Disease, B-K Mole Syndrome, Babinski-Froelich Syndrome, BADS, Baillarger's Syndrome, Balkan Disease, Baller-Gerold Syndrome, Ballooning Mitral Valve, Balo Disease Concentric Sclerosis, Baltic Myoclonus Epilepsy, Bannayan-Zonana 5 syndrome (BZS), Bannayan-Riley-Ruvalcaba syndrome, Banti's Disease, Bardet-Biedl Syndrome, Bare Lymphocyte Syndrome, Barlow's syndrome, Barraquer-Simons Disease, Barrett Esophagus, Barrett Ulcer, Barth Syndrome, Bartter's Syndrome, Basal Cell Nevus Syndrome, Basedow Disease, Bassen-Kornzweig Syndrome, Batten Disease, Batten Mayou Syndrome, Batten-Spielmeyer-Vogt's Disease, Batten Turner Syndrome, Batten 10 Turner Type Congenital myopathy, Batten-Vogt Syndrome, BBB Syndrome, BBB Syndrome (Opitz), BBB Syndrome, BBBG Syndrome, BCKD Deficiency, BD, BDLS, BE, Beals Syndrome, Beals Syndrome, Beals-Hecht Syndrome, Bean Syndrome, BEB, Bechterew Syndrome, Becker Disease, Becker Muscular Dystrophy, Becker Nevus, Beckwith Wiedemann Syndrome, Beckwith-Syndrome, Begnez-Cesar's Syndrome, 15 Behcet's syndrome, Behcet's Disease, Behr 1, Behr 2, Bell's Palsy, Benign Acanthosis Nigricans, Benign Astrocytoma, Benign Cranial Nerve Tumors, Benign Cystinosis, Benign Essential Blepharospasm, Benign Essential Tremor, Benign Familial Hematuria, Benign Focal Amyotrophy, Benign Focal Amyotrophy of ALS, Benign Hydrocephalus, Benign Hypermobility Syndrome, Benign Keratosis Nigricans, Benign Paroxysmal Peritonitis, 20 Benign Recurrent Hematuria, Benign Recurrent Intrahepatic Cholestasis, Benign Spinal Muscular Atrophy with Hypertrophy of the Calves, Benign Symmetrical Lipomatosis, Benign Tumors of the Central Nervous System, Berardinelli-Seip Syndrome, Berger's Disease, Beriberi, Berman Syndrome, Bernard-Horner Syndrome, Bernard-Soulier Syndrome, Besnier Prurigo, Best Disease, Beta-Alanine-Pyruvate Aminotransferase, Beta 25 Galactosidase Deficiency Morquio Syndrome, Beta-Glucuronidase Deficiency, Beta Oxidation Defects, Beta Thalassemia Major, Beta Thalassemia Minor, Betalipoprotein Deficiency, Bethlem myopathy, Beuren Syndrome, BH4 Deficiency, Biber-Haab-Dimmer Corneal Dystrophy, Bicuspid Aortic Valve, Biedl-Bardet, Bifid Cranium, Bifunctional Enzyme Deficiency, Bilateral Acoustic Neurofibromatosis, Bilateral Acoustic Neuroma, 30 Bilateral Right-Sidedness Sequence, Bilateral Renal Agenesis, Bilateral Temporal Lobe Disorder, Bilious Attacks, Bilirubin Glucuronosyltransferase Deficiency Type I, Binder Syndrome, Binswanger's Disease, Binswanger's Encephalopathy, Biotinidase deficiency, WO 2007/028212 PCT/AU2006/001325 -212 Bird-Headed Dwarfism Seckel Type, Birth Defects, Birthmark, Bitemporal Forceps Marks Syndrome, Biventricular Fibrosis, Bjornstad Syndrome, B-K Mole Syndrome, Black Locks-Albinism-Deafness of Sensoneural Type (BADS), Blackfan-Diamond Anemia, Blennorrheal Idiopathic Arthritis, Blepharophimosis, Ptosis, Epicanthus Inversus 5 Syndrome, Blepharospasm, Blepharospasm Benign Essential, Blepharospasm Oromandibular Dystonia, Blessig Cysts, BLFS, Blindness, Bloch-Siemens Incontinentia Pigmenti Melanoblastosis Cutis Linearis, Bloch-Siemens-Sulzberger Syndrome, Bloch Sulzberger Syndrome, Blood types, Blood type A, Blood type B, Blood type AB, Blood type 0, Bloom Syndrome, Bloom-Torre-Mackacek Syndrome, Blue Rubber Bleb Nevus, 10 Blue Baby, Blue Diaper Syndrome, BMD, BOD, BOFS, Bone Tumor-Epidermoid Cyst Polyposis, Bonnet-Dechaume-Blanc Syndrome, Bonnevie-Ulrich Syndrome, Book Syndrome, BOR Syndrome, BORJ, Borjeson Syndrome, Borjeson-Forssman-Lehmann Syndrome, Bowen Syndrome, Bowen-Conradi Syndrome, Bowen-Conradi Hutterite, Bowen-Conradi Type Hutterite Syndrome, Bowman's Layer, BPEI, BPES, Brachial 15 Neuritis, Brachial Neuritis Syndrome, Brachial Plexus Neuritis, Brachial-Plexus Neuropathy, Brachiocephalic Ischemia, Brachmann-de Lange Syndrome, Brachycephaly, Brachymorphic Type Congenital, Bradycardia, Brain Injury due to perinatal asphyxia, Brain Tumors, Brain Tumors Benign, Brain Tumors Malignant, Branched Chain Alpha Ketoacid Dehydrogenase Deficiency, Branched Chain Ketonuria I, Brancher Deficiency, 20 Branchio-Oculo-Facial Syndrome, Branchio-Oto-Renal Dysplasia, Branchio-Oto-Renal Syndrome, Branchiooculofacial Syndrome, Branchiootic Syndrome, Brandt Syndrome, Brandywine Type Dentinogenesis Imperfecta, Brandywine type Dentinogenesis Imperfecta, Breast Cancer, BRIC Syndrome, Brittle Bone Disease, Broad Beta Disease, Broad Thumb Syndrome, Broad Thumbs and Great Toes Characteristic Facies and Mental 25 Retardation, Broad Thumb-Hallux, Broca's Aphasia, Brocq-Duhring Disease, Bronze Diabetes, Bronze Schilder's Disease, Brown Albinism, Brown Enamel Hereditary, Brown Sequard Syndrome, Brown Syndrome, BRRS, Brueghel Syndrome, Bruton's Agammaglobulinemia Common, BS, BSS, Buchanan's Syndrome, Budd's Syndrome, Budd-Chiari Syndrome, Buerger-Gruetz Syndrome, Bulbospinal Muscular Atrophy-X 30 linked, Bulldog Syndrome, Bullosa Hereditaria, Bullous CIE, Bullous Congenital Ichthyosiform Erythroderma, Bullous Ichthyosis, Bullous Pemphigoid, Burkitt's Lymphoma, Burkitt's Lymphoma African type, Burkitt's Lymphoma Non-african type, WO 2007/028212 PCT/AU2006/001325 -213 BWS, Byler's Disease, C Syndrome, Cl Esterase Inhibitor Dysfunction Type II Angioedema, Cl-INH, C1 Esterase Inhibitor Deficiency Type I Angioedema, ClNH, Cacchi-Ricci Disease, CAD, CADASIL, CAH, Calcaneal Valgus, Calcaneovalgus, Calcium Pyrophosphate Dihydrate Deposits, Callosal Agenesis and Ocular Abnormalities, 5 Calves-Hypertrophy of Spinal Muscular Atrophy, Campomelic Dysplasia, Campomelic Dwarfism, Campomelic Syndrome, Camptodactyly-Cleft Palate-Clubfoot, Camptodactyly Limited Jaw Excursion, Camptomelic Dwarfism, Camptomelic Syndrome, Camptomelic Syndrome Long-Limb Type, Camurati-Engelmann Disease, Canada-Cronkhite Disease, Canavan disease, Canavan's Disease Included, Canavan's Leukodystrophy, Cancer, 10 Cancer Family Syndrome Lynch Type, Cantrell Syndrome, Cantrell-Haller-Ravich Syndrome, Cantrell Pentalogy, Carbamyl Phosphate Synthetase Deficiency, Carbohydrate Deficient Glycoprotein Syndrome, Carbohydrate-Deficient Glycoprotein Syndrome Type Ia, Carbohydrate-Induced Hyperlipemia, Carbohydrate Intolerance of Glucose Galactose, Carbon Dioxide Acidosis, Carboxylase Deficiency Multiple, Cardiac-Limb Syndrome, 15 Cardio-auditory Syndrome, Cardioauditory Syndrome of Jervell and and Lange-Nielsen, Cardiocutaneous Syndrome, Cardio-facial-cutaneous syndrome, Cardiofacial Syndrome Cayler Type, Cardiomegalia Glycogenica Diffusa, Cardiomyopathic Lentiginosis, Cardio myopathy, Cardio myopathy Associated with Desmin Storage myopathy, Cardio myopathy Due to Desmin Defect, Cardio myopathy-Neutropenia Syndrome, Cardio myopathy 20 Neutropenia Syndrome Lethal Infantile Cardio myopathy, Cardiopathic Amyloidosis, Cardiospasm, Cardocardiac Syndrome, Carnitine-Acylcarnitine Translocase Deficiency, Carnitine Deficiency and Disorders, Carnitine Deficiency Primary, Carnitine Deficiency Secondary, Carnitine Deficiency Secondary to MCAD Deficiency, Carnitine Deficiency Syndrome, Carnitine Palmitoyl Transferase I & II (CPT I & II), Carnitine 25 Palmitoyltransferase Deficiency, Carnitine Palmitoyltransferase Deficiency Type 1, Carnitine Palmitoyltransferase Deficiency Type 2 benign classical muscular form included severe infantile form included, Carnitine Transport Defect (Primary Carnitine Deficiency), Carnosinase Deficiency, Carnosinemia, Caroli Disease, Carpenter syndrome, Carpenter's, Cartilage-Hair Hypoplasia, Castleman's Disease, Castleman's Disease Hyaline Vascular 30 Type, Castleman's Disease Plasma Cell Type, Castleman Tumor, Cat Eye Syndrome, Cat's Cry Syndrome, Catalayse deficiency, Cataract-Dental Syndrome, Cataract X-Linked with Hutchinsonian Teeth, Catecholamine hormones, Catel-Manzke Syndrome, Catel-Manzke WO 2007/028212 PCT/AU2006/001325 -214 Type Palatodigital Syndrome, Caudal Dysplasia, Caudal Dysplasia Sequence, Caudal Regression Syndrome, Causalgia Syndrome Major, Cavernomas, Cavernous Angioma, Cavernous Hemangioma, Cavernous Lymphangioma, Cavernous Malformations, Cayler Syndrome, Cazenave's Vitiligo, CBGD, CBPS, CCA, CCD, CCHS, CCM Syndrome, 5 CCMS, CCO, CD, CDGla, CDG1A, CDGS Type Ia, CDGS, CDI, CdLS, Celiac Disease, Celiac sprue, Celiac Sprue-Dermatitis, Cellular Immunodeficiency with Purine Nucleoside Phosphorylase Deficiency, Celsus' Vitiligo, Central Apnea, Central Core Disease, Central Diabetes Insipidus, Central Form Neurofibromatosis, Central Hypoventilation, Central Sleep Apnea, Centrifugal Lipodystrophy, Centronuclear myopathy, CEP, Cephalocele, 10 Cephalothoracic Lipodystrophy, Ceramide Trihexosidase Deficiency, Cerebellar Agenesis, Cerebellar Aplasia, Cerebellar Hemiagenesis, Cerebellar Hypoplasia, Cerebellar Vermis Aplasia, Cerebellar Vermis Agenesis-Hypernea-Episodic Eye Moves-Ataxia-Retardation, Cerebellar Syndrome, Cerebellarparenchymal Disorder IV, Cerebellomedullary Malformation Syndrome, Cerebello-Oculocutaneous Telangiectasia, 15 Cerebelloparenchymal Disorder IV Familial, Cerebellopontine Angle Tumor, Cerebral Arachnoiditis, Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukodystrophy, Cerebral Beriberi, Cerebral Diplegia, Cerebral Gigantism, Cerebral Ischemia, Cerebral Malformations Vascular, Cerebral Palsy, Cerebro-Oculorenal Dystrophy, Cerebro-Oculo-Facio-Skeletal Syndrome, Cerebrocostomandibular syndrome, 20 Cerebrohepatorenal Syndrome, Cerebromacular Degeneration, Cerebromuscular Dystrophy Fukuyama Type, Cerebroocular Dysgenesis, Cerebroocular Dysplasia-Muscular Dystrophy Syndrome, Cerebrooculofacioskeletal Syndrome, Cerebroretinal Arteriovenous Aneurysm, Cerebroside Lipidosis, Cerebrosidosis, Cerebrotendinous Xanthomatosis, Cerebrovascular Ferrocalcinosis, Ceroid-Lipofuscinosis Adult form, Cervical Dystonia, 25 Cervical Dystonia, Cervico-Oculo-Acoustic Syndrome, Cervical Spinal Stenosis, Cervical Vertebral Fusion, CES, CF, CFC syndrome, CFIDS, CFND, CGD, CGF, Chalasodermia Generalized, Chanarin Dorfman Disease, Chanarin Dorfinan Syndrome, Chanarin Dorfinan Ichthyosis Syndrome, Chandler's Syndrome, Charcot's Disease, Charcot-Marie Tooth, Charcot-Marie-Tooth Disease, Charcot-Marie-Tooth Disease Variant, Charcot 30 Marie-Tooth-Roussy-Levy Disease, CHARGE Association, Charge Syndrome, CHARGE Syndrome, Chaund's Ectodermal Dysplasias, Chediak-Higashi Syndrome, Chediak Steinbrinck-Higashi Syndrome, Cheilitis Granulomatosa, Cheiloschisis, Chemke WO 2007/028212 PCT/AU2006/001325 -215 Syndrome, Cheney Syndrome, Cherry Red Spot and Myoclonus Syndrome, CHF, CHH, Chiari's Disease, Chiari Malformation I, Chiari Malformation, Chiari Type I (Chiari Malformation I), Chiari Type II (Chiari Malformation II), Chiari I Syndrome, Chiari-Budd Syndrome, Chiari-Frommel Syndrome, Chiari Malformation II, CHILD Syndrome, 5 CHILD Ichthyosis Syndrome, CHILD Syndrome Ichthyosis, Childhood Adrenoleukodystrophy, Childhood Dermatomyositis, Childhood-onset Dystonia, Childhood Cyclic Vomiting, Childhood Giant Axonal Neuropathy, Childhood Hypophosphatasia, Childhood Muscular Dystrophy, CHN, Cholestasis, Cholestasis Hereditary Norwegian Type, Cholestasis Intrahepatic, Cholestasis Neonatal, Cholestasis of 10 Oral Contraceptive Users, Cholestasis with Peripheral Pulmonary Stenosis, Cholestasis of Pregnancy, Cholesterol Desmolase Deficiency, Chondrodysplasia Punctata, Chondrodystrophia Calcificans Congenita, Chondrodystrophia Fetalis, Chondrodystrophic Myotonia, Chondrodystrophy, Chondrodystrophy with Clubfeet, Chondrodystrophy Epiphyseal, Chondrodystrophy Hyperplastic Form, Chondroectodermal Dysplasias, 15 Chondrogenesis Imperfecta, Chondrohystrophia, Chondroosteodystrophy, Choreoacanthocytosis, Chorionic Villi Sampling, Chorioretinal Anomalies, Chorioretinal Anomalies with ACC, Chorireninal Coloboma-Joubert Syndrome, Choroidal Sclerosis, Choroideremia, Chotzen Syndrome, Christ-Siemens-Touraine Syndrome, Christ-Siemans Touraine Syndrome, Christmas Disease, Christmas Tree Syndrome, Chromosome 3 20 Deletion of Distal 3p, Chromosome 3 Distal 3p Monosomy, Chromosome 3-Distal 3q2 Duplication, Chromosome 3-Distal 3q2 Trisomy, Chromosome 3 Monosomy 3p2, Chromosome 3q Partial Duplication Syndrome, Chromosome 3q, Partial Trisomy Syndrome, Chromosome 3-Trisomy 3q2, Chromosome 4 Deletion 4q31-qter Syndrome, Chromosome 4 Deletion 4q32-qter Syndrome, Chromosome 4 Deletion 4q33-qter 25 Syndrome, Chromosome 4 Long Arm Deletion, Chromosome 4 Long Arm Deletion, Chromosome 4 Monosomy 4q, Chromosome 4-Monosomy 4q, Chromosome 4 Monosomy Distal 4q, Chromosome 4 Partial Deletion 4p, Chromosome 4, Partial Deletion of the Short Arm, Chromosome 4 Partial Monosomy of Distal 4q, Chromosome 4 Partial Monosomy 4p, Chromosome 4 Partial Trisomy 4 (q25-qter), Chromosome 4 Partial Trisomy 4 (q26 or 30 q27-qter), Chromosome 4 Partial Trisomy 4 (q31 or 32-qter), Chromosome 4 Partial Trisomy 4p, Chromosome 4 Partial Trisomies 4q2 and 4q3, Chromosome 4 Partial Trisomy Distal 4, Chromosome 4 Ring, Chromosome 4 4q Terminal Deletion Syndrome, WO 2007/028212 PCT/AU2006/001325 -216 Chromosome 4 q- Syndrome, Chromosome 4q- Syndrome, Chromosome 4 Trisomy 4, Chromosome 4 Trisomy 4p, Chromosome 4 XY/47 XXY (Mosiac), Chromosome 5 Monosomy Sp, Chromosome 5, Partial Deletion of the Short Arm Syndrome, Chromosome 5 Trisomy 5p, Chromosome 5 Trisomy 5p Complete (5p1 1-pter), Chromosome 5 Trisomy 5 5p Partial (5p13 or 14-pter), Chromosome 5p-Syndrome, Chromosome 6 Partial Trisomy 6q, Chromosome 6 Ring, Chromosome 6 Trisomy 6q2, Chromosome 7 Monosomy 7p2, Chromosome 7 Partial Deletion of Short Arm (7p2-), Chromosome 7 Terminal 7p Deletion [del (7) (p2l-p22)], Chromosome 8 Monosomy 8p2, Chromosome 8 Monosomy 8p21-pter, Chromosome 8 Partial Deletion (short arm), Chromosome 8 Partial Monosomy 8p2, 10 Chromosome 9 Complete Trisomy 9P, Chromosome 9 Partial Deletion of Short Arm, Chromosome 9 Partial Monosomy 9p, Chromosome 9 Partial Monosomy 9p22, Chromosome 9 Partial Monosomy 9p22-pter, Chromosome 9 Partial Trisomy 9P Included, Chromosome 9 Ring, Chromosome 9 Tetrasomy 9p, Chromosome 9 Tetrasomy 9p Mosaicism, Chromosome 9 Trisomy 9p (Multiple Variants), Chromosome 9 Trisomy 9 15 (pter-p21 to q32) Included, Chromosome 9 Trisomy Mosaic, Chromosome 9 Trisomy Mosaic, Chromosome 10 Distal Trisomy 10q, Chromosome 10 Monosomy, Chromosome 10 Monosomy 10p, Chromosome 10, Partial Deletion (short arm), Choromsome 10, lOp Partial, Chromosome 10 Partial Trisomy 10q24-qter, Chromosome 10 Trisomy 10q2, Partial Monosomy of Long Ann of Chromosome 11, Chromosome 11 Partial Monosomy 20 11q, Chromosome 11 Partial Trisomy, Chromosome 11 Partial Trisomy 11ql3-qter, Chromosome 11 Partial Trisomy 11 q21 -qter, Chromosome 11 Partial Trisomy 11q23-qter, Chromosome 1lq,Partial Trisomy, Chromosome 12 Isochromosome 12p Mosaic, Chromosome 13 Partial Monosomy 13q, Chromosome 13, Partial Monosomy of the Long Arm, Chromosome 14 Ring, Chromosome 14 Trisomy, Chromosome 15 Distal Trisomy 25 15q, Chromosome r15, Chromosome 15 Ring, Chromosome 15 Trisomy 15q2, Chromosome 15q, Partial Duplication Syndrome, Chromosome 17 Interstitial Deletion 17p, Chromosome 18 Long Arm Deletion Syndrome, Chromosome 18 Monosomy 18p, Chromosome 18 Monosomy 18Q, Chromosome 18 Ring, Chromosome 18 Tetrasomy 18p, Chromosome 18q- Syndrome, Chromosome 21 Mosaic 21 Syndrome, Chromosome 21 30 Ring, Chromosome 21 Translocation 21 Syndrome, Chromosome 22 Inverted Duplication (22pter-22q11), Chromosome 22 Partial Trisomy (22pter-22q11), Chromosome 22 Ring, Chromosome 22 Trisomy Mosaic, Chromosome 48 XXYY, Chromosome 48 XXXY, WO 2007/028212 PCT/AU2006/001325 -217 Chromosome r15, Chromosomal Triplication, Chromosome Triplication, Chromosome Triploidy Syndrome, Chromosome X, Chromosome XXY, Chronic Acholuric Jaundice, Chronic Adhesive Arachnoiditis, Chronic Adrenocortical Insufficiency, Chronic Cavernositis, Chronic Congenital Aregenerative Anemia, Chronic Dysphagocytosis, 5 Chronic Familial Granulomatosis, Chronic Familial Icterus, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic Granulomatous Disease, Chronic Guillain-Barre Syndrome, Chronic Idiopathic Jaundice, Chronic Idiopathic Polyneuritis (CIP), Chronic Inflammatory Demyelinating Polyneuropathy, Chronic Inflammatory Demyelinating Polyradiculoneuropathy, Chronic Motor Tic, Chronic Mucocutaneous Candidiasis, 10 Chronic Multiple Tics, Chronic Non-Specific Ulcerative Colitis, Chronic Obliterative Cholangitis, Chronic Peptic Ulcer and Esophagitis Syndrome, Chronic Progressive Chorea, Chronic Progressive External Ophthalmoplegia Syndrome, Chronic Progressive External Ophthalmoplegia and myopathy, Chronic Progressive External Ophthalmoplegia with Ragged Red Fibers, Chronic Relapsing Polyneuropathy, Chronic Sarcoidosis, Chronic 15 Spasmodic Dysphonia, Chronic Vomiting in Childhood, CHS, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CIP, Cirrhosis Congenital Pigmentary, Cirrhosis, Cistinuria, Citrullinemia, CJD, Classic Schindler Disease, Classic Type Pfeiffer Syndrome, Classical Maple Syrup Urine Disease, Classical Hemophilia, Classical Form Cockayne Syndrome Type I (Type A), Classical Leigh's Disease, Classical Phenylketonuria, Classical X-Linked 20 Pelizaeus-Merzbacher Brain Sclerosis, CLE, Cleft Lip/Palate Mucous Cysts Lower Lip PP Digital and Genital Anomalies, Cleft Lip-Palate Blepharophimosis Lagophthalmos and Hypertelorism, Cleft Lip/Palate with Abnormal Thumbs and Microcephaly, Cleft palate joint contractures-dandy walker malformations, Cleft Palate and Cleft Lip, Cleidocranial Dysplasia w/ Micrognathia, Absent Thumbs, & Distal Aphalangia, Cleidocranial 25 Dysostosis, Cleidocranial Dysplasia, Click murmur syndrome, CLN1, Clonic Spasmodic, Cloustons Syndrome, Clubfoot, CMDI, CMM, CMT, CMTC, CMTX, COA Syndrome, Coarctation of the aorta, Coats' Disease, Cobblestone dysplasia, Cochin Jewish Disorder, Cockayne Syndrome, COD-MD Syndrome, COD, Coffin Lowry Syndrome, Coffin Syndrome, Coffin Siris Syndrome, COFS Syndrome, Cogan Corneal Dystrophy, Cogan 30 Reese Syndrome, Cohen Syndrome, Cold Agglutinin Disease, Cold Antibody Disease, Cold Antibody Hemolytic Anemia, Colitis Ulcerative, Colitis Gravis, Colitis Ulcerative Chronic Non-Specific Ulcerative Colitis, Collodion Baby, Coloboma Heart Defects Atresia WO 2007/028212 PCT/AU2006/001325 -218 of the Choanae Retardation of Growth and Development Genital and Urinary Anomalies and Ear Anomalies, Coloboma, Colonic Neurosis, Color blindness, Colour blindness, Colpocephaly, Columnar-Like Esophagus, Combined Cone-Rod Degeneration, Combined Immunodeficiency with Immunoglobulins, Combined Mesoectodermal Dysplasia, 5 Common Variable Hypogammaglobulinemia, Common Variable Immunodeficiency, Common Ventricle, Communicating Hydrocephalus, Complete Absense of Hypoxanthine Guanine Phosphoribosyltranferase, Complete Atrioventricular Septal Defect, Complement Component 1 Inhibitor Deficiciency, Complement Component Cl Regulatory Component Deficiency, Complete Heart Block, Complex Carbohydrate Intolerance, Complex Regional 10 Pain Syndrome, Complex V ATP Synthase Deficiency, Complex I, Complex I NADH dehydrogenase deficiency, Complex II, Complex II Succinate dehydrogenase deficiency, Complex III, Complex III Ubiquinone-cytochrome c oxidoreductase deficiency, Complex IV, Complex IV Cytochrome c oxidase deficiency, Complex IV Deficiency, Complex V, Concussive Brain Injury, Cone-Rod Degeneration, Cone-Rod Degeneration Progressive, 15 Cone Dystrophy, Cone-Rod Dystrophy, Confluent Reticular Papillomatosis, Congenital with low PK Kinetics, Congenital Absence of Abdominal Muscles, Congenital Absence of the Thymus and Parathyroids, Congenital Achromia, Congenital Addison's Disease, Congenital Adrenal Hyperplasia, Congenital Adreneal Hyperplasia, Congenital Afibrinogenemia, Congenital Alveolar Hypoventilation, Congenital Anemia of Newborn, 20 Congenital Bilateral Persylvian Syndrome, Congenital Brown Syndrome, Congenital Cardiovascular Defects, Congenital Central Hypoventilation Syndrome, Congenital Cerebral Palsy, Congenital Cervical Synostosis, Congenital Clasped Thumb with Mental Retardation, Congenital Contractural Arachnodactyly, Congenital Contractures Multiple with Arachnodactyly, Congenital Cyanosis, Congenital Defect of the Skull and Scalp, 25 Congenital Dilatation of Intrahepatic Bile Duct, Congenital Dysmyelinating Neuropathy, Congenital Dysphagocytosis, Congenital Dysplastic Angiectasia, Congenital Erythropoietic Porphyria, Congenital Factor XIII Deficiency, Congenital Failure of Autonomic Control of Respiration, Congenital Familial Nonhemolytic Jaundice Type I, Congenital Familial Protracted Diarrhea, Congenital Form Cockayne Syndrome Type II 30 (Type B), Congenital Generalized Fibromatosis, Congenital German Measles, Congenital Giant Axonal Neuropathy, Congenital Heart Block, Congenital Heart Defects, Congenital Hemidysplasia with Ichthyosis Erythroderma and Limb Defects, Congenital Hemolytic WO 2007/028212 PCT/AU2006/001325 -219 Jaundice, Congenital Hemolytic Anemia, Congenital Hepatic Fibrosis, Congenital Hereditary Corneal Dystrophy, Congenital Hereditary Lymphedema, Congenital Hyperchondroplasia, Congenital Hypomyelinating Polyneuropathy, Congenital Hypomyelination Neuropathy, Congenital Hypomyelination, Congenital Hypomyelination 5 (Onion Bulb) Polyneuropathy, Congenital Ichthyosiform Erythroderma, Congenital Keratoconus, Congenital Lactic Acidosis, Congenital Lactose Intolerance, Congenital Lipodystrophy, Congenital Liver Cirrhosis, Congenital Lobar Emphysema, Congenital Localized Emphysema, Congenital Macroglossia, Congenital Medullary Stenosis, Congenital Megacolon, Congenital Melanocytic Nevus, Congenital Mesodermal 10 Dysmorphodystrophy, Congenital Mesodermal Dystrophy, Congenital Microvillus Atrophy, Congenital Multiple Arthrogryposis, Congenital Myotonic Dystrophy, Congenital Neuropathy caused by Hypomyelination, Congenital Pancytopenia, Congenital Pernicious Anemia, Congenital Pernicious Anemia due to Defect of Intrinsic Factor, Congenital Pernicious Anemia due to Defect of Intrinsic Factor, Congenital Pigmentary 15 Cirrhosis, Congenital Porphyria, Congenital Proximal myopathy Associated with Desmin Storage myopathy, Congenital Pulmonary Emphysema, Congenital Pure Red Cell Anemia, Congenital Pure Red Cell Aplasia, Congenital Retinal Blindness, Congenital Retinal Cyst, Congenital Retinitis Pigmentosa, Congenital Retinoschisis, Congenital Rod Disease, Congenital Rubella Syndrome, Congenital Scalp Defects with Distal Limb Reduction 20 Anomalies, Congenital Sensory Neuropathy, Congenital SMA with arthrogryposis, Congenital Spherocytic Anemia, Congenital Spondyloepiphyseal Dysplasia, Congenital Tethered Cervical Spinal Cord Syndrome, Congenital Tyrosinosis, Congenital Varicella Syndrome, Congenital Vascular Cavernous Malformations, Congenital Vascular Veils in the Retina, Congenital Word Blindness, Congenital Wandering Spleen (Pediatric), 25 Congestive Cardio myopathy, Conical Cornea, Conjugated Hyperbilirubinemia, Conjunctivitis, Conjunctivitis Ligneous, Conjunctivo-Urethro-Synovial Syndrome, Conn's Syndrome, Connective Tissue Disease, Conradi Disease, Conradi Hunermann Syndrome, Constitutional Aplastic Anemia, Constitutional Erythroid Hypoplasia, Constitutional Eczema, Constitutional Liver Dysfunction, Constitutional Thrombopathy, Constricting 30 Bands Congenital, Constrictive Pericarditis with Dwarfism, Continuous Muscle Fiber Activity Syndrome, Contractural Arachnodactyly, Contractures of Feet Muscle Atrophy and Oculomotor Apraxia, Convulsions, Cooley's anemia, Copper Transport Disease, WO 2007/028212 PCT/AU2006/001325 - 220 Coproporphyria Porphyria Hepatica, Cor Triatriatum, Cor Triatriatum Sinistrum, Cor Triloculare Biatriatum, Cor Biloculare, Cori Disease, Cornea Dystrophy, Corneal Amyloidosis, Corneal Clouding-Cutis Laxa-Mental Retardation, Corneal Dystrophy, Cornelia de Lange Syndrome, Coronal Dentine Dysplasia, Coronary Artery Disease, 5 Coronary Heart Disease, Corpus Callosum Agenesis, Cortical-Basal Ganglionic Degeneration, Corticalis Deformaris, Cortico-Basal Ganglionic Degeneration (CBGD), Corticobasal Degeneration, Corticosterone Methloxidase Deficiency Type I, Corticosterone Methyloxidase Deficiency Type II, Cortisol, Costello Syndrome, Cot Death, COVESDEM Syndrome, COX, COX Deficiency, COX Deficiency French 10 Canadian Type, COX Deficiency Infantile Mitochondrial myopathy de Toni-Fanconi Debre included, COX Deficiency Type Benign Infantile Mitochondrial Myopathy, CP, CPEO, CPEO with myopathy, CPEO with Ragged-Red Fibers, CPPD Familial Form, CPT Deficiency, CPTD, Cranial Arteritis, Cranial Meningoencephalocele, Cranio-Oro-Digital Syndrome, Craniocarpotarsal dystrophy, Craniocele, Craniodigital Syndrome-Mental 15 Retardation Scott Type, Craniofacial Dysostosis, Craniofacial Dysostosis-PD Arteriosus Hypertrichosis-Hypoplasia of Labia, Craniofrontonasal Dysplasia, Craniometaphyseal Dysplasia, Cranioorodigital Syndrome, Cranioorodigital Syndrome Type II, Craniostenosis Crouzon Type, Craniostenosis, Craniosynostosis-Choanal Atresia-Radial Humeral Synostosis, Craniosynostosis-Hypertrichosis-Facial and Other Anomalies, 20 Craniosynostosis Midfacial Hypoplasia and Foot Abnormalities, Craniosynostosis Primary, Craniosynostosis-Radial Aplasia Syndrome, Craniosynostosis with Radial Defects, Cranium Bifidum, CREST Syndrome, Creutzfeldt Jakob Disease, Cri du Chat Syndrome, Crib Death, Crigler Najjar Syndrome Type I, Crohn's Disease, Cronkhite-Canada Syndrome, Cross Syndrome, Cross' Syndrome, Cross-McKusick-Breen Syndrome, 25 Crouzon, Crouzon Syndrome, Crouzon Craniofacial Dysostosis, Cryoglobulinemia Essential Mixed, Cryptophthalmos-Syndactyly Syndrome, Cryptorchidism-Dwarfism Subnormal Mentality, Crystalline Corneal Dystrophy of Schnyder, CS, CSD, CSID, CSO, CST Syndrome, Curly Hair-Ankyloblephanon-Nail Dysplasia, Curschmann-Batten Steinert Syndrome, Curth Macklin Type Ichthyosis Hystric, Curth-Macklin Type, 30 Cushing's, Cushing Syndrome, Cushing's III, Cutaneous Malignant Melanoma Hereditary, Cutaneous Porphyrias, Cutis Laxa, Cutis Laxa-Growth Deficiency Syndrome, Cutis Marmorata Telangiectatica Congenita, CVI, CVID, CVS, Cyclic vomiting syndrome, WO 2007/028212 PCT/AU2006/001325 -221 Cystic Disease of the Renal Medulla, Cystic Hygroma, Cystic Fibrosis, Cystic Lymphangioma, Cystine-Lysine-Arginine-Ornithinuria, Cystine Storage Disease, Cystinosis, Cystinuria, Cystinuria with Dibasic Aminoaciduria, Cystinuria Type I, Cystinuria Type II, Cystinuria Type III, Cysts of the Renal Medulla Congenital, 5 Cytochrome C Oxidase Deficiency, D.C., Dacryosialoadenopathy, Dacryosialoadenopathia, Dalpro, Dalton, Daltonism, Danbolt-Cross Syndrome, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dandy-Walker Cyst, Dandy Walker Deformity, Dandy Walker Malformation, Danish Cardiac Type Amyloidosis (Type III), Darier Disease, Davidson's Disease, Davies' Disease, DBA, DBS, DC, DD, De Barsy 10 Syndrome, De Barsy-Moens-Diercks Syndrome, de Lange Syndrome, De Morsier Syndrome, De Santis Cacchione Syndrome, de Toni-Fanconi Syndrome, Deafness Congenital and Functional Heart Disease, Deafness-Dwarfism-Retinal Atrophy, Deafness Functional Heart Disease, Deafness Onychodystrophy Osteodystrophy and Mental Retardation, Deafness and Pili Torti Bjornstad Type, Deafness Sensorineural with 15 Imperforate Anus and Hypoplastic Thumbs, Debrancher Deficiency, Deciduous Skin, Defect of Enterocyte Intrinsic Factor Receptor, Defect in Natural Killer Lymphocytes, Defect of Renal Reabsorption of Carnitine, Deficiency of Glycoprotein Neuraminidase, Deficiency of Mitochondrial Respiratory Chain Complex IV, Deficiency of Platelet Glycoprotein Ib, Deficiency of Von Willebrand Factor Receptor, Deficiency of Short 20 Chain Acyl-CoA Dehydrogenase (ACADS), Deformity with Mesomelic Dwarfism, Degenerative Chorea, Degenerative Lumbar Spinal Stenosis, Degos Disease, Degos Kohlmeier Disease, Degos Syndrome, DEH, Dejerine-Roussy Syndrome, Dejerine Sottas Disease, Deletion 9p Syndrome Partial, Deletion 1 lq Syndrome Partial, Deletion 13q Syndrome Partial, Delleman-Oorthuys Syndrome, Delleman Syndrome, Dementia with 25 Lobar Atrophy and Neuronal Cytoplasmic Inclusions, Demyelinating Disease, DeMyer Syndrome, Dentin Dysplasia Coronal, Dentin Dysplasia Radicular, Dentin Dysplasia Type I, Dentin Dysplasia Type II, Dentinogenesis Imperfecta Brandywine type, Dentinogenesis Imperfecta Shields Type, Dentinogenesis Imperfecta Type III, Dento-Oculo-Osseous Dysplasia, Dentooculocutaneous Syndrome, Denys-Drash Syndrome, Depakene, 30 DepakeneTM exposure, Depakote, Depakote Sprinkle, Depigmentation-Gingival Fibromatosis-Microphthalmia, Dercum Disease, Dermatitis Atopic, Dermatitis Exfoliativa, Dermatitis Herpetiformis, Dermatitis Multiformis, Dermatochalasia Generalized, WO 2007/028212 PCT/AU2006/001325 - 222 Dermatolysis Generalized, Dermatomegaly, Dermatomyositis sine myositis, Dermatomyositis, Dermatosparaxis, Dermatostomatitis Stevens Johnson Type, Desbuquois Syndrome, Desmin Storage myopathy, Desquamation of Newborn, Deuteranomaly, Developmental Reading Disorder, Developmental Gerstmann Syndrome, Devergie 5 Disease, Devic Disease, Devic Syndrome, Dextrocardia- Bronchiectasis and Sinusitis, Dextrocardia with Situs Inversus, DGS, DGSX Golabi-Rosen Syndrome Included, DH, DHAP alkyl transferase deficiency, DHBS Deficiency, DHOF, DHPR Deficiency, Diabetes Insipidus, Diabetes Insipidus Diabetes Mellitus Optic Atrophy and Deafness, Diabetes Insipidus Neurohypophyseal, Diabetes Insulin Dependent, Diabetes Mellitus, 10 Diabetes Mellitus Addison's Disease Myxedema, Diabetic Acidosis, Diabetic Bearded Woman Syndrome, Diabetic Neuropathy, Diamond-Blackfan Anemia, Diaphragmatic Apnea, Diaphyseal Aclasis, Diastrophic Dwarfism, Diastrophic Dysplasia, Diastrophic Nanism Syndrome, Dicarboxylic Aminoaciduria, Dicarboxylicaciduria Caused by Defect in Beta-Oxidation of Fatty Acids, Dicarboxylicaciduria due to Defect in Beta-Oxidation of 15 Fatty Acids, Dicarboxylicaciduria due to MCADH Deficiency, Dichromasy, Dicker-Opitz, DIDMOAD, Diencephalic Syndrome, Diencephalic Syndrome of Childhood, Diencephalic Syndrome of Emaciation, Dienoyl-CoA Reductase Deficiency, Diffuse Cerebral Degeneration in Infancy, Diffuse Degenerative Cerebral Disease, Diffuse Idiopathic Skeletal Hyperostosis, Diffusum-Glycopeptiduria, DiGeorge Syndrome, Digital-Oro 20 Cranio Syndrome, Digito-Oto-Palatal Syndrome, Digito-Oto-Palatal Syndrome Type I, Digito-Oto-Palatal Syndrome Type II, Dihydrobiopterin Synthetase Deficiency, Dihydropteridine Reductase Deficiency, Dihydroxyacetonephosphate synthase, Dilated (Congestive) Cardio myopathy, Dimitri Disease, Diplegia of Cerebral Palsy, Diplo-Y Syndrome, Disaccharidase Deficiency, Disaccharide Intolerance I, Discoid Lupus, Discoid 25 Lupus Erythematosus, DISH, Disorder of Cornification, Disorder of Cornification Type I, Disorder of Cornification 4, Disorder of Cornification 6, Disorder of Cornification 8, Disorder of Cornification 9 Netherton's Type, Disorder of Cornification 11 Phytanic Acid Type, Disorder of Cornification 12 (Neutral Lipid Storage Type), Disorder of Conification 13, Disorder of Cornification 14, Disorder of Cornification 14 Trichothiodystrophy Type, 30 Disorder of Cornification 15 (Keratitis Deafness Type), Disorder of Cornification 16, Disorder of Cornification 18 Erythrokeratodermia Variabilis Type, Disorder of Cornification 19, Disorder of Cornification 20, Disorder of Cornification 24, Displaced WO 2007/028212 PCT/AU2006/001325 - 223 Spleen, Disseminated Lupus Erythematosus, Disseminated Neurodermatitis, Disseminated Sclerosis, Distal 11 q Monosomy, Distal 11 q- Syndrome, Distal Arthrogryposis Multiplex Congenital Type IIA, Distal Artbrogryposis Multiplex Congenita Type IIA, Distal Arthrogryposis Type IIA, Distal Arthrogryposis Type 2A, Distal Duplication 6q, Distal 5 Duplication l0q, Dup(l0q) Syndrome, Distal Duplication 15q, Distal Monosomy 9p, Distal Trisomy 6q, Distal Trisomy 1 Oq Syndrome, Distal Trisomy 11 q, Divalproex, DJS, DKC, DLE, DLPIII, DM, DMC Syndrome, DMC Disease, DMD, DNS Hereditary, DOC I, DOC 2, DOC 4, DOC 6 (Harlequin Type), DOC 8 Curth-Macklin Type, DOC 11 Phytanic Acid Type, DOC 12 (Neutral Lipid Storage Type), DOC 13, DOC 14, DOC 14 10 Trichothiodystrophy Type, DOC 15 (Keratitis Deafness Type), DOC 16, DOC 16 Unilateral Hemidysplasia Type, DOC 18, DOC 19, DOC 20, DOC 24, Dohle's Bodies Myelopathy, Dolichospondylic Dysplasia, Dolichostenomelia, Dolichostenomelia Syndrome, Dominant Type Kenny-Caffe Syndrome, Dominant Type Myotonia Congenita, Donahue Syndrome, Donath-Landsteiner Hemolytic Anemia, Donath-Landsteiner 15 Syndrome, DOOR Syndrome, DOORS Syndrome, Dopa-responsive Dystonia (DRD), Dorfman Chanarin Syndrome, Dowling-Meara Syndrome, Down Syndrome, DR Syndrome, Drash Syndrome, DRD, Dreifuss-Emery Type Muscular Dystrophy with Contractures, Dressler Syndrome, Drifting Spleen, Drug-induced Acanthosis Nigricans, Drug-induced Lupus Erythematosus, Drug-related Adrenal Insufficiency, Drummond's 20 Syndrome, Dry Beriberi, Dry Eye, DTD, Duane's Retraction Syndrome, Duane Syndrome, Duane Syndrome Type IA 1B and 1C, Duane Syndrome Type 2A 2B and 2C, Duane Syndrome Type 3A 3B and 3C, Dubin Johnson Syndrome, Dubowitz Syndrome, Duchenne, Duchenne Muscular Dystrophy, Duchenne's Paralysis, Duhring's Disease, Duncan Disease, Duncan's Disease, Duodenal Atresia, Duodenal Stenosis, Duodenitis, 25 Duplication 4p Syndrome, Duplication 6q Partial, Dupuy's Syndrome, Dupuytren's Contracture, Dutch-Kennedy Syndrome, Dwarfism, Dwarfism Campomelic, Dwarfism Cortical Thickening of the Tubular Bones & Transient Hypocalcemia, Dwarfism Levi's Type, Dwarfism Metatropic, Dwarfism-Onychodysplasia, Dwarfism-Pericarditis, Dwarfism with Renal Atrophy and Deafness, Dwarfism with Rickets, DWM, Dyggve 30 Melchior Clausen Syndrome, Dysautonomia Familial, Dysbetalipoproteinemia Familial, Dyschondrodysplasia with Hemangiomas, Dyschondrosteosis, Dyschromatosis Universalis Hereditaria, Dysencephalia Splanchnocystica, Dyskeratosis Congenita, Dyskeratosis WO 2007/028212 PCT/AU2006/001325 - 224 Congenita Autosomal Recessive, Dyskeratosis Congenita Scoggins Type, Dyskeratosis Congenita Syndrome, Dyskeratosis Follicularis Vegetans, Dyslexia, Dysmyelogenic Leukodystrophy, Dysmyelogenic Leukodystrophy-Megalobare, Dysphonia Spastica, Dysplasia Epiphysialis Punctata, Dysplasia Epiphyseal. Hemimelica, Dysplasia of Nails 5 With Hypodontia, Dysplasia Cleidocranial, Dysplasia Fibrous, Dysplasia Gigantism SyndromeX-Linked, Dysplasia Osteodental, Dysplastic Nevus Syndrome, Dysplastic Nevus Type, Dyssynergia Cerebellaris Myoclonica, Dyssynergia Esophagus, Dystonia, Dystopia Canthorum, Dystrophia Adiposogenitalis, Dystrophia Endothelialis Cornea, Dystrophia Mesodermalis, Dystrophic Epidermolysis Bullosa, Dystrophy, Asphyxiating 10 Thoracic, Dystrophy Myotonic, E-D Syndrome, Eagle-Barrett Syndrome, Eales Retinopathy, Eales Disease, Ear Anomalies-Contractures-Dysplasia of Bone with Kyphoscoliosis, Ear Patella Short Stature Syndrome, Early Constraint Defects, Early Hypercalcemia Syndrome with Elfin Facie, Early-onset Dystonia, Eaton Lambert Syndrome, EB, Ebstein's anomaly, EBV Susceptibility (EBVS), EBVS, ECD, ECPSG, 15 Ectodermal Dysplasias, Ectodermal Dysplasia Anhidrotic with Cleft Lip and Cleft Palate, Ectodermal Dysplasia-Exocrine Pancreatic Insufficiency, Ectodermal Dysplasia Rapp Hodgkin type, Ectodermal and Mesodermal Dysplasia Congenital, Ectodermal and Mesodermal Dysplasia with Osseous Involvement, Ectodermosis Erosiva Pluriorificialis, Ectopia Lentis, Ectopia Vesicae, Ectopic ACTH Syndrome, Ectopic Adrenocorticotropic 20 Hormone Syndrome, Ectopic Anus, Ectrodactilia of the Hand, Ectrodactyly, Ectrodactyly Ectodermal Dysplasia-Clefting Syndrome, Ectrodactyly Ectodermal Dysplasias Clefting Syndrome, Ectrodactyly Ectodermal Dysplasia Cleft Lip/Cleft Palate, Eczema, Eczema Thrombocytopenia-Immunodeficiency Syndrome, EDA, EDMD, EDS, EDS Arterial Ecchymotic Type, EDS Arthrochalasia, EDS Classic Severe Form, EDS 25 Dysfibronectinemic, EDS Gravis Type, EDS Hypermobility, EDS Kyphoscoliotic, EDS Kyphoscoliosis, EDS Mitis Type, EDS Ocular-Scoliotic, EDS Progeroid, EDS Periodontosis, EDS Vascular, EEC Syndrome, EFE, EHBA, EHK, Ehlers Danlos Syndrome, Ehlers-Danlos syndrome, Ehlers Danlos IX, Eisemnenger Complex, Eisenmenger's complex, Eisenmenger Disease, Eisenmenger Reaction, Eisenmenger 30 Syndrome, Ekbom Syndrome, Ekman-Lobstein Disease, Ektrodactyly of the Hand, EKV, Elastin fiber disorders, Elastorrhexis Generalized, Elastosis Dystrophica Syndrome, Elective Mutism (obsolete), Elective Mutism, Electrocardiogram (ECG or EKG), Electron WO 2007/028212 PCT/AU2006/001325 -225 Transfer Flavoprotein (ETF) Dehydrogenase Deficiency: (GAII & MADD), Electrophysiologic study (EPS), Elephant Nails From Birth, Elephantiasis Congenita Angiomatosa, Hemangiectatic Hypertrophy, Elfin Facies with Hypercalcemia, Ellis-van Creveld Syndrome, Ellis Van Creveld Syndrome, Embryoma Kidney, Embryonal 5 Adenomyosarcoma Kidney, Embryonal Carcinosarcoma Kidney, Embryonal Mixed Tumor Kidney, EMC, Emery Dreyfus Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy, Emery-Dreifuss Syndrome, EMF, EMG Syndrome, Empty Sella Syndrome, Encephalitis Periaxialis Diffusa, Encephalitis Periaxialis Concentrica, Encephalocele, Encephalofacial Angiomatosis, Encephalopathy, Encephalotrigeminal Angiomatosis, 10 Enchondromatosis with Multiple Cavernous Hemangiomas, Endemic Polyneuritis, Endocardial Cushion Defect, Endocardial Cushion Defects, Endocardial Dysplasia, Endocardial Fibroelastosis (EFE), Endogenous Hypertriglyceridemia, Endolymphatic Hydrops, Endometrial Growths, Endometriosis, Endomyocardial Fibrosis, Endothelial Corneal Dystrophy Congenital, Endothelial Epithelial Corneal Dystrophy, Endothelium, 15 Engelmann Disease, Enlarged Tongue, Enterocolitis, Enterocyte Cobalamin Malabsorption, Eosinophia Syndrome, Eosinophilic Cellulitis, Eosinophilic Fasciitis, Eosinophilic Granuloma, Eosinophilic Syndrome, Epidermal Nevus Syndrome, Epidermolysis Bullosa, Epidermolysis Bullosa Acquisita, Epidermolysis Bullosa Hereditaria, Epidermolysis Bullosa Letalias, Epidermolysis Hereditaria Tarda, 20 Epidermolytic Hyperkeratosis, Epidermolytic Hyperkeratosis (Bullous CIE), Epilepsia Procursiva, Epilepsy, Epinephrine, Epiphyseal Changes and High Myopia, Epiphyseal Osteochondroma Benign, Epiphysealis Hemimelica Dysplasia, Episodic-Abnormal Eye Movement, Epithelial Basement Membrane Corneal Dystrophy, Epithelial Corneal Dystrophy of Meesmann Juvenile, Epitheliomatosis Multiplex with Nevus, Epithelium, 25 Epival, EPS, Epstein-Barr Virus-Induced Lymphoproliferative Disease in Males, Erb Goldflam syndrome, Erdheim Chester Disease, Erythema Multiforme Exudativum, Erythema Polymorphe Stevens Johnson Type, Erythroblastophthisis, Erythroblastosis Fetalis, Erythroblastosis Neonatorum, Erythroblastotic Anemia of Childhood, Erythrocyte Phosphoglycerate Kinase Deficiency, Erythrogenesis Imperfecta, Erythrokeratodermia 30 Progressiva Symmetrica, Erythrokeratodermia Progressiva Symmetrica Ichthyosis, Erythrokeratodermia Variabilis, Erythrokeratodermia Variabilis Type, Erythrokeratolysis Hiemalis, Erythropoietic Porphyrias, Erythropoietic Porphyria, Escobar Syndrome, WO 2007/028212 PCT/AU2006/001325 - 226 Esophageal Atresia, Esophageal Aperistalsis, Esophagitis-Peptic Ulcer, Esophagus Atresia and/or Tracheoesophageal Fistula, Essential Familial Hyperlipemia, Essential Fructosuria, Essential Hematuria, Essential Hemorrhagic Thrombocythemia, Essential Mixed Cryoglobulinemia, Essential Moschowitz Disease, Essential Thrombocythemia, Essential 5 Thrombocytopenia, Essential Thrombocytosis, Essential Tremor, Esterase Inhibitor Deficiency, Estren-Dameshek variant of Fanconi Anemia, Estrogen-related Cholestasis, ET, ETF, Ethylmalonic Adipicaciduria, Eulenburg Disease, pc, EVCS, Exaggerated Startle Reaction, Exencephaly, Exogenous Hypertriglyceridemia, Exomphalos-Macroglossia Gigantism Syndrom, Exoplithalmic Goiter, Expanded Rubella Syndrome, Exstrophy of the 10 Bladder, EXT, External Chondromatosis Syndrome, Extrahepatic Biliary Atresia, Extramedullary Plasmacytoma, Exudative Retinitis, Eye Retraction Syndrome, FA1, FAA, Fabry Disease, FAC, FACB, FACD, FACE, FACF, FACG, FACH, Facial Nerve Palsy, Facial Paralysis, Facial Ectodermal Dysplasias, Facial Ectodennal Dysplasia, Facio Scapulo-Humeral Dystrophy, Facio-Auriculo-Vertebral Spectrum, Facio-cardio-cutaneous 15 syndrome, Facio-Fronto-Nasal Dysplasia, Faciocutaneoskeletal Syndrome, Faciodigitogenital syndrome, Faciogenital dysplasia, Faciogenitopopliteal Syndrome, Faciopalatoosseous Syndrome, Faciopalatoosseous Syndrome Type II, Facioscapulohumeral muscular dystrophy, Factitious Hypoglycemia, Factor VIII Deficiency, Factor IX Deficiency, Factor XI Deficiency, Factor XII deficiency, Factor XIII 20 Deficiency, Fabr Disease, Fahr's Disease, Failure of Secretion Gastric Intrinsic Factor, Fairbank Disease, Fallot's Tetralogy, Familial Acrogeria, Familial Acromicria, Familial Adenomatous Colon Polyposis, Familial Adenomatous Polyposis with Extraintestinal Manifestations, Familial Alobar Holoprosencephaly, Familial Alpha-Lipoprotein Deficiency, Familial Amyotrophic Chorea with Acanthocytosis, Familial Arrhythmic 25 Myoclonus, Familial Articular Chondrocalcinosis, Familial Atypical Mole-Malignant Melanoma Syndrome, Familial Broad Beta Disease, Familial Calcium Gout, Familial Calcium Pyrophosphate Arthropathy, Familial Chronic Obstructive Lung Disease, Familial Continuous Skin Peeling, Familial Cutaneous Amyloidosis, Familial Dysproteinemia, Familial Emphysema, Familial Enteropathy Microvillus, Familial Foveal Retinoschisis, 30 Familial Hibernation Syndrome, Familial High Cholesterol, Familial Hemochromatosis, Familial High Blood Cholesterol, Familial High-Density Lipoprotein Deficiency, Familial High Serum Cholesterol, Familial Hyperlipidema, Familial Hypoproteinemia with WO 2007/028212 PCT/AU2006/001325 - 227 Lymphangietatic Enteropathy, Familial Jaundice, Familial Juvenile Nephronophtisis Associated Ocular Anomaly, Familial Lichen Amyloidosis (Type IX), Familial Lumbar Stenosis, Familial Lymphedema Praecox, Familial Mediterranean Fever, Familial Multiple Polyposis, Familial Nuchal Bleb, Familial Paroxysmal Polyserositis, Familial Polyposis 5 Coli, Familial Primary Pulmonary Hypertension, Familial Renal Glycosuria, Familial Splenic Anemia, Familial Startle Disease, Familial Visceral Amyloidosis (Type VIII), FAMMM, FANCA, FANCB, FANCC, FANCD, FANCE, Fanconi Panmyelopathy, Fanconi Pancytopenia, Fanconi II, Fanconi's Anemia, Fanconi's Anemia Type I, Fanconi's Anemia Complementation Group, Fanconi's Anemia Complementation Group A, 10 Fanconi's Anemia Complementation Group B, Fanconi's Anemia Complementation Group C, Fanconi's Anemia Complementation Group D, Fanconi's Anemia Complementation Group E, Fanconi's Anemia Complementation Group G, Fanconi's Anemia Complementation Group H, Fanconi's Anemia Estren-Dameshek Variant, FANF, FANG, FANH, FAP, FAPG, Farber's Disease, Farber's Lipogranulomatosis, FAS, Fasting 15 Hypoglycemia, Fat-Induced Hyperlipemia, Fatal Granulomatous Disease of Childhood, Fatty Oxidation Disorders, Fatty Liver with Encephalopathy, FAV, FCH, FCMD, FCS Syndrome, FD, FDH, Febrile Mucocutaneous Syndrome Stevens Johnson Type, Febrile Neutrophilic Dermatosis Acute, Febrile Seizures, Feinberg's syndrome, Feissinger-Leroy Reiter Syndrome, Female Pseudo-Turner Syndrome, Femoral Dysgenesis Bilateral-Robin 20 Anomaly, Femoral Dysgenesis Bilateral, Femoral Facial Syndrome, Femoral Hypoplasia Unusual Facies Syndrome, Fetal Alcohol Syndrome, Fetal Anti-Convulsant Syndrome, Fetal Cystic Hygroma, Fetal Effects of Alcohol, Fetal Effects of Chickenpox, Fetal Effects of Thalidomide, Fetal Effects of Varicella Zoster Virus, Fetal Endomyocardial Fibrosis, Fetal Face Syndrome, Fetal Iritis Syndrome, Fetal Transfusion Syndrome, Fetal Valproate 25 Syndrome, Fetal Valproic Acid Exposure Syndrome, Fetal Varicella Infection, Fetal Varicella Zoster Syndrome, FFDD Type II, FG Syndrome, FGDY, FHS, Fibrin Stabilizing Factor Deficiency, Fibrinase Deficiency, Fibrinoid Degeneration of Astrocytes, Fibrinoid Leukodystrophy, Fibrinoligase Deficiency, Fibroblastoma Perineural, Fibrocystic Disease of Pancreas, Fibrodysplasia Ossificans Progressiva, Fibroelastic Endocarditis, 30 Fibromyalgia, Fibromyalgia-Fibromyositis, Fibromyositis, Fibrosing Cholangitis, Fibrositis, Fibrous Ankylosis of Multiple Joints, Fibrous Cavernositis, Fibrous Dysplasia, Fibrous Plaques of the Penis, Fibrous Sclerosis of the Penis, Fickler-Winkler Type, Fiedler WO 2007/028212 PCT/AU2006/001325 -228 Disease, Fifth Digit Syndrome, Filippi Syndrome, Finnish Type Amyloidosis (Type V), First Degree Congenital Heart Block, First and Second Branchial Arch Syndrome, Fischer's Syndrome, Fish Odor Syndrome, Fissured Tongue, Flat Adenoma Syndrome, Flatau-Schilder Disease, Flavin Containing Monooxygenase 2, Floating Beta Disease, 5 Floating-Harbor Syndrome, Floating Spleen, Floppy Infant Syndrome, Floppy Valve Syndrome, Fluent aphasia, FMD, FMF, FMO Adult Liver Form, FMO2, FND, Focal Brain Ischemia, Focal Dermal Dysplasia Syndrome, Focal Dermal Hypoplasia, Focal Dermato Phalangeal Dysplasia, Focal Dystonia, Focal Epilepsy, Focal Facial Dermal Dysplasia Type II, Focal Neuromyotonia, FODH, Folling Syndrome, Fong Disease, FOP, Forbes 10 Disease, Forbes-Albright Syndrome, Forestier's Disease, Forsius-Eriksson Syndrome (X Linked), Fothergill Disease, Fountain Syndrome, Foveal Dystrophy Progressive, FPO Syndrome Type II, FPO, Fraccaro Type Achondrogenesis (Type IB), Fragile X syndrome, Franceschetti-Zwalen-Klein Syndrome, Francois Dyscephaly Syndrome, Francois-Neetens Speckled Dystrophy, Flecked Corneal Dystrophy, Fraser Syndrome, FRAXA, FRDA, 15 Fredrickson Type I Hyperlipoproteinemia, Freeman-Sheldon Syndrome, Freire-Maia Syndrome, Frey's Syndrome, Friedreich's Ataxia, Friedreich's Disease, Friedreich's Tabes, FRNS, Froelich's Syndrome, Frommel-Chiari Syndrome, Frommel-Chiari Syndrome Lactation-Uterus Atrophy, Frontodigital Syndrome, Frontofacionasal Dysostosis, Frontofacionasal Dysplasia, Frontonasal Dysplasia, Frontonasal Dysplasia 20 with Coronal Craniosynostosis, Fructose- 1-Phosphate Aldolase Deficiency, Fructosemia, Fructosuria, Fryns Syndrome, FSH, FSHD, FSS, Fuchs Dystrophy, Fucosidosis Type 1, Fucosidosis Type 2, Fucosidosis Type 3, Fukuhara Syndrome, Fukuyama Disease, Fukuyama Type Muscular Dystrophy, Fumarylacetoacetase deficiency, Furrowed Tongue, G Syndrome, G6PD Deficiency, G6PD, GA I, GA IIB, GA IIA, GA II, GAII & MADD, 25 Galactorrhea-Amenorrhea Syndrome Nonpuerperal, Galactorrhea-Amenorrhea without Pregnancy, Galactosamine-6-Sulfatase Deficiency, Galactose- 1-Phosphate Uridyl Transferase Deficiency, Galactosemia, GALB Deficiency, Galloway-Mowat Syndrome, Galloway Syndrome, GALT Deficiency, Gammaglobulin Deficiency, GAN, Ganglioside Neuraminidase Deficiency, Ganglioside Sialidase Deficiency, Gangliosidosis GM1 Type 30 1, Gangliosidosis GM2 Type 2, Gangliosidosis Beta Hexosaminidase B Defeciency, Gardner Syndrome, Gargoylism, Garies-Mason Syndrome, Gasser Syndrome, Gastric Intrinsic Factor Failure of Secretion, Enterocyte Cobalamin, Gastrinoma, Gastritis, WO 2007/028212 PCT/AU2006/001325 - 229 Gastroesophageal Laceration-Hemorrhage, Gastrointestinal Polyposis and Ectodermal Changes, Gastrointestinal ulcers, Gastroschisis, Gaucher Disease, Gaucher Schlagenhaufer, Gayet-Wernicke Syndrome, GBS, GCA, GCM Syndrome, GCPS, Gee Herter Disease, Gee-Thaysen Disease, Gehrig's Disease, Gelineau's Syndrome, Genee 5 Wiedemann Syndrome, Generalized Dystonia, Generalized Familial Neuromyotonia, Generalized Fibromatosis, Generalized Flexion Epilepsy, Generalized Glycogenosis, Generalized Hyperhidrosis, Generalized Lipofuscinosis, Generalized Myasthenia Gravis, Generalized Myotonia, Generalized Sporadic Neuromytonia, Genetic Disorders, Genital Defects, Genital and Urinary Tract Defects, Gerstmann Syndrome, Gerstmann Tetrad, 10 GHBP, GHD, GHR, Giant Axonal Disease, Giant Axonal Neuropathy, Giant Benign Lymphoma, Giant Cell Glioblastoma Astrocytoma, Giant Cell Arteritis, Giant Cell Disease of the Liver, Giant Cell Hepatitis, Giant Cell of Newborns Cirrhosis, Giant Cyst of the Retina, Giant Lymph Node Hyperplasia, Giant Platelet Syndrome Hereditary, Giant Tongue, gic Macular Dystrophy, Gilbert's Disease, Gilbert Syndrome, Gilbert-Dreyfus 15 Syndrome, Gilbert-Lereboullet Syndrome, Gilford Syndrome, Gilles de la Tourette's syndrome, Gillespie Syndrome, Gingival Fibromatosis-Abnormal Fingers Nails Nose Ear Splenomegaly, GLA Deficiency, GLA, GLB 1, Glaucoma, Glioma Retina, Global aphasia, Globoid Leukodystrophy, Glossoptosis Micrognathia and Cleft Palate, Glucocerebrosidase deficiency, Glucocerebrosidosis, Glucose-6-Phosphate Dehydrogenase Deficiency, 20 Glucose-6-Phosphate Tranport Defect, Glucose-6-Phospate Translocase Deficiency, Glucose-G-Phosphatase Deficiency, Glucose-Galactose Malabsorption, Glucosyl Ceramide Lipidosis, Glutaric Aciduria I, Glutaric Acidemia I, Glutaric Acidemia II, Glutaric Aciduria II, Glutaric Aciduria Type II, Glutaric Aciduria Type III, Glutaricacidemia I, Glutaricacidemia II, Glutaricaciduria I, Glutaricaciduria II, 25 Glutaricaciduria Type IIA, Glutaricaciduria Type IIB, Glutaryl-CoA Dehydrogenase Deficiency, Glutaurate-Aspartate Transport Defect, Gluten-Sensitive Enteropathy, Glycogen Disease of Muscle Type VII, Glycogen Storage Disease I, Glycogen Storage Disease III, Glycogen Storage Disease IV, Glycogen Storage Disease Type V, Glycogen Storage Disease VI, Glycogen Storage Disease VII, Glycogen Storage Disease VIII, 30 Glycogen Storage Disease Type II, Glycogen Storage Disease-Type II, Glycogenosis, Glycogenosis Type I, Glycogenosis Type IA, Glycogenosis Type IB, Glycogenosis Type II, Glycogenosis Type II, Glycogenosis Type III, Glycogenosis Type IV, Glycogenosis WO 2007/028212 PCT/AU2006/001325 -230 Type V, Glycogenosis Type VI, Glycogenosis Type VII, Glycogenosis Type VIII, Glycolic Aciduria, Glycolipid Lipidosis, GM2 Gangliosidosis Type 1, GM2 Gangliosidosis Type 1, GNPTA, Goitrous Autoimmune Thyroiditis, Goldenhar Syndrome, Goldenhar-Gorlin Syndrome, Goldscheider's Disease, Goltz Syndrome, Goltz-Gorlin 5 Syndrome, Gonadal Dysgenesis 45 X, Gonadal Dysgenesis XO, Goniodysgenesis Hypodontia, Goodman Syndrome, Goodman, Goodpasture Syndrome, Gordon Syndrome, Gorlin's Syndrome, Gorlin-Chaudhry-Moss Syndrome, Gottron Erythrokeratodermia Congenitalis Progressiva Symmetrica, Gottron's Syndrome, Gougerot-Carteaud Syndrome, Grand Mal Epilepsy, Granular Type Corneal Dystrophy, Granulomatous Arteritis, 10 Granulomatous Colitis, Granulomatous Dermatitis with Eosinophilia, Granulomatous Ileitis, Graves Disease, Graves' Hyperthyroidism, Graves' Disease, Greig Cephalopolysyndactyly Syndrome, Groenouw Type I Corneal Dystrophy, Groenouw Type II Corneal Dystrophy, Gronblad-Strandberg Syndrome, Grotton Syndrome, Growth Hormone Receptor Deficiency, Growth Hormone Binding Protein Deficiency, Growth 15 Hormone Deficiency, Growth-Mental Deficiency Syndrome of Myhre, Growth Retardation-Rieger Anomaly, GRS, Gruber Syndrome, GS, GSD6, GSD8, GTS, Guanosine Triphosphate-Cyclohydrolase Deficiency, Guanosine Triphosphate Cyclohydrolase Deficiency, Guenther Porphyria, Guerin-Stem Syndrome, Guillain-Barr6, Guillain-Barre Syndrome, Gunther Disease, H Disease, H. Gottron's Syndrome, Habit 20 Spasms, HAE, Hageman Factor Deficiency, Hageman factor, Haim-Munk Syndrome, Hajdu-Cheney Syndrome, Hajdu Cheney, HAL Deficiency, Hall-Pallister Syndrome, Hallermann-Streiff-Francois syndrome, Hallermann-Streiff Syndrome, Hallervorden-Spatz Disease, Hallervorden-Spatz Syndrome, Hallopeau-Siemens Disease, Hallux Duplication Postaxial Polydactyly and Absence of Corpus Callosum, Halushi-Behcet's Syndrome, 25 Hamartoma of the Lymphatics, Hand-Schueller-Christian Syndrome, HANE, Hanhart Syndrome, Happy Puppet Syndrome, Harada Syndrome, HARD +/-E Syndrome, HARD Syndrome, Hare Lip, Harlequin Fetus, Harlequin Type DOC 6, Harlequin Type Ichthyosis, Harley Syndrome, Harrington Syndrome, Hart Syndrome, Hartnup Disease, Hartnup Disorder, Hartnup Syndrome, Hashimoto's Disease, Hashimoto-Pritzker Syndrome, 30 Hashimoto's Syndrome, Hashimoto's Thyroiditis, Hashimoto-Pritzker Syndrome, Hay Well's Syndrome, Hay-Wells Syndrome of Ectodermal Dysplasia, HCMM, HCP, HCTD, HD, Heart-Hand Syndrome (Holt-Oram Type), Heart Disease, Hecht Syndrome, RED, WO 2007/028212 PCT/AU2006/001325 -231 Heerferdt-Waldenstrom and Lofgren's Syndromes, Hegglin's Disease, Heinrichsbauer Syndrome, Hemangiomas, Hemangioma Familial, Hemangioma-Thrombocytopenia Syndrome, Hemangiomatosis Chondrodystrophica, Hemangiomatous Branchial Clefts-Lip Pseudocleft Syndrome, Hemifacial Microsomia, Hemimegalencephaly, Hemiparesis of 5 Cerebral Palsy, Hemiplegia of Cerebral Palsy, Hemisection of the Spinal Cord, Hemochromatosis, Hemochromatosis Syndrome, Hemodialysis-Related Amyloidosis, Hemoglobin Lepore Syndromes, Hemolytic Anemia of Newborn, Hemolytic Cold Antibody Anemia, Hemolytic Disease of Newborn, Hemolytic-Uremic Syndrome, Hemophilia, Hemophilia A, Hemophilia B, Hemophilia B Factor IX, Hemophilia C, 10 Hemorrhagic Dystrophic Thrombocytopenia, Hemorrhagica Aleukia, Hemosiderosis, Hepatic Fructokinase Deficiency, Hepatic Phosphorylase Kinase Deficiency, Hepatic Porphyria, Hepatic Porphyrias, Hepatic Veno-Occlusive Diseas, Hepatitis C, Hepato-Renal Syndrome, Hepatolenticular Degeneration, Hepatophosphorylase Deficiency, Hepatorenal Glycogenosis, Hepatorenal Syndrome, Hepatorenal Tyrosinemia, Hereditary 15 Acromelalgia, Hereditary Alkaptonuria, Hereditary Amyloidosis, Hereditary Angioedema, Hereditary Areflexic Dystasia, Heredopathia Atactica Polyneuritiformis, Hereditary Ataxia, Hereditary Ataxia Friedrich's Type, Hereditary Benign Acanthosis Nigricans, Hereditary Cerebellar Ataxia, Hereditary Chorea, Hereditary Chronic Progressive Chorea, Hereditary Connective Tissue Disorders, Hereditary Coproporphyria, Hereditary 20 Coproporphyria Porphyria, Hereditary Cutaneous Malignant Melanoma, Hereditary Deafness-Retinitis Pigmentosa, Heritable Disorder of Zinc Deficiency, Hereditary DNS, Hereditary Dystopic Lipidosis, Hereditary Emphysema, Hereditary Fructose Intolerance, Hereditary Hemorrhagic Telangiectasia, Hereditary Hemorrhagic Telangiectasia Type I, Hereditary Hemorrhagic Telangiectasia Type II, Hereditary Hemorrhagic Telangiectasia 25 Type III, Hereditary Hyperuricemia and Choreoathetosis Syndrome, Hereditary Leptocytosis Major, Hereditary Leptocytosis Minor, Hereditary Lymphedema, Hereditary Lymphedema Tarda, Hereditary Lymphedema Type I, Hereditary Lymphedema Type II, Hereditary Motor Sensory Neuropathy, Hereditary Motor Sensory Neuropathy I, Hereditary Motor Sensory Neuropathy Type III, Hereditary Nephritis, Hereditary Nephritis 30 and Nerve Deafness, Hereditary Nephropathic Amyloidosis, Hereditary Nephropathy and Deafness, Hereditary Nonpolyposis Colorectal Cancer, Hereditary Nonpolyposis Colorectal Carcinoma, Hereditary Nonspherocytic Hemolytic Anemia, Hereditary WO 2007/028212 PCT/AU2006/001325 -232 Onychoosteodysplasia, Hereditary Optic Neuroretinopathy, Hereditary Polyposis Coli, Hereditary Sensory and Autonomic Neuropathy Type I, Hereditary Sensory and Autonomic Neuropathy Type II, Hereditary Sensory and Autonomic Neuropathy Type III, Hereditary Sensory Motor Neuropathy, Hereditary Sensory Neuropathy type I, Hereditary 5 Sensory Neuropathy Type I, Hereditary Sensory Neuropathy Type II, Hereditary Sensory Neuropathy Type III, Hereditary Sensory Radicular Neuropathy Type I, Hereditary Sensory Radicular Neuropathy Type I, Hereditary Sensory Radicular Neuropathy Type II, Hereditary Site Specific Cancer, Hereditary Spherocytic Hemolytic Anemia, Hereditary Spherocytosis, Hereditary Tyrosinemia Type 1, Heritable Connective Tissue Disorders, 10 Herlitz Syndrome, Hermans-Herzberg Phakomatosis, Hermansky-Pudlak Syndrome, Hermaphroditism, Herpes Zoster, Herpes Iris Stevens-Johnson Type, Hers Disease, Heterozygous Beta Thalassemia, Hexoaminidase Alpha-Subunit Deficiency (Variant B), Hexoaminidase Alpha-Subunit Deficiency (Variant B), HFA, HFM, HGPS, HH, HHHO, HHRH, HHT, Hiatal Hemia-Microcephaly-Nephrosis Galloway Type, Hidradenitis 15 Suppurativa, Hidrosadenitis Axillaris, Hidrosadenitis Suppurativa, Hidrotic Ectodermal Dysplasias, HIE Syndrome, High Imperforate Anus, High Potassium, High Scapula, HIM, Hirschsprung's Disease, Hirschsprung's Disease Acquired, Hirschsprung Disease Polydactyly of Ulnar & Big Toe and VSD, Hirschsprung Disease with Type D Brachydactyly, Hirsutism, HIS Deficiency, Histidine Ammonia-Lyase (HAL) Deficiency, 20 Histidase Deficiency, Histidinemia, Histiocytosis, Histiocytosis X, HLHS, HLP Type II, HMG, HMI, HMSN I, HNHA, HOCM, Hodgkin Disease, Hodgkin's Disease, Hodgkin's Lymphoma, Hollaender-Simons Disease, Holmes-Adie Syndrome, Holocarboxylase Synthetase Deficiency, Holoprosencephaly, Holoprosencephaly Malformation Complex, Holoprosencephaly Sequence, Holt-Oram Syndrome, Holt-Oram Type Heart-Hand 25 Syndrome, Homocystinemia, Homocystinuria, Homogentisic Acid Oxidase Deficiency, Homogentisic Acidura, Homozygous Alpha-i -Antitrypsin Deficiency, HOOD, Homer Syndrome, Horton's disease, HOS, HOS1, Houston-Harris Type Achrondrogenesis (Type IA), HPS, HRS, HS, HSAN Type I, HSAN Type II, HSAN-III, HSMN, HSMN Type III, HSN I, HSN-III, Huebner-Herter Disease, Hunner's Patch, Hunner's Ulcer, Hunter 30 Syndrome, Hunter-Thompson Type Acromesomelic Dysplasia, Huntington's Chorea, Huntington's Disease, Hurler Disease, Hurler Syndrome, Hurler-Scheie Syndrome, HUS, Hutchinson-Gilford Progeria Syndrome, Hutchinson-Gilford Syndrome, Hutchinson- WO 2007/028212 PCT/AU2006/001325 -233 Weber-Peutz Syndrome, Hutterite Syndrome Bowen-Conradi Type, Hyaline Panneuropathy, Hydranencephaly, Hydrocephalus, Hydrocephalus Agyria and Retinal Dysplasia, Hydrocephalus Internal Dandy-Walker Type, Hydrocephalus Noncommunicating Dandy-Walker Type, Hydrocephaly, Hydronephrosis With Peculiar 5 Facial Expression, Hydroxylase Deficiency, Hygroma Colli, Hyper-IgE Syndrome, Hyper IgM Syndrome, Hyperaldosteronism, Hyperaldosteronism With Hypokalemic Alkatosis, Hyperaldosteronism Without Hypertension, Hyperammonemia, Hyperammonemia Due to Carbamylphosphate Synthetase Deficiency, Hyperammonemia Due to Ornithine Transcarbamylase Deficiency, Hyperammonemia Type II, Hyper-Beta Carnosinemia, 10 Hyperbilirubinemia I, Hyperbilirubinemia II, Hypercalcemia Familial with Nephrocalcinosis and Indicanuria, Hypercalcemia-Supravalvar Aortic Stenosis, Hypercalciuric Rickets, Hypercapnic acidosis, Hypercatabolic Protein-Losing Enteropathy, Hyperchloremic acidosis, Hypercholesterolemia, Hypercholesterolemia Type IV, Hyperchylomicronemia, Hypercystinuria, Hyperekplexia, Hyperextensible joints, 15 Hyperglobulinemic Purpura, Hyperglycinemia with Ketoacidosis and Lactic Acidosis Propionic Type, Hyperglycinemia Nonketotic, Hypergonadotropic Hypogonadism, Hyperimmunoglobulin E Syndrome, Hyperimmunoglobulin E-Recurrent Infection Syndrome, Hyperimmunoglobulinemia E-Staphylococcal, Hyperkalemia, Hyperkinetic Syndrome, Hyperlipemic Retinitis, Hyperlipidemia I, Hyperlipidemia IV, 20 Hyperlipoproteinemia Type I, Hyperlipoproteinemia Type III, Hyperlipoproteinemia Type IV, Hyperoxaluria, Hyperphalangy-Clinodactyly of Index Finger with Pierre Robin Syndrome, Hyperphenylalanemia, Hyperplastic Epidermolysis Bullosa, Hyperpnea, Hyperpotassemia, Hyperprebeta-Lipoproteinemia, Hyperprolinemia Type I, Hyperprolinemia Type II, Hypersplenism, Hypertelorism with Esophageal Abnormalities 25 and Hypospadias, Hypertelorism-Hypospadias Syndrome, Hypertrophic Cardio myopathy, Hypertrophic Interstitial Neuropathy, Hypertrophic Interstitial Neuritis, Hypertrophic Interstitial Radiculoneuropathy, Hypertrophic Neuropathy of Refsum, Hypertrophic Obstructive Cardio myopathy, Hyperuricemia Choreoathetosis Self-multilation Syndrome, Hyperuricemia-Oligophrenia, Hypervalinemia, Hypocalcified (Hypomineralized) Type, 30 Hypochondrogenesis, Hypochrondroplasia, Hypogammaglobulinemia, Hypogammaglobulinemia Transient of Infancy, Hypogenital Dystrophy with Diabetic Tendency, Hypoglossia-Hypodactylia Syndrome, Hypoglycemia, Exogenous WO 2007/028212 PCT/AU2006/001325 -234 Hypoglycemia, Hypoglycemia with Macroglossia, Hypoglycosylation Syndrome Type I a, Hypoglycosylation Syndrome Type la, Hypogonadism with Anosmia, Hypogonadotropic Hypogonadism and Anosmia, Hypohidrotic Ectodermal Dysplasia, Hypohidrotic Ectodermal Dysplasia Autosomal Dominant type, Hypohidrotic Ectodermal Dysplasias 5 Autorecessive, Hypokalemia, Hypokalemic Alkalosis with Hypercalciuria, Hypokalemic Syndrome, Hypolactasia, Hypomaturation Type (Snow-Capped Teeth), Hypomelanosis of Ito, Hypomelia-Hypotrichosis-Facial Hemangioma Syndrome, Hypomyelination Neuropathy, Hypoparathyroidism, Hypophosphatasia, Hypophosphatemic Rickets with Hypercalcemia, Hypopigmentation, Hypopigmented macular lesion, Hypoplasia of the 10 Depressor Anguli Oris Muscle with Cardiac Defects, Hypoplastic Anemia, Hypoplastic Congenital Anemia, Hypoplastic Chondrodystrophy, Hypoplastic Enamel-Onycholysis Hypohidrosis, Hypoplastic (Hypoplastic-Explastic) Type, Hypoplastic Left Heart Syndrome, Hypoplastic-Triphalangeal Thumbs, Hypopotassemia Syndrome, Hypospadias Dysphagia Syndrome, Hyposmia, Hypothalamic Hamartoblastoma Hypopituitarism 15 Imperforate Anus Polydactyly, Hypothalamic Infantilism-Obesity, Hypothyroidism, Hypotonia-Hypomentia-Hypogonadism-Obesity Syndrome, Hypoxanthine-Guanine Phosphoribosyltranferase Defect (Complete Absense of), I-Cell Disease, Iatrogenic Hypoglycemia, IBGC, IBIDS Syndrome, IBM, IBS, IC, I-Cell Disease, ICD, ICE Syndrome Cogan-Reese Type, Icelandic Type Amyloidosis (Type VI), I-Cell Disease, 20 Ichthyosiform Erythroderma Corneal Involvement and Deafness, Ichthyosiform Erythroderma Hair Abnormality Growth and Men, Ichthyosiform Erythroderma with Leukocyte Vacuolation, Ichthyosis, Ichthyosis Congenita, Ichthyosis Congenital with Trichothiodystrophy, Ichthyosis Hystrix, Ichthyosis Hystrix Gravior, Ichthyosis Linearis Circumflexa, Ichthyosis Simplex, Ichthyosis Tay Syndrome, Ichthyosis Vulgaris, 25 Ichthyotic Neutral Lipid Storage Disease, Icteric Leptospirosis, Icterohemorrhagic Leptospirosis, Icterus (Chronic Familial), Icterus Gravis Neonatorum, Icterus Intermittens Juvenalis, Idiopathic Alveolar Hypoventilation, Idiopathic Amyloidosis, Idiopathic Arteritis of Takayasu, Idiopathic Basal Ganglia Calcification (IBGC), Idiopathic Brachial Plexus Neuropathy, Idiopathic Cervical Dystonia, Idiopathic Dilatation of the Pulmonary 30 Artery, Idiopathic Facial Palsy, Idiopathic Familial Hyperlipemia, Idiopathic Hypertrophic Subaortic Stenosis, Idiopathic Hypoproteinemia, Idiopathic Immunoglobulin Deficiency, Idiopathic Neonatal Hepatitis, Idiopathic Non-Specific Ulcerative Colitis, Idiopathic WO 2007/028212 PCT/AU2006/001325 -235 Peripheral Periphlebitis, Idiopathic Pulmonary Fibrosis, Idiopathic Refractory Sideroblastic Anemia, Idiopathic Renal Hematuria, Idiopathic Steatorrhea, Idiopathic Thrombocythemia, Idiopathic Thrombocytopenic Purpura, Idiopathic Thrombocytopenia Purpura (ITP), IDPA, IgA Nephropathy, IHSS, Ileitis, Ileocolitis, Illinois Type 5 Amyloidosis, ILS, IM, IMD2, IMD5, Immune Defect due to Absence of Thymus, Immune Hemolytic Anemia Paroxysmal Cold, Immunodeficiency with Ataxia Telangiectasia, Immunodeficiency Cellular with Abnormal Immunoglobulin Synthesis, Immunodeficiency Common Variable Unclassifiable, Immunodeficiency with Hyper-IgM, Immunodeficiency with Leukopenia, Immunodeficiency-2, Immunodeficiency-5 (IMD5), Immunoglobulin 10 Deficiency, Imperforate Anus, Imperforate Anus with Hand Foot and Ear Anomalies, Imperforate Nasolacrimal Duct and Premature Aging Syndrome, Impotent Neutrophil Syndrome, Inability To Open Mouth Completely And Short Finger-Flexor, INAD, Inborn Error of Urea Synthesis Arginase Type, Inborn Error of Urea Synthesis Arginino Succinic Type, Inborn Errors of Urea Synthesis Carbamyl Phosphate Type, Inborn Error of Urea 15 Synthesis Citrullinemia Type, Inborn Errors of Urea Synthesis Glutamate Synthetase Type, INCL, Inclusion body myositis, Incomplete Atrioventricular Septal Defect, Incomplete Testicular Feminization, Incontinentia Pigmenti, Incontinenti Pigmenti Achromians, Index Finger Anomaly with Pierre Robin Syndrome, Indiana Type Amyloidosis (Type II), Indolent systemic mastocytosis, Infantile Acquired Aphasia, Infantile Autosomal 20 Recessive Polycystic Kidney Disease, Infantile Beriberi, Infantile Cerebral Ganglioside, Infantile Cerebral Paralysis, Infantile Cystinosis, Infantile Epileptic, Infantile Fanconi Syndrome with Cystinosis, Infantile Finnish Type Neuronal Ceroid Lipofuscinosis, Infantile Gaucher Disease, Infantile Hypoglycemia, Infantile Hypophasphatasia, Infantile Lobar Emphysema, Infantile Myoclonic Encephalopathy, Infantile Myoclonic 25 Encephalopathy and Polymyoclonia, Infantile Myofibromatosis, Infantile Necrotizing Encephalopathy, Infantile Neuronal Ceroid Lipofuscinosis, Infantile Neuroaxonal Dystrophy, Infantile Onset Schindler Disease, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease (IRD), Infantile Sipoidosis GM-2 Gangliosideosis (Type S), Infantile Sleep Apnea, Infantile Spasms, Infantile Spinal Muscular Atrophy (all types), 30 Infantile Spinal Muscular Atrophy ALS, Infantile Spinal Muscular Atrophy Type I, Infantile Type Neuronal Ceroid Lipofuscinosis, Infectious Jaundice, Inflammatory Bowel Disease, Inflammatory Breast Cancer, Inflammatory Linear Nevus Sebaceous Syndrome, WO 2007/028212 PCT/AU2006/001325 -236 Iniencephaly, Insulin Resistant Acanthosis Nigricans, Insulin Lipodystrophy, Insulin dependent Diabetes, Intention Myoclonus, Intermediate Cystinosis, Intermediate Maple Syrup Urine Disease, Intermittent Ataxia with Pyruvate Dehydrogenase Deficiency, Intermittent Maple Syrup Urine Disease, Internal Hydrocephalus, Interstitial Cystitis, 5 Interstitial Deletion of 4q Included, Intestinal Lipodystrophy, Intestinal Lipophagic Granulomatosis, Intestinal Lymphangiectasia, Intestinal Polyposis I, Intestinal Polyposis II, Intestinal Polyposis III, Intestinal Polyposis-Cutaneous Pigmentation Syndrome, Intestinal Pseudoobstruction with External Ophthalmoplegia, Intracranial Neoplasm, Intracranial Tumors, Intracranial Vascular Malformations, Intrauterine Dwarfism, 10 Intrauterine Synechiae, Inverted Smile And Occult Neuropathic Bladder, Iowa Type Amyloidosis (Type IV), IP, IPA, Iridocorneal Endothelial Syndrome, Iridocorneal Endothelial (ICE) Syndrome Cogan-Resse Type, Iridogoniodysgenesis With Somatic Anomalies, Iris Atrophy with Corneal Edema and Glaucoma, Iris Nevus Syndrome, Iron Overload Anemia, Iron Overload Disease, Irritable Bowel Syndrome, Irritable Colon 15 Syndrome, Isaacs Syndrome, Isaacs-Merten Syndrome, Ischemic Cardio myopathy, Isolated Lissencephaly Sequence, Isoleucine 33 Amyloidosis, Isovaleric Acid CoA Dehydrogenase Deficiency, Isovaleric Acidaemia, Isovalericacidemia, Isovaleryl CoA Carboxylase Deficiency, ITO Hypomelanosis, ITO, ITP, IVA, Ivemark Syndrome, Iwanoff Cysts, Jackknife Convulsion, Jackson-Weiss Craniosynostosis, Jackson-Weiss Syndrome, 20 Jacksonian Epilepsy, Jacobsen Syndrome, Jadassohn-Lewandowsky Syndrome, Jaffe Lichenstein Disease, Jakob's Disease, Jakob-Creutzfeldt Disease, Janeway I, Janeway Dysgammaglobulinemia, Jansen Metaphyseal Dysostosis, Jansen Type Metaphyseal Chondrodysplasia, Jarcho-Levin Syndrome, Jaw-Winking, JBS, JDMS, Jegher's Syndrome, Jejunal Atresia, Jejunitis, Jejunoileitis, Jervell and Lange-Nielsen Syndrome, 25 Jeune Syndrome, JMS, Job Syndrome, Job-Buckley Syndrome, Johanson-Blizzard Syndrome, John Dalton, Johnson-Stevens Disease, Jonston's Alopecia, Joseph's Disease, Joseph's Disease Type I, Joseph's Disease Type II, Joseph's Disease Type III, Joubert Syndrome, Joubert-Bolthauser Syndrome, JRA, Juberg Hayward Syndrome, Juberg Marsidi Syndrome, Juberg-Marsidi Mental Retardation Syndrome, Jumping Frenchmen, 30 Jumping Frenchmen of Maine, Juvenile Arthritis, Juvenile Autosomal Recessive Polycystic Kidney Disease, Juvenile Cystinosis, Juvenile (Childhood) Dermatomyositis (JDMS), Juvenile Diabetes, Juvenile Gaucher Disease, Juvenile Gout Choreoathetosis and WO 2007/028212 PCT/AU2006/001325 -237 Mental Retardation Syndrome, Juvenile Intestinal Malabsorption of Vit B12, Juvenile Intestinal Malabsorption of Vitamin B12, Juvenile Macular Degeneration, Juvenile Pernicious Anemia, Juvenile Retinoschisis, Juvenile Rheumatoid Arthritis, Juvenile Spinal Muscular Atrophy Included, Juvenile Spinal Muscular Atrophy ALS Included, Juvenile 5 Spinal Muscular Atrophy Type III, Juxta-Articular Adiposis Dolorosa, Juxtaglomerular Hyperplasia, Kabuki Make-Up Syndrome, Kahler Disease, Kallmann Syndrome, Kanner Syndrome, Kanzaki Disease, Kaposi Disease (not Kaposi Sarcoma), Kappa Light Chain Deficiency, Karsch-Neugebauer Syndrome, Kartagener Syndrome-Chronic Sinobronchial Disease and Dextrocardia, Kartagener Triad, Kasabach-Merritt Syndrome, Kast Syndrome, 10 Kawasaki Disease, Kawasaki Syndrome, KBG Syndrome, KD, Kearns-Sayre Disease, Kearns-Sayre Syndrome, Kennedy Disease, Kennedy Syndrome, Kennedy Type Spinal and Bulbar Muscular Atrophy, Kennedy-Stefanis Disease, Kenny Disease, Kenny Syndrome, Kenny Type Tubular Stenosis, Kenny-Caffe Syndrome, Kera. Palmoplant. Con. Pes Planus Ony. Periodon. Arach., Keratitis Ichthyosis Deafness Syndrome, Keratoconus, 15 Keratoconus Posticus Circumscriptus, Keratolysis, Keratolysis Exfoliativa Congenita, Keratolytic Winter Erythema, Keratomalacia, Keratosis Follicularis, Keratosis Follicularis Spinulosa Decalvans, Keratosis Follicularis Spinulosa Decalvans Ichthyosis, Keratosis Nigricans, Keratosis Palmoplantaris with Periodontopathia and Onychogryposis, Keratosis Palmoplantaris Congenital Pes Planus Onychogryposis Periodontosis Arachnodactyly, 20 Keratosis Palmoplantaris Congenital, Pes Planus, Onychogryphosis, Periodontosis, Arachnodactyly, Acroosteolysis, Keratosis Rubra Figurata, Keratosis Seborrheica, Ketoacid Decarboxylase Deficiency, Ketoaciduria, Ketotic Glycinemia, KFS, KID Syndrome, Kidney Agenesis, Kidneys Cystic-Retinal Aplasia Joubert Syndrome, Killian Syndrome, Killian/Teschler-Nicola Syndrome, Kiloh-Nevin syndrome III, Kinky Hair 25 Disease, Kinsbourne Syndrome, Kleeblattschadel Deformity, Kleine-Levin Syndrome, Kleine-Levin Hibernation Syndrome, Klinefelter, Klippel-Feil Syndrome, Klippel-Feil Syndrome Type I, Klippel-Feil Syndrome Type II, Klippel-Feil Syndrome Type III, Klippel Trenaunay Syndrome, Klippel-Trenaunay-Weber Syndrome, Kluver-Bucy Syndrome, KMS, Kniest Dysplasia, Kniest Syndrome, Kobner's Disease, Koebberling 30 Dunnigan Syndrome, Kohlmeier-Degos Disease, Kok Disease, Korsakoff Psychosis, Korsakoff s Syndrome, Krabbe's Disease Included, Krabbe's Leukodystrophy, Kramer Syndrome, KSS, KTS, KTW Syndrome, Kufs Disease, Kugelberg-Welander Disease, WO 2007/028212 PCT/AU2006/001325 -238 Kugelberg-Welander Syndrome, Kussmaul-Landry Paralysis, KWS, L-3-Hydroxy-Acyl CoA Dehydrogenase (LCHAD) Deficiency, Laband Syndrome, Labhart-Willi Syndrome, Labyrinthine Syndrome, Labyrinthine Hydrops, Lacrimo-Auriculo-Dento-Digital Syndrome, Lactase Isolated Intolerance, Lactase Deficiency, Lactation-Uterus Atrophy, 5 Lactic Acidosis Leber Hereditary Optic Neuropathy, Lactic and Pyruvate Acidemia with Carbohydrate Sensitivity, Lactic and Pyruvate Acidemia with Episodic Ataxia and Weakness, Lactic and Pyruvate, Lactic acidosis, Lactose Intolerance of Adulthood, Lactose Intolerance, Lactose Intolerance of Childhood, LADD Syndrome, LADD, Lafora Disease Included, Lafora Body Disease, Laki-Lorand Factor Deficiency, LAM, Lambert 10 Type Ichthyosis, Lambert-Eaton Syndrome, Lambert-Eaton Myasthenic Syndrome, Lamellar Recessive Ichthyosis, Lamellar Ichthyosis, Lancereaux-Mathieu-Weil Spirochetosis, Landau-Kleffner Syndrome, Landouzy Dejerine Muscular Dystrophy, Landry Ascending Paralysis, Langer-Salidino Type Achondrogensis (Type II), Langer Giedion Syndrome, Langerhans-Cell Granulomatosis, Langerhans-Cell Histiocytosis 15 (LCH), Large Atrial and Ventricular Defect, Laron Dwarfism, Laron Type Pituitary Dwarfism, Larsen Syndrome, Laryngeal Dystonia, Latah (Observed in Malaysia), Late Infantile Neuroaxonal Dystrophy, Late Infantile Neuroaxonal Dystrophy, Late Onset Cockayne Syndrome Type III (Type C), Late-Onset Dystonia, Late-Onset Immunoglobulin Deficiency, Late Onset Pelizaeus-Merzbacher Brain Sclerosis, Lattice Corneal Dystrophy, 20 Lattice Dystrophy, Launois-Bensaude, Launois-Cleret Syndrome, Laurence Syndrome, Laurence-Moon Syndrome, Laurence-Moon/Bardet-Biedl, Lawrence-Seip Syndrome, LCA, LCAD Deficiency, LCAD, LCAD, LCADH Deficiency, LCH, LCHAD, LCPD, Le Jeune Syndrome, Leband Syndrome, Leber's Amaurosis, Leber's Congenital Amaurosis,Congenital Absence of the Rods and Cones, Leber's Congenital Tapetoretinal 25 Degeneration, Leber's Congenital Tapetoretinal Dysplasia, Leber's Disease, Leber's Optic Atrophy, Leber's Optic Neuropathy, Left Ventricular Fibrosis, Leg Ulcer, Legg-Calve Perthes Disease, Leigh's Disease, Leigh's Syndrome, Leigh's Syndrome (Subacute Necrotizing Encephalomyelopathy), Leigh Necrotizing Encephalopathy, Lennox-Gastaut Syndrome, Lentigio-Polypose-Digestive Syndrome, Lenz Dysmorphogenetic Syndrome, 30 Lenz Dysplasia, Lenz Microphthalmia Syndrome, Lenz Syndrome, LEOPARD Syndrome, Leprechaunism, Leptomeningeal Angiomatosis, Leptospiral Jaundice, Leri-Weill Disease, Leri-Weil Dyschondrosteosis, Leri-Weil Syndrome, Lermoyez Syndrome, Leroy Disease, WO 2007/028212 PCT/AU2006/001325 -239 Lesch Nyhan Syndrome, Lethal Infantile Cardio myopathy, Lethal Neonatal Dwarfism, Lethal Osteochondrodysplasia, Letterer-Siwe Disease, Leukocytic Anomaly Albinism, Leukocytic Inclusions with Platelet Abnormality, Leukodystrophy, Leukodystrophy with Rosenthal Fibers, Leukoencephalitis Periaxialis Concentric, Levine-Critchley Syndrome, 5 Levulosuria, Levy-Hollister Syndrome, LGMD, LGS, LHON, LIC, Lichen Ruber Acuminatus, Lichen Acuminatus, Lichen Amyloidosis, Lichen Planus, Lichen Psoriasis, Lignac-Debre-Fanconi Syndrome, Lignac-Fanconi Syndrome, Ligneous Conjunctivitis, Limb-Girdle Muscular Dystrophy, Limb Malformations-Dento-Digital Syndrome, Limit Dextrinosis, Linear Nevoid Hypermelanosis, Linear Nevus Sebacous Syndrome, Linear 10 Scleroderma, Linear Sebaceous Nevus Sequence, Linear Sebaceous Nevus Syndrome, Lingua Fissurata, Lingua Plicata, Lingua Scrotalis, Linguofacial Dyskinesia, Lip Pseudocleft-hemangiomatous Branchial Cyst Syndrome, Lipid Granulomatosis, Lipid Histiocytosis, Lipid Kerasin Type, Lipid Storage Disease, Lipid-Storage myopathy Associated with SCAD Deficiency, Lipidosis Ganglioside Infantile, Lipoatrophic Diabetes 15 Mellitus, Lipodystrophy, Lipoid Corneal Dystrophy, Lipoid Hyperplasia-Male Pseudohermaphroditism, Lipomatosis of Pancreas Congenital, Lipomucopolysaccharidosis Type I, Lipomyelomeningocele, Lipoprotein Lipase Deficiency Familial, LIS, LIS1, Lissencephaly 1, Lissencephaly Type I, Lissencephaly variants with agenesis of the corpus callosum cerebellar hypoplasia or other anomalies, Little Disease, Liver Phosphorylase 20 Deficiency, LKS, LM Syndrome, Lobar Atrophy, Lobar Atrophy of the Brain, Lobar Holoprosencephaly, Lobar Tension Emphysema in Infancy, Lobstein Disease (Type I), Lobster Claw Deformity, Localized Epidermolysis Bullosa, Localized Lipodystrophy, Localized Neuritis of the Shoulder Girdle, Loeffler's Disease, Loeffler Endomyocardial Fibrosis with Eosinophilia, Loeffler Fibroplastic Parietal Endocarditis, Loken Syndrome, 25 Loken-Senior Syndrome, Long-Chain 3-hydroxyacyl-CoA Dehydrogenase (LCHAD), Long Chain Acyl CoA Dehydrogenase Deficiency, Long-Chain Acyl-CoA Dehydrogenase (ACADL), Long-Chain Acyl-CoA Dehydrogenase Deficiency, Long QT Syndrome without Deafness, Lou Gehrig's Disease, Lou Gehrig's Disease Included, Louis-Bar Syndrome, Low Blood Sugar, Low-Density Beta Lipoprotein Deficiency, Low Imperforate 30 Anus, Low Potassium Syndrome, Lowe syndrome, Lowe's Syndrome, Lowe-Bickel Syndrome, Lowe-Terry-MacLachlan Syndrome, Lower Back Pain, LS, LTD, Lubs Syndrome, Luft Disease, Lumbar Canal Stenosis, Lumbar Spinal Stenosis, Lumbosacral WO 2007/028212 PCT/AU2006/001325 - 240 Spinal Stenosis, Lundborg-Unverricht Disease, Lundborg-Unverricht Disease Included, Lupus, Lupus, Lupus Erythematosus, Luschka-Magendie Foramina Atresia, Lyell Syndrome, Lyelles Syndrome, Lymphadenoid Goiter, Lymphangiectatic Protein-Losing Enteropathy, Lymphangioleiomatosis, Lymphangioleimyomatosis, Lymphangiomas, 5 Lymphatic Malformations, Lynch Syndromes, Lynch Syndrome I, Lynch Syndrome II, Lysosomal Alpha-N-Acetylgalactosaminidase Deficiency Schindler Type, Lysosomal Glycoaminoacid Storage Disease-Angiokeratoma Corporis Diffusum, Lysosomal Glucosidase Deficiency, MAA, Machado Disease, Machado-Joseph Disease, Macrencephaly, Macrocephaly, Macrocephaly Hemihypertrophy, Macrocephaly with 10 Multiple Lipomas and Hemangiomata, Macrocephaly with Pseudopapilledema and Multiple Hemangiomata, Macroglobulinemia, Macroglossia, Macroglossia-Omphalocele Visceromegaly Syndrome, Macrostomia Ablepheron Syndrome, Macrothrombocytopenia Familial Bernard-Soulier Type, Macula Lutea degeneration, Macular Amyloidosis, Macular Degeneration, Macular Degeneration Disciform, Macular Degeneration Senile, 15 Macular Dystrophy, Macular Type Corneal Dystrophy, MAD, Madelung's Disease, Maffucci Syndrome, Major Epilepsy, Malabsorption, Malabsorption-Ectodermal Dysplasia-Nasal Alar Hypoplasia, Maladie de Roger, Maladie de Tics, Malaria, Male Malformation of Limbs and Kidneys, Male Turner Syndrome, Malignant Acanthosis, Malignant Acanthosis Nigricans, Malignant Astrocytoma, Malignant Atrophic Papulosis, 20 Malignant Fever, Malignant Hyperphenylalaninemia, Malignant Hyperpyrexia, Malignant Hyperthermia, Malignant Melanoma, Malignant Tumors of the Central Nervous System, Mallory-Weiss Laceration, Mallory-Weiss Tear, Mallory-Weiss Syndrome, Mammary Paget's Disease, Mandibular Ameloblastoma, Mandibulofacial Dysostosis, Mannosidosis, Map-Dot-Fingerprint Type Corneal Dystrophy, Maple Syrup Urine Disease, Marble 25 Bones, Marchiafava-Micheli Syndrome, Marcus Gunn Jaw-Winking Syndrome, Marcus Gunn Phenomenon, Marcus Gunn Ptosis with jaw-winking, Marcus Gunn Syndrome, Marcus Gunn (Jaw-Winking) Syndrome, Marcus Gunn Ptosis (with jaw-winking), Marden-Walker Syndrome, Marden-Walker Type Connective Tissue Disorder, Marfan's Abiotrophy, Marfan-Achard syndrome, Marfan Syndrome, Marfan's Syndrome I, 30 Marfan's Variant, Marfanoid Hypermobility Syndrome, Marginal Corneal Dystrophy, Marie's Ataxia, Marie Disease, Marie-Sainton Disease, Marie Strumpell Disease, Marie Strumpell Spondylitis, Marinesco-Sjogren Syndrome, Marinesco-Sjogren-Gorland WO 2007/028212 PCT/AU2006/001325 -241 Syndrome, Marker X Syndrome, Maroteaux Lamy Syndrome, Maroteaux Type Acromesomelic Dysplasia, Marshall's Ectodermal Dysplasias With Ocular and Hearing Defects, Marshall-Smith Syndrome, Marshall Syndrome, Marshall Type Deafness Myopia-Cataract-Saddle Nose, Martin-Albright Syndrome, Martin-Bell Syndrome, 5 Martorell Syndrome, MASA Syndrome, Massive Myoclonia, Mast Cell Leukemia, Mastocytosis, Mastocytosis With an Associated Hematologic Disorder, Maumenee Corneal Dystrophy, Maxillary Ameloblastoma, Maxillofacial Dysostosis, Maxillonasal Dysplasia, Maxillonasal Dysplasia Binder Type, Maxillopalpebral Synkinesis, May Hegglin Anomaly, MCAD Deficiency, MCAD, McArdle Disease, McCune-Albright, 10 MCD, McKusick Type Metaphyseal Chondrodysplasia, MCR, MCTD, Meckel Syndrome, Meckel-Gruber Syndrome, Median Cleft Face Syndrome, Mediterranean Anemia, Medium-Chain Acyl-CoA dehydrogenase (ACADM), Medium Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency, Medium-Chain Acyl-CoA Dehydrogenase Deficiency, Medullary Cystic Disease, Medullary Sponge Kidney, MEF, Megaesophagus, 15 Megalencephaly, Megalencephaly with Hyaline Inclusion, Megalencephaly with Hyaline Panneuropathy, Megaloblastic Anemia, Megaloblastic Anemia of Pregnancy, Megalocornea-Mental Retardation Syndrome, Meier-Gorlin Syndrome, Meige's Lymphedema, Meige's Syndrome, Melanodermic Leukodystrophy, Melanoplakia Intestinal Polyposis, Melanoplakia-Intestinal Polyposis, MELAS Syndrome, MELAS, 20 Melkersson Syndrome, Melnick-Fraser Syndrome, Melnick-Needles Osteodysplasty, Melnick-Needles Syndrome, Membranous Lipodystrophy, Mendes Da Costa Syndrome, Meniere Disease, M6niere's Disease, Meningeal Capillary Angiomatosis, Menkes Disease, Menke's Syndrome I, Mental Retardation Aphasia Shuffling Gait Adducted Thumbs (MASA), Mental Retardation-Deafness-Skeletal Abnormalities-Coarse Face with Full 25 Lips, Mental Retardation with Hypoplastic 5th Fingernails and Toenails, Mental Retardation with Osteocartilaginous Abnormalities, Mental Retradation-X-linked with Growth Delay-Deafness-Microgenitalism, Menzel Type OPCA, Mermaid Syndrome, MERRF, MERRF Syndrome, Merten-Singleton Syndrome, MES, Mesangial IGA Nephropathy, Mesenteric Lipodystrophy, Mesiodens-Cataract Syndrome, Mesodermal 30 Dysmorphodystrophy, Mesomelic Dwarfism-Madelung Deformity, Metabolic Acidosis, Metachromatic Leukodystrophy, Metatarsus Varus, Metatropic Dwarfism Syndrome, Metatropic Dysplasia, Metatropic Dysplasia I, Metatropic Dysplasia II, Methylmalonic WO 2007/028212 PCT/AU2006/001325 - 242 Acidemia, Methylmalonic Aciduria, Meulengracht's Disease, MFD1, MG, MH, MHA, Micrencephaly, Microcephalic Primordial Dwarfism I, Microcephaly, Microcephaly-Hiatal Hernia-Nephrosis Galloway Type, Microcephaly-Hiatal Hernia-Nephrotic Syndrome, Microcystic Corneal Dystrophy, Microcythemia, Microlissencephaly, Microphthalmia, 5 Microplithalmia or Anophthalmos with Associated Anomalies, Micropolygyria With Muscular Dystrophy, Microtia Absent Patellae Micrognathia Syndrome, Microvillus Inclusion Disease, MID, Midsystolic-click-late systolic murmur syndrome, Miescher's Type I Syndrome, Mikulicz Syndrome, Mikulicz-Radecki Syndrome, Mikulicz-Sjogren Syndrome, Mild Autosomal Recessive, Mild Intermediate Maple Syrup Urine Disease, 10 Mild Maple Syrup Urine Disease, Miller Syndrome, Miller-Dieker Syndrome, Miller Fisher Syndrome, Milroy Disease, Minkowski-Chauffard Syndrome, Minor Epilepsy, Minot-Von Willebrand Disease, Mirror-Image Dextrocardia, Mitochondrial Beta Oxidation Disorders, Mitrochondrial and Cytosolic, Mitochondrial Cytopathy, Mitochondrial Cytopathy, Kearn-Sayre Type, Mitochondrial Encephalopathy, 15 Mitochondrial Encephalo myopathy Lactic Acidosis and Strokelike Episodes, Mitochondrial myopathy, Mitochondrial myopathy Encephalopathy Lactic Acidosis Stroke-Like Episode, Mitochondrial PEPCK Deficiency, Mitral-valve prolapse, Mixed Apnea, Mixed Connective Tissue Disease, Mixed Hepatic Porphyria, Mixed Non-Fluent Aphasia, Mixed Sleep Apnea, Mixed Tonic and Clonic Torticollis, MJD, MKS, ML I, ML 20 II, ML III, ML IV, ML Disorder Type I, ML Disorder Type II, ML Disorder Type III, ML Disorder Type IV, MLNS, MMR Syndrome, MND, MNGIE, MNS, Mobitz I, Mobitz II, Mobius Syndrome, Moebius Syndrome, Moersch-Woltmann Syndrome, Mohr Syndrome, Monilethrix, Monomodal Visual Amnesia, Mononeuritis Multiplex, Mononeuritis Peripheral, Mononeuropathy Peripheral, Monosomy 3p2, Monosomy 9p Partial, 25 Monosomy 11 q Partial, Monosomy 13q Partial, Monosomy 18q Syndrome, Monosomy X, Monostotic Fibrous Dysplasia, Morgagni-Turner-Albright Syndrome, Morphea, Morquio Disease, Morquio Syndrome, Morquio Syndrome A, Morquio Syndrome B, Morquio Brailsford Syndrome, Morvan Disease, Mosaic Tetrasomy 9p, Motor Neuron Disease, Motor Neuron Syndrome, Motor Neurone Disease, Motoneuron Disease, Motoneurone 30 Disease, Motor System Disease (Focal and Slow), Moya-moya Disease, Moyamoya Disease, MPS, MPS I, MPS I H, MPS 1 H/S Hurler/Scheie Syndrome, MPS I S Scheic Syndrome, MPS II, MPS IIA, MPS IIB, MPS II-AR Autosomal Recessive Hunter WO 2007/028212 PCT/AU2006/001325 - 243 Syndrome, MPS II-XR, MPS II-XR Severe Autosomal Recessive, MPS III, MPS III A B C and D Sanfiloppo A, MPS IV, MPS IV A and B Morquio A, MPS V, MPS VI, MPS VI Severe Intermediate Mild Maroteaux-Lamy, MPS VII, MPS VII Sly Syndrome, MPS VIII, MPS Disorder, MPS Disorder I, MPS Disorder II, MPS Disorder III, MPS Disorder VI, 5 MPS Disorder Type VII, MRS, MS, MSA, MSD, MSL, MSS, MSUD, MSUD, MSUD Type Ib, MSUD Type II, Mucocutaneous Lymph Node Syndrome, Mucolipidosis I, Mucolipidosis II, Mucolipidosis III, Mucolipidosis IV, Mucopolysaccharidosis, Mucopolysaccharidosis I-H, Mucopolysaccharidosis I-S, Mucopolysaccharidosis II, Mucopolysaccharidosis III, Mucopolysaccharidosis IV, Mucopolysaccharidosis VI, 10 Mucopolysaccharidosis VII, Mucopolysaccharidosis Type 1, Mucopolysaccharidosis Type II, Mucopolysaccharidosis Type III, Mucopolysaccharidosis Type VII, Mucosis, Mucosulfatidosis, Mucous Colitis, Mucoviscidosis, Mulibrey Dwarfism, Mulibrey Nanism Syndrome, Mullerian Duct Aplasia-Renal Aplasia-Cervicothoracic Somite Dysplasia, Mullerian Duct-Renal-Cervicothoracic-Upper Limb Defects, Mullerian Duct and Renal 15 Agenesis with Upper Limb and Rib Anomalies, Mullerian-Renal-Cervicothoracic Somite Abnormalities, Multi-Infaret Dementia Binswanger's Type, Multicentric Castleman's Disease, Multifocal Eosinophilic Granuloma, Multiple Acyl-CoA Dehydrogenase Deficiency, Multiple Acyl-CoA Dehydrogenase Deficiency / Glutaric Aciduria Type II, Multiple Angiomas and Endochondromas, Multiple Carboxylase Deficiency, Multiple 20 Cartilaginous Enchondroses, Multiple Cartilaginous Exostoses, Multiple Enchondromatosis, Multiple Endocrine Deficiency Syndrome Type II, Multiple Epiphyseal Dysplasia, Multiple Exostoses, Multiple Exostoses Syndrome, Multiple Familial Polyposis, Multiple Lentigines Syndrome, Multiple Myeloma, Multiple Neuritis of the Shoulder Girdle, Multiple Osteochondromatosis, Multiple Peripheral Neuritis, 25 Multiple Polyposis of the Colon, Multiple Pterygium Syndrome, Multiple Sclerosis, Multiple Sulfatase Deficiency, Multiple Symmetric Lipomatosis, Multiple System Atrophy, Multisynostotic Osteodysgenesis, Multisynostotic Osteodysgenesis with Long Bone Fractures, Mulvihill-Smith Syndrome, MURCS Association, Murk Jansen Type Metaphyseal Chondrodysplasia, Muscle Carnitine Deficiency, Muscle Core Disease, 30 Muscle Phosphofructokinase Deficiency, Muscular Central Core Disease, Muscular Dystrophy, Muscular Dystrophy Classic X-linked Recessive, Muscular Dystrophy Congenital With Central Nervous System Involvement, Muscular Dystrophy Congenital WO 2007/028212 PCT/AU2006/001325 - 244 Progressive with Mental Retardation, Muscular Dystrophy Facioscapulohumeral, Muscular Rheumatism, Muscular Rigidity - Progressive Spasm, Musculoskeletal Pain Syndrome, Mutilating Acropathy, Mutism, mvp, MVP, MWS, Myasthenia Gravis, Myasthenia Gravis Pseudoparalytica, Myasthenic Syndrome of Lambert-Eaton, Myelinoclastic Diffuse 5 Sclerosis, Myelomatosis, Myhre Syndrome, Myoclonic Astatic Petit Mal Epilepsy, Myoclonic Dystonia, Myoclonic Encephalopathy of Infants, Myoclonic Epilepsy, Myoclonic Epilepsy Hartung Type, Myoclonus Epilepsy Associated with Ragged Red Fibers, Myoclonic Epilepsy and Ragged-Red Fiber Disease, Myoclonic Progressive Familial Epilepsy, Myoclonic Progressive Familial Epilepsy, Myoclonic Seizure, 10 Myoclonus, Myoclonus Epilepsy, Myoencephalopathy Ragged-Red Fiber Disease, Myofibromatosis, Myofibromatosis Congenital, Myogenic Facio-Scapulo-Peroneal Syndrome, Myoneurogastointestinal Disorder and Encephalopathy, Myopathic Arthrogryposis Multiplex Congenita, Myopathic Carnitine Deficiency, Myopathy Central Fibrillar, myopathy Congenital Nonprogressive, myopathy Congenital Nonprogressive 15 with Central Axis, myopathy with Deficiency of Camitine Palmitoyltransferase, myopathy-Marinesco-Sjogren Syndrome, myopathy-Metabolic Carnitine Palmitoyltransderase Deficiency, myopathy Mitochondrial-Encephalopathy-Lactic Acidosis-Stroke, myopathy with Sarcoplasmic Bodies and Intermediate Filaments, Myophosphorylase Deficiency, Myositis Ossificans Progressiv, Myotonia Atrophica, 20 Myotonia Congenita, Myotonia Congenita Intermittens, Myotonic Dystrophy, Myotonic myopathy Dwarfism Chondrodystrophy Ocular and Facial Anomalies, Myotubular myopathy, Myotubular myopathy X-linked, Myproic Acid, Myriachit (Observed in Siberia), Myxedema, N-Acetylglucosamine-1-Phosphotransferase Deficiency, N-Acetyl Glutamate Synthetase Deficiency, NADH-CoQ reductase deficiency, Naegeli Ectodermal 25 Dysplasias, Nager Syndrome, Nager Acrofacial Dysostosis Syndrome, Nager Syndrome, NAGS Deficiency, Nail Dystrophy-Deafness Syndrome, Nail Dysgenesis and Hypodontia, Nail-Patella Syndrome, Nance-Horan Syndrome, Nanocephalic Dwarfism, Nanocephaly, Nanophthalmia, Narcolepsy, Narcoleptic syndrome, NARP, Nasal-fronto-faciodysplasia, Nasal Alar Hypoplasia Hypothyroidism Pancreatic Achylia Congenital Deafness, 30 Nasomaxillary Hypoplasia, Nasu Lipodystrophy, NBIA1, ND, NDI, NDP, Necrotizing Encephalomyclopathy of Leigh's, Necrotizing Respiratory Granulomatosis, Neill Dingwall Syndrome, Nelson Syndrome, Nemaline myopathy, Neonatal WO 2007/028212 PCT/AU2006/001325 - 245 Adrenoleukodystrophy, Neonatal Adrenoleukodystrophy (NALD), Neonatal Adrenoleukodystrophy (ALD), Neonatal Autosomal Recessive Polycystic Kidney Disease, Neonatal Dwarfism, Neonatal Hepatitis, Neonatal Hypoglycemia, Neonatal Lactose Intolerance, Neonatal Lymphedema due to Exudative Enteropathy, Neonatal Necrotizing 5 Enterocolitis, Neonatal Progeroid Syndrome, Neonatal Pseudo-Hydrocephalic Progeroid Syndrome of Wiedemann-Rautenstrauch, Neoplastic Arachnoiditis, Nephroblastom, Nephrogenic Diabetes Insipidus, Nepbronophthesis Familial Juvenile, Nephropathic Cystinosis, Nephropathy-Pseudohermaphroditism-Wilms Tumor, Nephrosis-Microcephaly Syndrome, Nephrosis-Neuronal Dysmigration Syndrome, Nephrotic-Glycosuric 10 Dwarfism-Rickets-Hypophosphatemic Syndrome, Netherton Disease, Netherton Syndrome, Netherton Syndrome Ichthyosis, Nettleship Falls Syndrome (X-Linked), Neu Laxova Syndrome, Neuhauser Syndrome, Neural-tube defects, Neuralgic Amyotrophy, Neuraminidase Deficiency, Neuraocutaneous melanosis, Neurinoma of the Acoustic Nerve, Neurinoma, Neuroacanthocytosis, Neuroaxonal Dystrophy Schindler Type, 15 Neurodegeneration with brain iron accumulation type 1 (NBIAl), Neurofibroma of the Acoustic Nerve, Neurogenic Arthrogryposis Multiplex Congenita, Neuromyelitis Optica, Neuromyotonia, Neuromyotonia, Focal, Neuromyotonia, Generalized, Familial, Neuromyotonia, Generalized, Sporadic, Neuronal Axonal Dystrophy Schindler Type, Neuronal Ceroid Lipofuscinosis Adult Type, Neuronal Ceroid Lipofuscinosis Juvenile 20 Type, Neuronal Ceroid Lipofuscinosis Type 1, Neuronopathic Acute Gaucher Disease, Neuropathic Amyloidosis, Neuropathic Beriberi, Neuropathy Ataxia and Retinitis Pigmentosa, Neuropathy of Brachialpelxus Syndrome, Neuropathy Hereditary Sensory Type I, Neuropathy Hereditary Sensory Type II, Neuropsychiatric Porphyria, Neutral Lipid Storage Disease, Nevii, Nevoid Basal Cell Carcinoma Syndrome, Nevus, Nevus 25 Cavernosus, Nevus Comedonicus, Nevus Depigmentosus, Nevus Sebaceous of Jadassohn, Nezelof's Syndrome, Nezelof's Thymic Aplasia, Nezelof Type Severe Combined Immunodeficiency, NF, NFl, NF2, NF-1, NF-2, NHS, Niemann Pick Disease, Nieman Pick disease Type A (acute neuronopathic form), Nieman Pick disease Type B, Nieman Pick Disease Type C (chronic neuronopathic form), Nieman Pick disease Type D (Nova 30 Scotia variant), Nieman Pick disease Type E, Nieman Pick disease Type F (sea-blue histiocyte disease), Night Blindness, Nigrospinodentatal Degeneration, Niikawakuroki Syndrome, NLS, NM, Noack Syndrome Type I, Nocturnal Myoclonus Hereditary WO 2007/028212 PCT/AU2006/001325 - 246 Essential Myoclonus, Nodular Cornea Degeneration, Non-Bullous CIE, Non-Bullous Congenital Ichthyosiform Erythroderma, Non-Communicating Hydrocephalus, Non Deletion Type Alpha-Thalassemia / Mental Retardation syndrome, Non-Ketonic Hyperglycinemia Type I (NKHI), Non-Ketotic Hyperglycinemia, Non-Lipid 5 Reticuloendotheliosis, Non-Neuronopathic Chronic Adult Gaucher Disease, Non-Scarring Epidermolysis Bullosa, Nonarteriosclerotic Cerebral Calcifications, Nonarticular Rheumatism, Noncerebral,Juvenile Gaucher Disease, Nondiabetic Glycosuria, Nonischemic Cardio myopathy, Nonketotic Hypoglycemia and Carnitine Deficiency due to MCAD Deficiency, Nonketotic Hypoglycemia Caused by Deficiency of Acyl-CoA 10 Dehydrogenase, Nonketotic Glycinemia, Nonne's Syndrome, Nonne-Milroy-Meige Syndrome, Nonopalescent Opalescent Dentine, Nonpuerperal Galactorrhea-Amenorrhea, Nonsecretory Myeloma, Nonspherocytic Hemolytic Anemia, Nontropical Sprue, Noonan Syndrome, Norepinephrine, Normal Pressure Hydrocephalus, Norman-Roberts Syndrome, Norrbottnian Gaucher Disease, Norrie Disease, Norwegian Type Hereditary Cholestasis, 15 NPD, NPS, NS, NSA, Nuchal Dystonia Dementia Syndrome, Nutritional Neuropathy, Nyhan Syndrome, OAV Spectrum, Obstructive Apnea, Obstructive Hydrocephalus, Obstructive Sleep Apnea, OCC Syndrome, Occlusive Thromboaortopathy, OCCS, Occult Intracranial Vascular Malformations, Occult Spinal Dysraphism Sequence, Ochoa Syndrome, Ochronosis, Ochronotic Arthritis, OCR, OCRL, Octocephaly, Ocular Albinism, 20 Ocular Herpes, Ocular Myasthenia Gravis, Oculo-Auriculo-Vertebral Dysplasia, Oculo Auriculo-Vertebral Spectrum, Oculo-Bucco-Genital Syndrome, Oculocerebral Syndrome with Hypopigmentation, Oculocerebrocutaneous Syndrome, Oculo-Cerebro-Renal, Oculocerebrorenal Dystrophy, Oculocerebrorenal Syndrome, Oculocraniosomatic Syndrome (obsolete), Oculocutaneous Albinism, Oculocutaneous Albinism Chediak 25 Higashi Type, Oculo-Dento-Digital Dysplasia, Oculodentodigital Syndrome, Oculo-Dento Osseous Dysplasia, Oculo Gastrointestinal Muscular Dystrophy, Oculo Gastrointestinal Muscular Dystrophy, Oculomandibulodyscephaly with hypotrichosis, Oculomandibulofacial Syndrome, Oculomotor with Congenital Contractures and Muscle Atrophy, Oculosympathetic Palsy, ODD Syndrome, ODOD, Odontogenic Tumor, 30 Odontotrichomelic Syndrome, OFD, OFD Syndrome, Ohio Type Amyloidosis (Type VII), 01, 01 Congenita, 01 Tarda, Oldfield Syndrome, Oligohydramnios Sequence, Oligophrenia Microphthalmos, Oligophrenic Polydystrophy, Olivopontocerebellar WO 2007/028212 PCT/AU2006/001325 - 247 Atrophy, Olivopontocerebellar Atrophy with Dementia and Extrapyramidal Signs, Olivopontocerebellar Atrophy with Retinal Degeneration, Olivopontocerebellar Atrophy I, Olivopontocerebellar Atrophy II, Olivopontocerebellar Atrophy III, Olivopontocerebellar Atrophy IV, Olivopontocerebellar Atrophy V, Ollier Disease, Ollier Osteochondromatosis, 5 Omphalocele-Visceromegaly-Macroglossia Syndrome, Ondine's Curse, Onion-Bulb Neuropathy, Onion Bulb Polyneuropathy, Onychoosteodysplasia, Onychotrichodysplasia with Neutropenia, OPCA, OPCA I, OPCA II, OPCA III, OPCA IV, OPCA V, OPD Syndrome, OPD Syndrome Type I, OPD Syndrome Type II, OPD I Syndrome, OPD II Syndrome, Ophthalmoarthropathy, Ophthalmoplegia-Intestinal Pseudoobstruction, 10 Ophthalmoplegia, Pigmentary Degeneration of the Retina and Cadio myopathy, Ophthalmoplegia Plus Syndrome, Ophthalmoplegia Syndrome, Opitz BBB Syndrome, Opitz BBB/G Compound Syndrome, Opitz BBBG Syndrome, Opitz-Frias Syndrome, Opitz G Syndrome, Opitz G/BBB Syndrome, Opitz Hypertelorism-Hypospadias Syndrome, Opitz-Kaveggia Syndrome, Opitz Oculogenitolaryngeal Syndrome, Opitz 15 Trigonocephaly Syndrome, Opitz Syndrome, Opsoclonus, Opsoclonus-Myoclonus, Opthalmoneuromyelitis, Optic Atrophy Polyneuropathy and Deafness, Optic Neuroencephalomyelopathy, Optic Neuromyelitis, Opticomyelitis, Optochiasmatic Arachnoiditis, Oral-Facial Clefts, Oral-facial Dyskinesia, Oral Facial Dystonia, Oral Facial-Digital Syndrome, Oral-Facial-Digital Syndrome Type I, Oral-Facial-Digital 20 Syndrome I, Oral-Facial-Digital Syndrome II, Oral-Facial-Digital Syndrome III, Oral Facial-Digital Syndrome IV, Orbital Cyst with Cerebral and Focal Dermal Malformations, Ornithine Carbamyl Transferase Deficiency, Ornithine Transcarbamylase Deficiency, Orocraniodigital Syndrome, Orofaciodigital Syndrome, Oromandibular Dystonia, Orthostatic Hypotension, Osler-Weber-Rendu disease, Osseous-Oculo-Dento Dysplasia, 25 Osseous-Oculo-Dento Dysplasia, Osteitis deformans, Osteochondrodystrophy Deformans, Osteochondroplasia, Osteodysplasty of Melnick and Needles, Osteogenesis Imperfect, Osteogenesis Imperfecta, Osteogenesis Imperfecta Congenita, Osteogenesis Imperfecta Tarda, Osteohypertrophic Nevus Flammeus, Osteopathia Hyperostotica Scleroticans Multiplex Infantalis, Osteopathia Hyperostotica Scleroticans Multiplex Infantalis, 30 Osteopathyrosis, Osteopetrosis, Osteopetrosis Autosomal Dominant Adult Type, Osteopetrosis Autosomal Recessive Malignant Infantile Type, Osteopetrosis Mild Autosomal Recessive Intermediate Typ, Osteosclerosis Fragilis Generalisata, WO 2007/028212 PCT/AU2006/001325 - 248 Osteosclerotic Myeloma, Ostium Primum Defect (endocardial cushion defects included), Ostium Secundum Defect, OTC Deficiency, Oto Palato Digital Syndrome, Oto-Palato Digital Syndrome Type I, Oto-Palatal-Digital Syndrome Type II, Otodental Dysplasia, Otopalatodigital Syndrome, Otopalataldigital Syndrome Type II, Oudtshoorn Skin, 5 Ovarian Dwarfism Turner Type, Ovary Aplasia Turner Type, OWR, Oxalosis, Oxidase deficiency, Oxycephaly, Oxycephaly-Acrocephaly, P-V, PA, PAC, Pachyonychia Ichtyosiforme, Pachyonychia Congenita with Natal Teeth, Pachyonychia Congenita, Pachyonychia Congenita Keratosis Disseminata Circumscripta (follicularis), Pachyonychia Congenita Jadassohn-Lewandowsky Type, PAF with MSA, Paget's Disease, Paget's 10 Disease of Bone, Paget's Disease of the Breast, Paget's Disease of the Nipple, Paget's Disease of the Nipple and Areola, Pagon Syndrome, Painful Ophthalmoplegia, PAIS, Palatal Myoclonus, Palato-Oto-Digital Syndrome, Palatal-Oto-Digital Syndrome Type I, Palatal-Oto-Digital Syndrome Type II, Pallister Syndrome, Pallister-Hall Syndrome, Pallister-Killian Mosaic Syndrome, Pallister Mosaic Aneuploidy, Pallister Mosaic 15 Syndrome, Pallister Mosaic Syndrome Tetrasomy 12p, Pallister-W Syndrome, Palmoplantar Hyperkeratosis and Alopecia, Palsy, Pancreatic Fibrosis, Pancreatic Insufficiency and Bone Marrow Dysfunction, Pancreatic Ulcerogenic Tumor Syndrome, Panmyelophthisis, Pamnyelopathy, Pantothenate kinase associated neurodegeneration (PKAN), Papillon-Lefevre Syndrome, Papillotonic Psuedotabes, Paralysis Periodica 20 Paramyotonica, Paralytic Beriberi, Paralytic Brachial Neuritis, Paramedian Lower Lip Pits Popliteal Pyerygium Syndrome, Paramedian Diencephalic Syndrome, Paramyeloidosis, Paramyoclonus Multiple, Paramyotonia Congenita, Paramyotonia Congenita of Von Eulenburg, Parkinson's disease, Paroxysmal Atrial Tachycardia, Paroxysmal Cold Hemoglobinuria, Paroxysmal Dystonia, Paroxysmal Dystonia Choreathetosis, Paroxysmal 25 Kinesigenic Dystonia, Paroxysmal Nocturnal Hemoglobinuria, Paroxysmal Normal Hemoglobinuria, Paroxysmal Sleep, Parrot Syndrome, Parry Disease, Parry-Romberg Syndrome, Parsonage-Turner Syndrome, Partial Androgen Insensitivity Syndrome, Partial Deletion of the Short Arm of Chromosome 4, Partial Deletion of the Short Ann of Chromosome 5, Partial Deletion of Short Arm of Chromosome 9, Partial Duplication 3q 30 Syndrome, Partial Duplication 15q Syndrome, Partial Facial Palsy With Urinary Abnormalities, Partial Gigantism of Hands and Feet- Nevi-Hemihypertrophy Macrocephaly, Partial Lipodystrophy, Partial Monosomy of Long Ann of Chromosome WO 2007/028212 PCT/AU2006/001325 - 249 11, Partial Monosomy of the Long Arm of Chromosome 13, Partial Spinal Sensory Syndrome, Partial Trisomy 11q, Partington Syndrome, PAT, Patent Ductus Arteriosus, Pathological Myoclonus, Pauciarticular-Onset Juvenile Arthritis, Paulitis, PBC, PBS, PC Deficiency, PC Deficiency Group A, PC Deficiency Group B, PC, Eulenburg Disease, 5 PCC Deficiency, PCH, PCLD, PCT, PD, PDA, PDH Deficiency, Pearson Syndrome Pyruvate Carboxylase Deficiency, Pediatric Obstructive Sleep Apnea, Peeling Skin Syndrome, Pelizaeus-Merzbacher Disease, Pelizaeus-Merzbacher Brain Sclerosis, Pellagra-Cerebellar Ataxia-Renal Aminoaciduria Syndrome, Pelvic Pain Syndrome, Pemphigus Vulgaris, Pena Shokeir II Syndrome, Pena Shokeir Syndrome Type II, Penile 10 Fibromatosis, Penile Fibrosis, Penile Induration, Penta X Syndrome, Pentalogy of Cantrell, Pentalogy Syndrome, Pentasomy X, PEPCK Deficiency, Pepper Syndrome, Perheentupa Syndrome, Periarticular Fibrositis, Pericardial Constriction with Growth Failure, Pericollagen Amyloidosis, Perinatal Polycystic Kidney Diseases, Perineal Anus, Periodic Amyloid Syndrome, Periodic Peritonitis Syndrome, Periodic Somnolence and Morbid 15 Hunger, Periodic Syndrome, Peripheral Cystoid Degeneration of the Retina, Peripheral Dysostosis-Nasal Hypoplasia-Mental Retardation, Peripheral Neuritis, Peripheral Neuropathy, Peritoneopericardial Diaphragmatic Hernia, Pernicious Anemia, Peromelia with Micrognathia, Peroneal Muscular Atrophy, Peroneal Nerve Palsy, Peroutka Sneeze, Peroxisomal Acyl-CoA Oxidase, Peroxisomal Beta-Oxidation Disorders, Peroxisomal 20 Bifunctional Enzyme, Peroxisomal Thiolase, Peroxisomal Thiolase Deficiency, Persistent Truncus Arteriosus, Perthes Disease, Petit Mal Epilepsy, Petit Mal Variant, Peutz-Jeghers Syndrome, Peutz-Touraine Syndrome, Peyronie Disease, Pfeiffer, Pfeiffer Syndrome Type I, PGA I, PGA II, PGA III, PGK, PH Type I, PH Type I, Pharyngeal Pouch Syndrome, PHD Short-Chain Acyl-CoA Dehydrogenase Deficiency, Phenylalanine Hydroxylase 25 Deficiency, Phenylalaninemia, Phenylketonuria, Phenylpyruvic Oligophrenia, Phocomelia, Phocomelia Syndrome, Phosphoenolpyruvate Carboxykinase Deficiency, Phosphofructokinase Deficiency, Phosphoglycerate Kinase Deficiency, Phosphoglycerokinase, Phosphorylase 6 Kinase Deficiency, Phosphorylase Deficiency Glycogen Storage Disease, Phosphorylase Kinase Deficiency of Liver, Photic Sneeze 30 Reflex, Photic Sneezing, Phototherapeutic keratectomy, PHS, Physicist John Dalton, Phytanic Acid Storage Disease, Pi Phenotype ZZ, PI, Pick Disease of the Brain, Pick's Disease, Pickwickian Syndrome, Pierre Robin Anomalad, Pierre Robin Complex, Pierre WO 2007/028212 PCT/AU2006/001325 -250 Robin Sequence, Pierre Robin Syndrome, Pierre Robin Syndrome with Hyperphalangy and Clinodactyly, Pierre-Marie's Disease, Pigmentary Degeneration of Globus Pallidus Substantia Nigra Red Nucleus, Pili Torti and Nerve Deafness, Pili Torti-Sensorineural Hearing Loss, Pituitary Dwarfism II, Pituitary Tumor after Adrenalectomy, Pityriasis 5 Pilaris, Pityriasis Rubra Pilaris, PJS, PKAN, PKD, PKD1, PKD2, PKD3, PKU, PKU1, Plagiocephaly, Plasma Cell Myeloma, Plasma Cell Leukemia, Plasma Thromboplastin Component Deficiency, Plasma Transglutaminase Deficiency, Plastic Induration Corpora Cavernosa, Plastic Induration of the Penis, PLD, Plicated Tongue, PLS, PMD, Pneumorenal Syndrome, PNH, PNM, PNP Deficiency, POD, POH, Poikiloderma 10 Atrophicans and Cataract, Poikiloderma Congenitale, Poland Anomaly, Poland Sequence, Poland Syndactyly, Poland Syndrome, Poliodystrophia Cerebri Progressiva, Polyarthritis Enterica, Polyarteritis Nodosa, Polyarticular-Onset Juvenile Arthritis Type I, Polyarticular Onset Juvenile Arthritis Type II, Polyarticular-Onset Juvenile Arthritis Types I and II, Polychondritis, Polycystic Kidney Disease, Polycystic Kidney Disease Medullary Type, 15 Polycystic Liver Disease, Polycystic Ovary Disease, Polycystic Renal Diseases, Polydactyly-Joubert Syndrome, Polydysplastic Epidermolysis Bullosa, Polydystrophia Oligophrenia, Polydystrophic Dwarfism, Polyglandular Autoimmune Syndrome Type III, Polyglandular Autoimmune Syndrome Type II, Polyglandular Autoimmune Syndrome Type I, Polyglandular Autoimmune Syndrome Type II, Polyglandular Deficiency 20 Syndrome Type II, Polyglandular Syndromes, Polymorphic Macula Lutea Degeneration, Polymorphic Macular Degeneration, Polymorphism of Platelet Glycoprotien Lb, Polymorphous Corneal Dystrophy Hereditary, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, Polyneuritis Peripheral, Polyneuropathy-Deafness-Optic Atrophy, Polyneuropathy Peripheral, Polyneuropathy and 25 Polyradiculoneuropathy, Polyostotic Fibrous Dysplasia, Polyostotic Sclerosing Histiocytosis, Polyposis Familial, Polyposis Gardner Type, Polyposis Hamartomatous Intestinal, Polyposis-Osteomatosis-Epidermoid Cyst Syndrome, Polyposis Skin Pigmentation Alopecia and Fingernail Changes, Polyps and Spots Syndrome, Polyserositis Recurrent, Polysomy Y, Polysyndactyly with Peculiar Skull Shape, Polysyndactyly 30 Dysmorphic Craniofacies Greig Type, Pompe Disease, Pompe Disease, Popliteal Pterygium Syndrome, Porcupine Man, Porencephaly, Porencephaly, Porphobilinogen deaminase (PBG-D), Porphyria, Porphyria Acute Intermittent, Porphyria ALA-D, WO 2007/028212 PCT/AU2006/001325 -251 Porphyria Cutanea Tarda, Porphyria Cutanea Tarda Hereditaria, Porphyria Cutanea Tarda Symptomatica, Porphyria Hepatica Variegate, Porphyria Swedish Type, Porphyria Variegate, Porphyriam Acute Intermittent, Porphyrins, Porrigo Decalvans, Port Wine Stains, Portuguese Type Amyloidosis, Post-Infective Polyneuritis, Postanoxic Intention 5 Myoclonus, Postaxial Acrofacial Dysostosis, Postaxial Polydactyly, Postencephalitic Intention Myoclonus, Posterior Corneal Dystrophy Hereditary, Posterior Thalamic Syndrome, Postmyelographic Arachnoiditis, Postnatal Cerebral Palsy, Postoperative Cholestasis, Postpartum Galactorrhea-Amenorrhea Syndrome, Postpartum Hypopituitarism, Postpartum Panhypopituitary Syndrome, Postpartum Panhypopituitarism, 10 Postpartum Pituitary Necrosis, Postural Hypotension, Potassium-Losing Nephritis, Potassium Loss Syndrome, Potter Type I Infantile Polycystic Kidney Diseases, Potter Type III Polycystic Kidney Disease, PPH, PPS, Prader-Willi Syndrome, Prader-Labhart-Willi Fancone Syndrome, Prealbumin Tyr-77 Amyloidosis, Preexcitation Syndrome, Pregnenolone Deficiency, Premature Atrial Contractions, Premature Senility Syndrome, 15 Premature Supraventricular Contractions, Premature Ventricular Complexes, Prenatal or Connatal Neuroaxonal Dystrophy, Presenile Dementia, Presenile Macula Lutea Retinae Degeneration, Primary Adrenal Insufficiency, Primary Agammaglobulinemias, Primary Aldosteronism, Primary Alveolar Hypoventilation, Primary Amyloidosis, Primary Anemia, Primary Beriberi, Primary Biliary, Primary Biliary Cirrhosis, Primary Brown Syndrome, 20 Primary Carnitine Deficiency, Primary Central Hypoventilation Syndrome, Primary Ciliary Dyskinesia Kartagener Type, Primary Cutaneous Amyloidosis, Primary Dystonia, Primary Failure Adrenocortical Insufficiency, Primary Familial Hypoplasia of the Maxilla, Primary Hemochromatosis, Primary Hyperhidrosis, Primary Hyperoxaluria [Type I], Primary Hyperoxaluria Type 1 (PHI), Primary Hyperoxaluria Type 1, Primary 25 Hyperoxaluria Type II, Primary Hyperoxaluria Type III, Primary Hypogonadism, Primary Intestinal Lymphangiectasia, Primary Lateral Sclerosis, Primary Nonhereditary Amyloidosis, Primary Obliterative Pulmonary Vascular Disease, Primary Progressive Multiple Sclerosis, Primary Pulmonary Hypertension, Primary Reading Disability, Primary Renal Glycosuria, Primary Sclerosing Cholangitis, Primary Thrombocythemia, Primary 30 Tumors of Central Nervous System, Primary Visual Agnosia, Proctocolitis Idiopathic, Proctocolitis Idiopathic, Progeria of Adulthood, Progeria of Childhood, Progeroid Nanism, Progeriod Short Stature with Pigmented Nevi, Progeroid Syndrome of De Barsy, WO 2007/028212 PCT/AU2006/001325 - 252 Progressive Autonomic Failure with Multiple System Atrophy, Progressive Bulbar Palsy, Progressive Bulbar Palsy Included, Progressive Cardiomyopathic Lentiginosis, Progressive Cerebellar Ataxia Familial, Progressive Cerebral Poliodystrophy, Progressive Choroidal Atrophy, Progressive Diaphyseal Dysplasia, Progressive Facial Hemiatrophy, Progressive 5 Familial Myoclonic Epilepsy, Progressive Hemifacial Atrophy, Progressive Hypoerythemia, Progressive Infantile Poliodystrophy, Progressive Lenticular Degeneration, Progressive Lipodystrophy, Progressive Muscular Dystrophy of Childhood, Progressive Myoclonic Epilepsy, Progressive Osseous Heteroplasia, Progressive Pallid Degeneration Syndrome, Progressive Spinobulbar Muscular Atrophy, Progressive 10 Supranuclear Palsy, Progressive Systemic Sclerosis, Progressive Tapetochoroidal Dystrophy, Proline Oxidase Deficiency, Propionic Acidemia, Propionic Acidemia Type I (PCCA Deficiency), Propionic Acidemia Type II (PCCB Deficiency), Propionyl CoA Carboxylase Deficiency, Protanomaly, Protanopia, Protein-Losing Enteropathy Secondary to Congestive Heart Failure, Proteus Syndrome, Proximal Deletion of 4q Included, PRP, 15 PRS, Prune Belly Syndrome, PS, Pseudo-Hurler Polydystrophy, Pseudo-Polydystrophy, Pseudoacanthosis Nigricans, Pseudoachondroplasia, Pseudocholinesterase Deficiency, Pseudogout Familial, Pseudohemophilia, Pseudohermaphroditism, Pseudohermaphroditism-Nephron Disorder-Wilm's Tumor, Pseudohypertrophic Muscular Dystrophy, Pseudohypoparathyroidism, Pseudohypophosphatasia, Pseudopolydystrophy, 20 Pseudothalidomide Syndrome, Pseudoxanthoma Elasticum, Psoriasis, Psorospermosis Follicularis, PSP, PSS, Psychomotor Convulsion, Psychomotor Epilepsy, Psychomotor Equivalent Epilepsy, PTC Deficiency, Pterygium, Pterygium Colli Syndrome, Pterygium Universale, Pterygolymphangiectasia, Pulmonary Atresia, Pulmonary Lymphangiomyomatosis, Pulmonary Stenosis, Pulmonic Stenosis-Ventricular Septal 25 Defect, Pulp Stones, Pulpal Dysplasia, Pulseless Disease, Pure Alymphocytosis, Pure Cutaneous Histiocytosis, Purine Nucleoside Phosphorylase Deficiency, Purpura Hemorrhagica, Purtilo Syndrome, PXE, PXE Dominant Type, PXE Recessive Type, Pycnodysostosis, Pyknodysostosis, Pyknoepilepsy, Pyroglutamic Aciduria, Pyroglutamicaciduria, Pyrroline Carboxylate Dehydrogenase Deficiency, Pyruvate 30 Carboxylase Deficiency, Pyruvate Carboxylase Deficiency Group A, Pyruvate Carboxylase Deficiency Group B, Pyruvate Dehydrogenase Deficiency, Pyruvate Kinase Deficiency, q25-qter, q26 or q27-qter, q31 or 32-qter, QT Prolongation with Extracellular WO 2007/028212 PCT/AU2006/001325 -253 Hypohypocalcinemia, QT Prolongation without Congenital Deafness, QT Prolonged with Congenital Deafness, Quadriparesis of Cerebral Palsy, Quadriplegia of Cerebral Palsy, Quantal Squander, Quantal Squander, r4, r6, r14, r 18, r21, r22, Rachischisis Posterior, Radial Aplasia-Amegakaryocytic Thrombocytopenia, Radial Aplasia-Thrombocytopenia 5 Syndrome, Radial Nerve Palsy, Radicular Neuropathy Sensory, Radicular Neuropathy Sensory Recessive, Radicular Dentin Dysplasia, Rapid-onset Dystonia-parkinsonism, Rapp-Hodgkin Syndrome, Rapp-Hodgkin (hypohidrotic) Ectodermal Dysplasia syndrome, Rapp-Hodgkin Hypohidrotic Ectodermal Dysplasias, Rare hereditary ataxia with polyneuritic changes and deafness caused by a defect in the enzyme phytanic acid 10 hydroxylase, Rautenstrauch-Wiedemann Syndrome, Rautenstrauch-Wiedemann Type Neonatal Progeria, Raynaud's Phenomenon, RDP, Reactive Functional Hypoglycemia, Reactive Hypoglycemia Secondary to Mild Diabetes, Recessive Type Kenny-Caffe Syndrome, Recklin Recessive Type Myotonia Congenita, Recklinghausen Disease, Rectoperineal Fistula, Recurrent Vomiting, Reflex Neurovascular Dystrophy, Reflex 15 Sympathetic Dystrophy Syndrome, Refractive Errors, Refractory Anemia, Refrigeration Palsy, Refsum Disease, Refsum's Disease, Regional Enteritis, Reid-Barlow's syndrome, Reifenstein Syndrome, Reiger Anomaly-Growth Retardation, Reiger Syndrome, Reimann Periodic Disease, Reimann's Syndrome, Reis-Bucklers Corneal Dystrophy, Reiter's Syndrome, Relapsing Guillain-Barre Syndrome, Relapsing-Remitting Multiple Sclerosis, 20 Renal Agenesis, Renal Dysplasia-Blindness Hereditary, Renal Dysplasia-Retinal Aplasia Loken-Senior Type, Renal Glycosuria, Renal Glycosuria Type A, Renal Glycosuria Type B, Renal Glycosuria Type 0, Renal-Oculocerebrodystrophy, Renal-Retinal Dysplasia with Medullary Cystic Disease, Renal-Retinal Dystrophy Familial, Renal-Retinal Syndrome, Rendu-Osler-Weber Syndrome, Respiratory Acidosis, Respiratory Chain Disorders, 25 Respiratory Myoclonus, Restless Legs Syndrome, Restrictive Cardio myopathy, Retention Hyperlipemia, Rethore Syndrome (obsolete), Reticular Dysgenesis, Retinal Aplastic Cystic Kidneys-Joubert Syndrome, Retinal Cone Degeneration, Retinal Cone Dystrophy, Retinal Cone-Rod Dystrophy, Retinitis Pigmentosa, Retinitis Pigmentosa and Congenital Deafness, Retinoblastoma, Retinol Deficiency, Retinoschisis, Retinoschisis Juvenile, 30 Retraction Syndrome, Retrobulbar Neuropathy, Retrolenticular Syndrome, Rett Syndrome, Reverse Coarction, Reye Syndrome, Reye's Syndrome, RGS, Rh Blood Factors, Rh Disease, Rh Factor Incompatibility, Rh Incompatibility, Rhesus Incompatibility, WO 2007/028212 PCT/AU2006/001325 -254 Rheumatic Fever, Rheumatoid Arthritis, Rheumatoid Myositis, Rhinosinusogenic Cerebral Arachnoiditis, Rhizomelic Chondrodysplasia Punctata (RCDP),Acatalasemia,Classical Refsum disease, RHS, Rhythmical Myoclonus, Rib Gap Defects with Micrognathia, Ribbing Disease (obsolete), Ribbing Disease, Richner-Hanhart Syndrome, Rieger 5 Syndrome, Rieter's Syndrome, Right Ventricular Fibrosis, Riley-Day Syndrome, Riley Smith syndrome, Ring Chromosome 14, Ring Chromosome 18, Ring 4, Ring 4 Chromosome, Ring 6, Ring 6 Chromosome, Ring 9, Ring 9 Chromosome R9, Ring 14, Ring 15, Ring 15 Chromosome (mosaic pattern), Ring 18, Ring Chromosome 18, Ring 21, Ring 21 Chromosome, Ring 22, Ring 22 Chromosome, Ritter Disease, Ritter-Lyell 10 Syndrome, RLS, RMSS, Roberts SC-Phocomelia Syndrome, Roberts Syndrome, Roberts Tetraphocomelia Syndrome, Robertson's Ectodermal Dysplasias, Robin Anomalad, Robin Sequence, Robin Syndrome, Robinow Dwarfism, Robinow Syndrome, Robinow Syndrome Dominant Form, Robinow Syndrome Recessive Form, Rod myopathy, Roger Disease, Rokitansky's Disease, Romano-Ward Syndrome, Romberg Syndrome, Rootless 15 Teeth, Rosenberg-Chutorian Syndrome, Rosewater Syndrome, Rosselli-Gulienatti Syndrome, Rothmund-Thomson Syndrome, Roussy-Levy Syndrome, RP, RS X-Linked, RS, RSDS, RSH Syndrome, RSS, RSTS, RTS, Rubella Congenital, Rubinstein Syndrome, Rubinstein-Taybi Syndrome, Rubinstein Taybi Broad Thumb-Hallux syndrome, Rufous Albinism, Ruhr's Syndrome, Russell's Diencephalic Cachexia, Russell's Syndrome, 20 Russell Syndrome, Russell-Silver Dwarfism, Russell-Silver Syndrome, Russell-Silver Syndrome X-linked, Ruvalcaba-Myhre-Smith syndrome (RMSS), Ruvalcaba Syndrome, Ruvalcaba Type Osseous Dysplasia with Mental Retardation, Sacral Regression, Sacral Agenesis Congenital, SAE, Saethre-Chotzen Syndrome, Sakati, Sakati Syndrome, Sakati Nyhan Syndrome, Salaam Spasms, Salivosudoriparous Syndrome, Salzman Nodular 25 Corneal Dystrophy, Sandhoff Disease, Sanfilippo Syndrome, Sanfilippo Type A, Sanfilippo Type B, Santavuori Disease, Santavuori-Haltia Disease, Sarcoid of Boeck, Sarcoidosis, Sathre-chotzen, Saturday Night Palsy, SBMA, SC Phocomelia Syndrome, SC Syndrome, SCA 3, SCAD Deficiency, SCAD Deficiency Adult-Onset Localized, SCAD Deficiency Congenital Generalized, SCAD, SCADH Deficiency, Scalded Skin Syndrome, 30 Scalp Defect Congenital, Scaphocephaly, Scapula Elevata, Scapuloperoneal myopathy, Scapuloperoneal Muscular Dystrophy, Scapuloperoneal Syndrome Myopathic Type, Scarring Bullosa, SCHAD, Schaumann's Disease, Scheie Syndrome, Schereshevkii-Turner WO 2007/028212 PCT/AU2006/001325 - 255 Syndrome, Schilder Disease, Schilder Encephalitis, Schilder's Disease, Schindler Disease Type I (Infantile Onset), Schindler Disease Infantile Onset, Schindler Disease, Schindler Disease Type II (Adult Onset), Schinzel Syndrome, Schinzel-Giedion Syndrome, Schinzel Acrocallosal Syndrome, Schinzel-Giedion Midface-Retraction Syndrome, Schizencephaly, 5 Schizophrenia, Schmid Type Metaphyseal Chondrodysplasia, Schmid Metaphyseal Dysostosis, Schmid-Fraccaro Syndrome, Schmidt Syndrome, Schopf-Schultz-Passarge Syndrome, Schueller-Christian Disease, Schut-Haymaker Type, Schwartz-Jampel Aberfeld Syndrome, Schwartz-Jampel Syndrome Types 1A and 1B, Schwartz-Jampel Syndrome, Schwartz-Jampel Syndrome Type 2, SCID, Scleroderma, Sclerosis Familial 10 Progressive Systemic, Sclerosis Diffuse Familial Brain, Sciatic Nerve Crush, Scott Craniodigital Syndrome With Mental Retardation, Scrotal Tongue, SCS, SD, SDS, SDYS, Seasonal Conjunctivitis, Sebaceous Nevus Syndrome, Sebaceous nevus, Seborrheic Keratosis, Seborrheic Warts, Seckel Syndrome, Seckel Type Dwarfism, Second Degree Congenital Heart Block, Secondary Amyloidosis, Secondary Blepharospasm, Secondary 15 Non-tropical Sprue, Secondary Brown Syndrome, Secondary Beriberi, Secondary Generalized Amyloidosis, Secondary Dystonia, Secretory Component Deficiency, Secretory IgA Deficiency, SED Tarda, SED Congenital, SEDC, Segmental linear achromic nevus, Segmental Dystonia, Segmental Myoclonus, Seip Syndrome, Seitelberger Disease, Seizures, Selective Deficiency of IgG Subclasses, Selective Mutism, Selective Deficiency 20 of IgG Subclass, Selective IgM Deficiency, Selective Mutism, Selective IgA Deficiency, Self-Healing Histiocytosis, Semilobar Holoprosencephaly, Seminiferous Tubule Dysgenesis, Senile Retinoschisis, Senile Warts, Senior-Loken Syndrome, Sensory Neuropathy Hereditary Type I, Sensory Neuropathy Hereditary Type II, Sensory Neuropathy Hereditary Type I, Sensory Radicular Neuropathy, Sensory Radicular 25 Neuropathy Recessive, Septic Progressive Granulomatosis, Septo-Optic Dysplasia, Serous Circumscribed Meningitis, Serum Protease Inhibitor Deficiency, Serum Carnosinase Deficiency, Setleis Syndrome, Severe Combined Immunodeficiency, Severe Combined Immunodeficiency with Adenosine Deaminase Deficiency, Severe Combined Immunodeficiency (SCID), Sex Reversal, Sexual Infantilism, SGB Syndrome, Sheehan 30 Syndrome, Shields Type Dentinogenesis Imperfecta, Shingles,varicella-zoster virus, Ship Beriberi, SHORT Syndrome, Short Arm 18 Deletion Syndrome, Short Chain Acyl CoA Dehydrogenase Deficiency, Short Chain Acyl-CoA Dehydrogenase (SCAD) Deficiency, WO 2007/028212 PCT/AU2006/001325 - 256 Short Stature and Facial Telangiectasis, Short Stature Facial/Skeletal Anomalies Retardation-Macrodontia, Short Stature-Hyperextensibility-Rieger Anomaly-Teething Delay, Short Stature-Onychodysplasia, Short Stature Telangiectatic Erythema of the Face, SHORT Syndrome, Shoshin Beriberi, Shoulder girdle syndrome, Shprintzen-Goldberg 5 Syndrome, Shulman Syndrome, Shwachman-Bodian Syndrome, Shwachman-Diamond Syndrome, Shwachman Syndrome, Shwacbman-Diamond-Oski Syndrome, Shwachmann Syndrome, Shy Drager Syndrome, Shy-Magee Syndrome, SI Deficiency, Sialidase Deficiency, Sialidosis Type I Juvenile, Sialidosis Type II Infantile, Sialidosis, Sialolipidosis, Sick Sinus Syndrome, Sickle Cell Anemia, Sickle Cell Disease, Sickle Cell 10 Hemoglobin C Disease, Sickle Cell-Hemoglobin D Disease, Sickle Cell-Thalassemia Disease, Sickle Cell Trait, Sideroblastic Anemias, Sideroblastic Anemia, Sideroblastosis, SIDS, Siegel-Cattan-Mamou Syndrome, Siemens-Bloch type Pigmented Dermatosis, Siemens Syndrome, Siewerling-Creutzfeldt Disease, Siewert Syndrome, Silver Syndrome, Silver-Russell Dwarfism, Silver-Russell Syndrome, Simmond's Disease, Simons 15 Syndrome, Simplex Epidennolysis Bullosa, Simpson Dysmorphia Syndrome, Simpson Golabi-Behmel Syndrome, Sinding-Larsen-Johansson Disease, Singleton-Merten Syndrome, Sinus Arrhythmia, Sinus Venosus, Sinus tachycardia, Sirenomelia Sequence, Sirenomelus, Situs Inversus Bronchiectasis and Sinusitis, SJA Syndrome, Sjogren Larsson Syndrome Ichthyosis, Sjogren Syndrome, Sjgren's Syndrome, SJS, Skeletal dysplasia, 20 Skeletal Dysplasia Weismann Netter Stuhl Type, Skin Peeling Syndrome, Skin Neoplasms, Skull Asymmetry and Mild Retardation, Skull Asymmetry and Mild Syndactyly, SLE, Sleep Epilepsy, Sleep Apnea, SLO, Sly Syndrome, SMA, SMA Infantile Acute Form, SMA I, SMA III, SMA type I, SMA type II, SMA type III, SMA3, SMAX1, SMCR, Smith Lemli Opitz Syndrome, Smith Magenis Syndrome, Smith-Magenis Chromosome Region, 25 Smith-McCort Dwarfism, Smith-Opitz-Inbom Syndrome, Smith Disease, Smoldering Myeloma, SMS, SNE, Sneezing From Light Exposure, Sodium valproate, Solitary Plasmacytoma of Bone, Sorsby Disease, Sotos Syndrome, Souques-Charcot Syndrome, South African Genetic Porphyria, Spasmodic Dysphonia, Spasmodic Torticollis, Spasmodic Wryneck, Spastic Cerebral Palsy, Spastic Colon, Spastic Dysphonia, Spastic 30 Paraplegia, SPD Calcinosis, Specific Antibody Deficiency with Normal Immunoglobulins, Specific Reading Disability, SPH2, Spherocytic Anemia, Spherocytosis, Spherophakia Brachymorphia Syndrome, Sphingomyelin Lipidosis, Sphingomyelinase Deficiency, WO 2007/028212 PCT/AU2006/001325 -257 Spider fingers, Spielmeyer-Vogt Disease, Spielmeyer-Vogt-Batten Syndrome, Spina Bifida, Spina Bifida Aperta, Spinal Arachnoiditis, Spinal Arteriovenous Malformation, Spinal Ataxia Hereditofamilial, Spinal and Bulbar Muscular Atrophy, Spinal Cord Crush, Spinal Diffuse Idiopathic Skeletal Hyperostosis, Spinal DISH, Spinal Muscular Atrophy, 5 Spinal Muscular Atrophy All Types, Spinal Muscular Atrophy Type ALS, Spinal Muscular Atrophy-Hypertrophy of the Calves, Spinal Muscular Atrophy Type I, Spinal Muscular Atrophy Type III, Spinal Muscular Atrophy type 3, Spinal Muscular Atrophy-Hypertrophy of the Calves, Spinal Ossifying Arachnoiditis, Spinal Stenosis, Spino Cerebellar Ataxia, Spinocerebellar Atrophy Type I, Spinocerebellar Ataxia Type I (SCAI), Spinocerebellar 10 Ataxia Type II (SCAII), Spinocerebellar Ataxia Type III (SCAIII), Spinocerebellar Ataxia Type III (SCA 3), Spinocerebellar Ataxia Type IV (SCAIV), Spinocerebellar Ataxia Type V (SCAV), Spinocerebellar Ataxia Type VI (SCAVI), Spinocerebellar Ataxia Type VII (SCAVII), Spirochetal Jaundice, Splenic Agenesis Syndrome, Splenic Ptosis, Splenoptosis, Split Hand Deformity-Mandibulofacial Dysostosis, Split Hand Deformity, 15 Spondyloarthritis, Spondylocostal Dysplasia - Type I, Spondyloepiphyseal Dysplasia Tarda, Spondylothoracic Dysplasia, Spondylotic Caudal Radiculopathy, Sponge Kidney, Spongioblastoma Multiforme, Spontaneous Hypoglycemia, Sprengel Deformity, Spring Ophthalmia, SRS, ST, Stale Fish Syndrome, Staphyloccal Scalded Skin Syndrome, Stargardt's Disease, Startle Disease, Status Epilepticus, Steele-Richardson-Olszewski 20 Syndrome, Steely Hair Disease, Stein-Leventhal Syndrome, Steinert Disease, Stengel's Syndrome, Stengel-Batten-Mayou-Spielmeyer-Vogt-Stock Disease, Stenosing Cholangitis, Stenosis of the Lumbar Vertebral Canal, Stenosis, Steroid Sulfatase Deficiency, Stevanovic's Ectoderial Dysplasias, Stevens Johnson Syndrome, STGD, Stickler Syndrome, Stiff-Man Syndrome, Stiff Person Syndrome, Still's Disease, Stilling-Turk 25 Duane Syndrome, Stillis Disease, Stimulus-Sensitive Myoclonus, Stone Man Syndrome, Stone Man, Streeter Anomaly, Striatonigral Degeneration Autosomal Dominant Type, Striopallidodentate Calcinosis, Stroma, Descemet's Membrane, Stromal Corneal Dystrophy, Struma Lymphomatosa, Sturge-Kalischer-Weber Syndrome, Sturge Weber Syndrome, Sturge-Weber Phakomatosis, Subacute Necrotizing Encephalomyelopathy, 30 Subacute Spongiform Encephalopathy, Subacute Necrotizing Encephalopathy, Subacute Sarcoidosis, Subacute Neuronopathic, Subaortic Stenosis, Subcortical Arteriosclerotic Encephalopathy, Subendocardial Sclerosis, Succinylcholine Sensitivity, Sucrase- WO 2007/028212 PCT/AU2006/001325 -258 Isomaltase Deficiency Congenital, Sucrose-Isomaltose Malabsorption Congenital, Sucrose Intolerance Congenital, Sudanophilic Leukodystrophy ADL, Sudanophilic Leukodystrophy Pelizaeus-Merzbacher Type, Sudanophilic Leukodystrophy Included, Sudden Infant Death Syndrome, Sudeck's Atrophy, Sugio-Kajii Syndrome, Summerskill 5 Syndrome, Summit Acrocephalosyndactyly, Summitt's Acrocephalosyndactyly, Summitt Syndrome, Superior Oblique Tendon Sheath Syndrome, Suprarenal glands, Supravalvular Aortic Stenosis, Supraventricular tachycardia, Surdicardiac Syndrome, Surdocardiac Syndrome, SVT, Sweat Gland Abscess, Sweating Gustatory Syndrome, Sweet Syndrome, Swiss Cheese Cartilage Syndrome, Syndactylic Oxycephaly, Syndactyly Type I with 10 Microcephaly and Mental Retardation, Syndromatic Hepatic Ductular Hypoplasia, Syringomyelia, Systemic Aleukemic Reticuloendotheliosis, Systemic Amyloidosis, Systemic Carnitine Deficiency, Systemic Elastorrhexis, Systemic Lupus Erythematosus, Systemic Mast Cell Disease, Systemic Mastocytosis, Systemic-Onset Juvenile Arthritis, Systemic Sclerosis, Systopic Spleen, T-Lymphocyte Deficiency, Tachyalimentation 15 Hypoglycemia, Tachycardia, Takahara syndrome, Takayasu Disease, Takayasu Arteritis, Talipes Calcaneus, Talipes Equinovarus, Talipes Equinus, Talipes Varus, Talipes Valgus, Tandem Spinal Stenosis, Tangier Disease, Tapetoretinal Degeneration, TAR Syndrome, Tardive Dystonia, Tardive Muscular Dystrophy, Tardive Dyskinesia, Tardive Oral Dyskinesia, Tardive Dystonia, Tardy Ulnar Palsy, Target Cell Anemia, Tarsomegaly, Tarui 20 Disease, TAS Midline Defects Included, TAS Midline Defect, Tay Sachs Sphingolipidosis, Tay Sachs Disease, Tay Syndrome Ichthyosis, Tay Sachs Sphingolipidosis, Tay Syndrome Ichthyosis, Taybi Syndrome Type I, Taybi Syndrome, TCD, TCOF1, TCS, TD, TDO Syndrome, TDO-I, TDO-II, TDO-III, Telangiectasis, Telecanthus with Associated Abnormalities, Telecanthus-Hypospadias Syndrome, Temporal Lobe Epilepsy, Temporal 25 Arteritis/Giant Cell Arteritis, Temporal Arteritis, TEN, Tendon Sheath Adherence Superior Obliqu, Tension Myalgia, Terminal Deletion of 4q Included, Terrian Corneal Dystrophy, Teschler-Nicola/Killian Syndrome, Tethered Spinal Cord Syndrome, Tethered Cord Malformation Sequence, Tethered Cord Syndrome, Tethered Cervical Spinal Cord Syndrome, Tetrahydrobiopterin Deficiencies, Tetrahydrobiopterin Deficiencies, Tetralogy 30 of Fallot, Tetraphocomelia-Thrombocytopenia Syndrome, Tetrasomy Short Arm of Chromosome 9, Tetrasomy 9p, Tetrasomy Short Arm of Chromosome 18, Thalamic Syndrome, Thalamic Pain Syndrome, Thalamic Hyperesthetic Anesthesia, Thalassemia WO 2007/028212 PCT/AU2006/001325 -259 Intermedia, Thalassemia Minor, Thalassemia Major, Thiamine Deficiency, Thiamine Responsive Maple Syrup Urine Disease, Thin-Basement-Membrane Nephropathy, Thiolase deficiency,RCDP,Acyl-CoA dihydroxyacetonephosphate acyltransferase, Third and Fourth Pharyngeal Pouch Syndrome, Third Degree Congenital (Complete) Heart 5 Block, Thomsen Disease, Thoracic-Pelvic-Phalangeal Dystrophy, Thoracic Spinal Canal, Thoracoabdominal Syndrome, Thoracoabdominal Ectopia Cordis Syndrome, Three M Syndrome, Three-M Slender-Boned Nanism, Thrombasthenia of Glanzmann and Naegeli, Thrombocythemia Essential, Thrombocytopenia-Absent Radius Syndrome, Thrombocytopenia-Hemangioma Syndrome, Thrombocytopenia-Absent Radii Syndrome, 10 Thrombophilia Hereditary Due to AT III, Thrombotic Thrombocytopenic Purpura, Thromboulcerative Colitis, Thymic Dysplasia with Normal Immunoglobulins, Thymic Agenesis,Thymic Aplasia DiGeorge Type, Thymic Hypoplasia Agammaglobulinemias Primary Included, Thymic Hypoplasia DiGeorge Type, Thymus Congenital Aplasia, Tic Douloureux, Tics, Tinel's syndrome, Tolosa Hunt Syndrome, Tonic Spasmodic Torticollis, 15 Tonic Pupil Syndrome, Tooth and Nail Syndrome, Torch Infection, TORCH Syndrome, Torsion Dystonia, Torticollis, Total Lipodystrophy, Total anomalous pulmonary venous connection, Touraine's Aphthosis, Tourette Syndrome, Tourette's disorder, Townes Brocks Syndrome, Townes Syndrome, Toxic Paralytic Anemia, Toxic Epidermal Necrolysis, Toxopachyosteose Diaphysaire Tibio-Peroniere, Toxopachyosteose, 20 Toxoplasmosis Other Agents Rubella Cytomegalovirus Herpes Simplex, Tracheoesophageal Fistula with or without Esophageal Atresia, Tracheoesophageal Fistula, Transient neonatal myasthenia gravis, Transitional Atrioventricular Septal Defect, Transposition of the great arteries, Transtelephonic Monitoring, Transthyretin Methionine 30 Amyloidosis (Type I), Trapezoidocephaly-Multiple Synostosis Syndrome, Treacher 25 Collins Syndrome, Treacher Collins-Franceschetti Syndrome 1, Trevor Disease, Triatrial Heart, Tricho-Dento-Osseous Syndrome, Trichodento Osseous Syndrome, Trichopoliodystrophy, Trichorhinophalangeal Syndrome, Trichorhinophalangeal Syndrome, Tricuspid atresia, Trifunctional Protein Deficiency, Trigeminal Neuralgia, Triglyceride Storage Disease Impaired Long-Chain Fatty Acid Oxidation, Trigonitis, 30 Trigonocephaly, Trigonocephaly Syndrome, Trigonocephaly "C" Syndrome, Trimethylaminuria, Triphalangeal Thumbs-Hypoplastic Distal Phalanges Onychodystrophy, Triphalangeal Thumb Syndrome, Triple Symptom Complex of Beheet, WO 2007/028212 PCT/AU2006/001325 -260 Triple X Syndrome, Triplo X Syndrome, Triploid Syndrome, Triploidy, Triploidy Syndrome, Trismus-Pseudocamptodactyly Syndrome, Trisomy, Trisomy G Syndrome, Trisomy X, Trisomy 6q Partial, Trisomy 6q Syndrome Partial, Trisomy 9 Mosaic, Trisomy 9P Syndrome (Partial) Included, Trisomy 1lq Partial, Trisomy 14 Mosaic, Trisomy 14 5 Mosaicism Syndrome, Trisomy 21 Syndrome, Trisomy 22 Mosaic, Trisomy 22 Mosaicism Syndrome, TRPS, TRPS1, TRPS2, TRPS3, True Hermaphroditism, Truncus arteriosus, Tryptophan Malabsorption, Tryptophan Pyrrolase Deficiency, TS, TTP, TTTS, Tuberous Sclerosis, Tubular Ectasia, Turcot Syndrome, Turner Syndrome, Turner-Kieser Syndrome, Turner Phenotype with Normal Chromosomes (Karyotype), Turner-Varny Syndrome, 10 Turricephaly, Twin-Twin Transfusion Syndrome, Twin-to-Twin Transfusion Syndrome, Type A, Type B, Type AB, Type 0, Type I Diabetes, Type I Familial Incomplete Male, Type I Familial Incomplete Male Pseudohermaphroditism, Type I Gaucher Disease, Type I (PCCA Deficiency), Type I Tyrosinemia, Type II Gaucher Disease, Type II Histiocytosis, Type II (PCCB Deficiency), Type II Tyrosinnemia, Type IIA Distal Arthrogryposis 15 Multiplex Congenita, Type III Gaucher Disease, Type III Tyrosinemia, Type III Dentinogenesis Imperfecta, Typical Retinoschisis, Tyrosinase Negative Albinism (Type I), Tyrosinase Positive Albinism (Type II), Tyrosinemia type 1 acute form, Tyrosinemia type 1 chronic form, Tyrosinosis, UCE, Ulcerative Colitis, Ulcerative Colitis Chronic Non Specific, Ulnar-Mammary Syndrome, Ulnar-Mammary Syndrome of Pallister, Ulnar 20 Nerve Palsy, UMS, Unclassified FODs, Unconjugated Benign Bilirubinemiav, Underactivity of Parathyroid, Unilateral Ichthyosiform Erythroderma with Ipsilateral Malformations Limb, Unilateral Chondromatosis, Unilateral Defect of Pectoralis Muscle and Syndactyly of the Hand, Unilateral Hemidysplasia Type, Unilateral Megalencephaly, Unilateral Partial Lipodystrophy, Unilateral Renal Agenesis, Unstable Colon, Unverricht 25 Disease, Unverricht-Lundborg Disease, Unverricht-Lundborg-Laf Disease, Unverricht Syndrome, Upper Limb - Cardiovascular Syndrome (Holt-Oram), Upper Motor Neuron Disease, Upper Airway Apnea, Urea Cycle Defects or Disorders, Urea Cycle Disorder Arginase Type, Urea Cycle Disorder Arginino Succinase Type, Urea Cycle Disorders Carbamyl Phosphate Synthetase Type, Urea Cycle Disorder Citrullinemia Type, Urea 30 Cycle Disorders N-Acrtyl Glutamate Synthetase Typ, Urea Cycle Disorder OTC Type, Urethral Syndrome, Urethro-Oculo-Articular Syndrome, Uridine Diphosphate Glucuronosyltransferase Severe Def. Type I, Urinary Tract Defects, Urofacial Syndrome, WO 2007/028212 PCT/AU2006/001325 -261 Uroporphyrinogen III cosynthase, Urticaria pigmentosa, Usher Syndrome, Usher Type I, Usher Type II, Usher Type III, Usher Type IV, Uterine Synechiae, Uoporphyrinogen I synthase, Uveitis, Uveomeningitis Syndrome, V-CJD, VACTEL Association, VACTERL Association, VACTERL Syndrome, Valgus Calcaneus, Valine Transaminase Deficiency, 5 Valinemia, Valproic Acid, Valproate acid exposure, Valproic acid exposure, Valproic acid, Van Buren's Disease, Van der Hoeve-Habertsma-Waardenburg-Gauldi Syndrome, Variable Onset Immunoglobulin Deficiency Dysgammaglobulinemia, Variant Creutzfeldt Jakob Disease (V-CJD), Varicella Embryopathy, Variegate Porphyria, Vascular Birthmarks, Vascular Dementia Binswanger's Type, Vascular Erectile Tumor, Vascular 10 Hemophilia, Vascular Malformations, Vascular Malformations of the Brain, Vasculitis, Vasomotor Ataxia, Vasopressin-Resistant Diabetes Insipidus, Vasopressin-Sensitive Diabetes Insipidus, VATER Association, Vcf syndrome, Vcfs, Velocardiofacial Syndrome, VeloCardioFacial Syndrome, Venereal Arthritis, Venous Malformations, Ventricular Fibrillation, Ventricular Septal Defects, Congenital Ventricular Defects, Ventricular Septal 15 Defect, Ventricular Tachycardia, Venual Malformations, VEOHD, Vermis Aplasia, Vermis Cerebellar Agenesis, Vernal Keratoconjunctivitis, Verruca, Vertebral Anal Tracheoesophageal Esophageal Radial, Vertebral Ankylosing Hyperostosis, Very Early Onset Huntington's Disease, Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) Deficiency, Vestibular Schwannoma, Vestibular Schwannoma Neurofibromatosis, 20 Vestibulocerebellar, Virchow's Oxycephaly, Visceral Xanthogranulomatosis, Visceral Xantho-Granulomatosis, Visceral myopathy-External Ophthalmoplegia, Visceromegaly Umbilical Hernia-Macroglossia Syndrome, Visual Amnesia, Vitamin A Deficiency, Vitamin B-1 Deficiency, Vitelline Macular Dystrophy, Vitiligo, Vitiligo Capitis, Vitreoretinal Dystrophy, VKC, VKH Syndrome, VLCAD, Vogt Syndrome, Vogt 25 Cephalosyndactyly, Vogt Koyanagi Harada Syndrome, Von Bechterew-Strumpell Syndrome, Von Eulenburg Paramyotonia Congenita, Von Frey's Syndrome, Von Gierke Disease, Von Hippel-Lindau Syndrome, Von Mikulicz Syndrome, Von Recklinghausen Disease, Von Willebrandt Disease, VP, Vrolik Disease (Type II), VSD, Vulgaris Type Disorder of Cornification, Vulgaris Type Ichthyosis, W Syndrome, Waardenburg 30 Syndrome, Waardenburg-Klein Syndrome, Waardenburg Syndrome Type I (WS1), Waardenburg Syndrome Type II (WS2), Waardenburg Syndrome Type IA (WS2A), Waardenburg Syndrome Type IIB (WS2B), Waardenburg Syndrome -Type III (WS3), WO 2007/028212 PCT/AU2006/001325 - 262 Waardenburg Syndrome Type IV (WS4), Waelsch's Syndrome, WAGR Complex, WAGR Syndrome, Waldenstroem's Macroglobulinemia, Waldenstrom's Purpura, Waldenstrom's Syndrome, Waldmann Disease, Walker-Warburg Syndrome, Wandering Spleen, Warburg Syndrome, Warm Antibody Hemolytic Anemia, Warm Reacting Antibody Disease, 5 Wartenberg Syndrome, WAS, Water on the Brain, Watson Syndrome, Watson-Alagille Syndrome, Waterhouse-Friderichsen syndrome, Waxy Disease, WBS, Weaver Syndrome, Weaver-Smith Syndrome, Weber-Cockayne Disease, Wegener's Granulomatosis, Weil Disease, Weil Syndrome, Weill-Marchesani, Weill-Marchesani Syndrome, Weill-Reyes Syndrome, Weismann-Netter-Stuhl Syndrome, Weissenbacher-Zweymuller Syndrome, 10 Wells Syndrome, Wenckebach, Werdnig-Hoffman Disease, Werdnig-Hoffman Paralysis, Werlhof's Disease, Werner Syndrome, Wemicke's (C) I Syndrome, Wernicke's aphasia, Wernicke-Korsakoff Syndrome, West Syndrome, Wet Beriberi, WHCR, Whipple's Disease, Whipple Disease, Whistling face syndrome, Whistling Face-Windmill Vane Hand Syndrome, White-Darier Disease, Whitnall-Norman Syndrome, Whorled nevoid 15 hypermelanosis, WHS, Wieacker Syndrome, Wieacher Syndrome, Wieacker-Wolff Syndrome, Wiedmann-Beckwith Syndrome, Wiedemann-Rautenstrauch Syndrome, Wildervanck Syndrome, Willebrand-Juergens Disease, Willi-Prader Syndrome, Williams Syndrome, Williams-Beuren Syndrome, Wilms' Tumor, Wilms' Tumor-Aniridia Gonadoblastoma-Mental Retardation Syndrome, Wilms Tumor Aniridia Gonadoblastoma 20 Mental Retardation, Wilms' Tumor-Aniridia-Genitourinary Anomalies-Mental Retardation Syndrome, Wilms Tumor-Pseudohermapbroditism-Nephropathy, Wilms Tumor and Pseudohermaphroditism, Wilms Tumor-Pseuodohermaphroditism-Glomerulopathy, Wilson's Disease, Winchester Syndrome, Winchester-Grossman Syndrome, Wiskott Aldrich Syndrome, Wiskott-Aldrich Type Immunodeficiency, Witkop Ectodermal 25 Dysplasias, Witkop Tooth-Nail Syndrome, Wittmaack-Ekbom Syndrome, WM Syndrome, WMS, WNS, Wohlfart-Disease, Wohlfart-Kugelberg-Welander Disease, Wolf Syndrome, Wolf-Hirschhorn Chromosome Region (WHCR), Wolf-Hirschhorn Syndrome, Wolff Parkinson-White Syndrome, Wolfram Syndrome, Wolman Disease (Lysomal Acid Lypase Deficiency), Woody Guthrie's Disease, WPW Syndrome, Writer's Cramp, WS, WSS, 30 WWS, Wyburn-Mason Syndrome, X-Linked Addison's Disease, X-linked Adrenoleukodystrophy (X-ALD), X-linked Adult Onset Spinobulbar Muscular Atrophy, X-linked Adult Spinal Muscular Atrophy, X-Linked Agammaglobulinemia with Growth WO 2007/028212 PCT/AU2006/001325 -263 Hormone Deficiency, X-Linked Agammaglobulinemia, Lymphoproliferate X-Linked Syndrome, X-linked Cardio myopathy and Neutropenia, X-Linked Centronuclear myopathy, X-linked Copper Deficiency, X-linked Copper Malabsorption, X-Linked Dominant Conradi-Hunermann Syndrome, X-Linked Dominant Inheritance Agenesis of 5 Corpus Callosum, X-Linked Dystonia-parkinsonism, X Linked Ichthyosis, X-Linked Infantile Agammaglobulinemia, X-Linked Infantile Nectrotizing Encephalopathy, X linked Juvenile Retinoschisis, X-linked Lissencephaly, X-linked Lymphoproliferative Syndrome, X-linked Mental Retardation-Clasped Thumb Syndrome, X-Linked Mental Retardation with Hypotonia, X-linked Mental Retardation and Macroorchidism, X-Linked 10 Progressive Combined Variable Immunodeficiency, X-Linked Recessive Conradi Hunermann Syndrome, X-Linked Recessive Severe Combined Immunodeficiency, X Linked Retinoschisis, X-linked Spondyloepiphyseal Dysplasia, Xanthine Oxidase Deficiency (Xanthinuria Deficiency, Hereditary), Xanthinuria Deficiency, Hereditary (Xanthine Oxidase Deficiency), Xanthogranulomatosis Generalized, Xanthoma 15 Tuberosum, Xeroderma Pigmentosum, Xeroderma Pigmentosum Dominant Type, Xeroderma Pigmentosum Type A I XPA Classical Form, Xeroderma Pigmentosum Type B II XPB, Xeroderma Pigmentosum Type E V XPE, Xeroderma Pigmentosum Type C III XPC, Xeroderma Pigmentosum Type D IV XPD, Xeroderma Pigmentosum Type F VI XPF, Xeroderma Pigmentosum Type G VII XPG, Xeroderma Pigmentosum Variant Type 20 XP-V, Xerodenna-Talipes-and Enamel Defect, Xerodermic Idiocy, Xerophthalmia, Xerotic Keratitis, XLP, XO Syndrome, XP, XX Male Syndrome,Sex Reversal, XXXXX Syndrome, XXY Syndrome, XYY Syndrome, XYY Chromosome Pattern, Yellow Mutant Albinism, Yellow Nail Syndrome, YKL, Young Female Arteritis, Yunis-Varon Syndrome, YY Syndrome, Z-E Syndrome, Z- and -Protease Inhibitor Deficiency, Zellweger 25 Syndrome, Zellweger cerebro-hepato-renal syndrome, ZES, Ziehen-Oppenheim Disease (Torsion Dystonia), Zimmermann-Laband Syndrome, Zinc Deficiency Congenital, Zinsser-Cole-Engman Syndrome, ZLS, Zollinger-Ellison Syndrome. In another embodiment, the pharmaceutical composition of the present invention can be 30 used, alone or in conjunction with other drugs or therapies, in the treatment of diseases associated with abnormal bone growth for example, fibrodysplasia Ossificans progressive (FOP). Noggin may also be used to prevent disorders characterized by mutations in the WO 2007/028212 PCT/AU2006/001325 - 264 gene encoding noggin resulting in skeletal anomalies such as the fusion of bones, eg tarsal carpal coalition syndrome, congenital stapes ankylosis and proximal symphalangism. However, the pharmaceutical composition of this invention has higher therapeutic efficacy, 5 increased thermal stability, increased serum half-life or higher solubility in the bloodstream when compared with noggin or chimeric noggin molecule expressed in non human cell lines.. The present invention also shows reduced risks for immune-related clearance or related side effects. Because of these improved properties, the composition of the present invention can be administered at a lower frequency than a noggin or chimeric 10 noggin molecule expressed in non-human cell lines. Decreased frequency of administration is anticipated to enhance patient compliance resulting in improved treatment outcomes. The quality of life of the patient is also elevated. Accordingly, in one embodiment, the composition of the invention can be administered in 15 a therapeutically effective amount to patients in the same way noggin or chimeric noggin molecule expressed in non-human cell lines is administered. The therapeutic amount is that amount of the composition necessary for the desired in vivo activity. The exact amount of composition administered is a matter of preference subject to such factors as the exact type of condition being treated, the condition of the patient being treated and the other 20 ingredients in the composition. The pharmaceutical compositions containing the isoforms of noggin or chimeric noggin molecule of the present invention may be formulated at a strength effective for administration by various means to a human patient experiencing one or more of the above disease conditions. Average therapeutically effective amounts of the composition may vary. Effective doses are anticipated to range from 0.lng/kg body weight 25 to 20pjg/kg body weight; or based upon the recommendations and prescription of a qualified physician. The present invention further extends to uses of the isolated noggin or chimeric noggin molecule comprising at least part of a noggin or chimeric noggin molecule and a 30 composition comprising same in a variety of therapeutic and/or diagnostic applications. More particularly, the present invention extends to a method of treating or preventing a WO 2007/028212 PCT/AU2006/001325 -265 condition in a mammalian subject, wherein the condition can be ameliorated by increasing the amount or activity of noggin or chimeric noggin molecule, the method comprising administering to said mammalian subject an effective amount of an isolated noggin or chimeric noggin molecule, a chimeric molecule comprising the noggin or chimeric noggin 5 molecule or an extracellular domain thereof or a composition comprising the isolated noggin or chimeric noggin molecule. The subject invention also provides a pharmaceutical composition comprising a human derived noggin or chimeric noggin molecule for use, alone or in combination with other 10 proteins, such as bFGF, in culturing hES, reducing the potential risks of transferring animal-derived infectious agents to hES, thus allowing hES to be applicable for therapeutic applications. The present invention is further described by the following non-limiting examples. 15 WO 2007/028212 PCT/AU2006/001325 - 266 EXAMPLE 1 (a) Production of a pIRESbleo3-Fc 5 The DNA sequence for the Fe domain of human IgGI was amplified from EST cDNA library (Clone ID 6277773, Invitrogen) by Polymerase Chain Reaction (PCR), using forward primer (SEQ ID NO: 33) and reverse primer (SEQ ID NO: 34) that incorporated restriction enzyme sites for BamHl and BstX1, respectively. This amplicon was cloned into the corresponding enzyme sites of pIRESbleo3 (Cat. No. 6989-1, BD Biosciences) to 10 produce the construct pIRESbleo3-Fc. Digest of pIRESbleo3-Fc with BamH1 and BstX1 resulted in a correct size insert of 780bp. (b) Production of a DNA construct expressing Noggin 15 The DNA sequence for human noggin was amplified from EST cDNA library (Clone ID: 5261433, Open Biosystems) by PCR, using forward primer (SEQ ID NO: 21) and reverse primer (SEQ ID NO: 22) that incorporated restriction enzyme sites for EcoRV and BamHI, respectively. After amplification, the sequence was digested using EcoRV and BamHI enzymes and cloned into the corresponding restriction sites of expression vector 20 pIRESbleo3 (Cat. No. 6989-1, BD Biosciences) to produce the vector pIRESbleo3 noggin. Digestion of pIRESbleo3-noggin using EcoRV and BamHI resulted in an expected size fragment of 722 bp. Alternatively, the sequence of noggin is amplified with primers that incorporate restriction 25 sites allowing the cloning of the noggin sequence upstream of the Fe sequence in pIRESbleo-Fc, such that the two sequences are fused in-frame directly or by a linker. (c) Preparation of pIRESbleo3-noggin 30 750ml of sterile LB broth containing ampicillin (100 micrograms/ml) was inoculated with 750p1 of overnight culture of E. coli transformed with pIRESbleo3-noggin. The culture WO 2007/028212 PCT/AU2006/001325 - 267 was incubated at 370C with shaking for 16 hours. Plasmid was prepared in accordance with a Qiagen Endofree Plasmid Mega Kit (Qiagen Mega Prep Kit #12381). EXAMPLE 2 5 Production, Isolation and Purification of Noggin of the Present Invention (a) Production of Noggin At day 0, five 500 cm 2 tissue culture dishes (Coming) were seeded with 3 x 107 cells of 10 transformed embryonal human kidney cell line, for example HEK 293 or derivatives thereof, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD 293 (Stratagene), or 293A (Invitrogen). Cells were seeded in 90 ml per plate of Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12 (DMEM/F 12) (JRH Biosciences), the medium being supplemented with 10% (v/v) donor calf serum (DCS, JRH Biosciences) 15 4 mM L-glutamine (Amresco) and 1% (v/v) Penicillin-Streptomycin (Penicillin G 5000 U/ml, Streptomycin Sulphate 5000 ptg/ml) (JRH Biosciences). The plates were incubated at 37 'C and 5% C02 overnight. At day 1, transfection was performed using calcium phosphate. Before transfection, the 20 medium in each plate was replaced with 120 ml of fresh DMEM/F12 supplemented with 10% (v/v) DCS, 4 mM L-glutamine, and 1% (v/v) Penicillin-Streptomycin. Calcium phosphate / DNA precipitate was prepared by adding 1200 pg of plasmid DNA harbouring the gene for human noggin and 3000 ji of 2.5 M CaC12 in sterile 1xTE to a final volume of 30 ml (solution A). Solution A was added drop-wise to 30 ml of 2 x HEPES Buffered 25 Saline (HBS) (solution B) with a 10 ml pipette. During the course of addition, bubbles were gently blown through solution B. The mixture was incubated at 250C for 20 minutes and vortexed. 12 ml of the mixture was added drop-wise to each plate. After 4 hours the medium containing the transfection mixture was removed and 100 ml of DMEM/F 12 supplemented with 10% (v/v) DCS, 4 mM L-glutamine, 1% (v/v) Penicillin-Streptomycin, 30 and a final concentration of 3.5 mM HCl, with the medium having a final pH of 7, was added to each plate. The plates were incubated at 37C and 5% C02 overnight.
WO 2007/028212 PCT/AU2006/001325 -268 At day 2, the cell culture supernatant was discarded, The contents in the plates were washed twice with 50 ml of DMEM/F12 medium per plate and 100 ml of fresh serum-free DMEM/F12 medium supplemented with 40 mM N-acetyl-D-mannosamine (New Zealand Pharmaceuticals), 10 mM L-Glutamine (Amresco), 0.5 g/L Mannose (Sigma) and 1% (v/v) 5 Penicillin-Streptomycin was added to each plate. The plates were incubated at 37 *C and 5% CO 2 overnight. At day 3, the cell culture supernatant was collected and 100 ml fresh serum-free DMEM/F12 medium supplemented with 40 mM N-acetyl-D-mannosamine, 10 mM L 10 Glutamine, 4.1 g/L Mannose, and 1% (v/v) Penicillin-Streptomycin was added to each plate. The plates were incubated at 37 *C and 5% CO 2 overnight. 100 mM PMSF (1% (v/v)) and 500 mM EDTA (1% (v/v)) were added to the collected cell culture supernatant and the mixture was stored at 4 'C. 15 At day 4, the cell culture supernatant was collected. 100 mM PMSF (1% (v/v)) and 500 mM EDTA (1% (v/v)) was added to the collected cell culture supernatant and combined with the day 3 collection before particulate removal using a 0.45 micron low-protein binding filter (Durapore, Millipore). The mixture was either stored at 4'C or used immediately. For long-term storage, the supernatant was kept at -70*C. 20 (b) Isolation and Purification of Noggin One litre of medium containing noggin was passed by gravity flow over SP Sepharose 4 fast flow beads (Pharmacia) with a 3 ml bed volume that had been pre-equilibrated to pH 7 25 with 50 mM HEPES pH 7 (Sigma). After washing with 10 column volumes of column buffer 50 mM HEPES pH 7, Noggin was eluted in 3 ml fractions by using four Elution Buffers in the following order: Buffer 1: 50 mM HEPES pH 7/ 0.25 M NaCl 30 Buffer 2: 50 mM HEPES pH 7 / 0.5 M NaCl Buffer 3: 50 mM HEPES pH 7/ 0.75 M NaCl WO 2007/028212 PCT/AU2006/001325 - 269 Buffer 4: 50 mM HEPES pH 7/ 1 M NaCl The eluted fractions were analysed by silver-stained SDS-PAGE using 4 - 20% Tris Glycine gels (Invitrogen) and by Western blot analysis using an anti-human noggin 5 antibody (Santa Cruz Biotechnology, Inc.). Noggin was found to bind to SP sepharose and to elute in Buffer 3. The SP Sepharose fractions containing noggin were pooled and concentrated to less than 1 ml for size exclusion chromatography using a centrifugal filter device (Amicon Ultra, Millipore). 10 Size exclusion chromatography was performed on the concentrated sample using Superdex 75 prep grade 16/70 (Pharmacia, Uppsala, Sweden) column. An isocratic flow of 1%(w/v) Ammonium Bicarbonate was used at a flow rate of 1 ml/min. Total run time was 120 min with peaks eluting between 40 and 110 minutes. The eluted fractions were assayed by silver-stained SDS-PAGE using 4 - 20 % Tris-Glycine gels (Invitrogen). The peak eluting 15 at approximately 50 minutes was found to contain noggin. Fractions containing noggin were pooled and concentrated to less than 2 ml using a centrifugal filter device (Amicon Ultra, Millipore). The purified noggin was found to have an apparent MW of around 30-40 kDa and to be at 20 least 95 % pure as assessed by silver-stained SDS-PAGE using 4 - 20 % Tris-Glycine gels (Invitrogen). The final concentration of the noggin was determined by absorption at 280 nm using a molar extinction co-efficient of 40950 M'cm 1 . EXAMPLE 3 25 (a) Characterization of Noggin by Two-Dimensional Polyacrylamide Electrophoresis The collected sample from Example 2 underwent TCA precipitation and was then re solubilized in 200 microliters of 2D sample buffer (7M urea, 2M thiourea, 65mM DTT, 30 4% (w/v) CHAPS , 0.2% (v/v) carrier ampholytes, 40mM Tris, 0.002% (w/v) bromophenol blue). The sample was reduced and alkylated with 5mM Tributylphosphine and 15mM acrylamide for 1.5 hours then centrifuged at 20000 x g for 10 minutes.
WO 2007/028212 PCT/AU2006/001325 -270 Isoelectric focusing (IEF) was performed using precast 11 cm pH 3-10 immobolised pH gradient IPG strips (BioRad, catalogue number: 163-2014). The IPG strips were re hydrated with 200gL of the sample in a sealed tube at room temperature for at least 6 5 hours. The IPG strips were placed into the focusing chamber and covered with paraffin oil. IEF was carried out at 100 V for 3 hours, 300V for 3 hours, 1000V for 2 hours, 25000 V for 1 hours, 5000 V for 12 hours. The strips were equilibrated in 1 x Tris/HCl pH 8.8, 6M urea, 2% (w/v) SDS, 20% (v/v) 10 glycerol, 5mM tributylphosphine (TBP), 2.5% (v/v) acrylamide solution for 20 minutes. The proteins were separated in the second dimension using Criterion pre cast (11 x Scm x 1mm thick) 10-20% Tris glycine gradient gels (BioRad).. Precision molecular weight markers (BioRad catalogue number: 161-0363) were also run on the gel. The strip was set 15 into place using 0.5% Agarose containing bromophenol blue as a tracking dye. The SDS-PAGE was run using a Criterion electrophoresis system (BioRad) at 200 V until the buffer front was about to run off the end of the gel. The buffer used was 192 mM glycine, 0.1% (w/v) SDS, 24.8 mM Tris base at pH 8.3. 20 The completed second dimension gels were fixed overnight in 30% methanol (MeOH) and 7.5% acetic acid (HAc) then basified with 200 mM Na 2
CO
3 (2 x 15 minute washes). The gel was then stained using Deep Purple (Fluorotechnics product number RPN6306V) as per manufacturers instructions for at least 1 hour and destained with two 15 minute washes 25 of 30% MeOH and 7% HAc. The gel was imaged using a Typhoon Trio Variable Mode Imager (Amersham Biosciences) using the appropriate filter. The software ImageJ (http://rsb.info.nih.gov/ij/) was used to analyse the relative intensities of the protein spots on the gel. Densitometry was performed on the spots within a selected 30 area of the gel and a background subtraction was conducted using the appropriate region of the gel lacking protein spots. A volume integration was performed on each protein spot of interest from which the centre of mass for the spot was calculated. Relative percentage WO 2007/028212 PCT/AU2006/001325 -271 intensities were calculated for each protein spot and by normalising the combined value of the intensities of all spots to 100%, the intensity of each protein spot relative to the other spots in the gel was determined. 5 The charge of the isoforms (pKa values) were detennined by measuring the respective distance of the spots from the left side of the gel using ImageJ. Since the relationship between the pI values of the strip and the physical distance of the gel is linear, the pI values corresponding to the different pKa values of the isoform spots were readily determined. 10 The major protein spots in the resulting gel correspond to isoforms of noggin. The low intensity spots may be noggin or low level contaminants, however, these cannot be confirmed by PMF due to the low intensity. Examination of the gels revealed that noggin of the present invention contains 1 to 16 isoforms. Table 8 shows the pI values (± 1.0) of 15 these isoforms: The values listed correspond to the intensity weighted center within the selected area of gel containing the spot and hence, are only reflective of the pI of the protein at one particular reading within the selected area of the gel. Taking into consideration the inherent variability of size and position of protein spots within 2D gels, the pI values for the noggin of the present invention were determined to range from about 20 8 to 14 based on the values listed in Table 8. TABLE 8 pI values of isoforms of noggin Spot NoIsoelectric Point (131) Range 2 8.32 +1.00 3 8.38 ±1.00 4 8.58 :1.00 5 8.70 ±1.00 WO 2007/028212 PCT/AU2006/001325 - 272 Spot No Isoelectric Point (pI) Range 6 8.76 +1.00 7 8.94 1.00 8 9.03 1.00 9 9.12 +1.00 10 9.21 1.00 11 9.27 ±1.00 12 9.32 ±1.00 13 9.41 i1.00 14 9.59 ±1.00 15 9.74 ±1.00 16 9.91 ±1.00 17 9.76 ±1.00 (b) One-Dimensional Polyacrylamide Electrophoresis The collected sample from Example 2 underwent TCA precipitation and then re 5 solubilised into 60pL of 1D sample buffer (10% glycerol, 0.1% SDS, 10mM DTT, 63mM tris-HCl) and heated at 100'C for 5 minutes. For PNGaseF treatment, an aliquot of the sample was taken, and NP40 added to a final concentration of 0.5 %. 5 tL of PNGaseF was added and the sample was incubated at 37'C for 3 hours. For glycosidase cocktail treatment of the sample, an aliquot was taken then NP40 was added to a final concentration 10 of 0.5%. 1 L of PNGaseF, and 1 pL each of Sialidase A (neuramidase), 0-Glycanase, P (1-4)-Galactosidase and 1-N-Acetylglucosaminidase was added. The sample was incubated at 37 'C for 3 hours then run on ID gel. Precision molecular weight markers (BioRad catalogue number 161-0363) were also applied to the gel. Criterion 4-20% or 18% gels were used for 1D SDS-PAGE (BioRad catalogue numbers: 345-0033 or 345-0024). 15 WO 2007/028212 PCT/AU2006/001325 - 273 The SDS-PAGE was run using either a Criterion electrophoresis system (BioRad) at 200 V until the buffer front was about to run off the end of the gel. The buffer used was 192 mM glycine, 0.1% (w/v) SDS, 24.8 mM Tris base at pH 8.3. 5 The completed gels were fixed for 30 minutes- overnight in 30% methanol (MeOH) and 7.5% acetic acid (HAc) then basified with 200 mM Na 2
CO
3 (2 x 15 minute washes). The gel was then stained using Deep Purple (Fluorotechnics product number RPN6306V) as per manufacturers instructions for at least 1 hour and destained with 30% MeOH and 7% HAc for at least 30 minutes. The gel was imaged using a Typhoon Trio Variable Mode 10 Imager (Amersham Biosciences) and the appropriate filter. The apparent molecular weight of the molecule was found to be between 25 and 29 kDa. The apparent molecular weight of the molecule (as observed by SDS-PAGE) following the 15 release of N-linked oligosaccharides (by PNGase treatment) was between 24 and 26 kDa. The apparent molecular weight of the molecule (as observed by SDS-PAGE) following the release of N-linked oligosaccharides (by PNGase treatment) and O-linked oligosaccharides (by p-elimination) was between 23 and 24 kDa. 20 (c) N-Terminal Sequencing of Proteins Protein bands are cut from the gel prepared above (either from a two-dimensional gel or a one-dimensional gel) and are placed into a 0.5ml tube and 1 00ml extraction buffer is added (100mM Sodium acetate, 0.1%SDS, 50mM DTT pH 5.5). The gel slices are incubated at 25 37'C for 16 hours with shaking. The supernatant is applied to a ProSorb membrane (ABI) as per the manufacturers instruction and sequenced using an automated 494 Protein Sequencer (Applied Biosystems) as per the manufacturers instructions. The sequence generated is used to confirm the identity of the noggin.
WO 2007/028212 PCT/AU2006/001325 - 274 (d) Peptide Mass Fingerprinting Protein bands were cut from the one-dimensional gel prepared above and washed with 25d of wash buffer (50% acetonitrile in 50mM NH 4
HCO
3 ). The gel pieces were left at 5 room temperature for at least 1 hour and dried by vacuum centrifugation for 30 minutes. The gel pieces and 12[d of trypsin solution (20[Lg trypsin, 1200pl NH 4
HCO
3 ) was placed in each sample well and incubated at 4*C for 1 hour. The remaining trypsin solution was removed and 20pl 50mM NH 4
HCO
3 was added. The mixture was incubated overnight at 37'C with gentle shaking. The peptide samples were concentrated and desalted using C18 10 Zip-Tips (Millipore, Bedford, MA) or pre-fabricated micro-columns containing Poros R2 (Perseptive Biosystems, Framingham, MA) chromatography resin. Bound peptides were eluted in 0.8 R1 of matrix solution (a-cyano-4-hydroxy cinnamic acid (Sigma), 8 mg/ml in 70% acetonitrile / 1% formic acid) directly onto a target plate. Peptide mass fingerprints of tryptic peptides were generated by matrix-assisted laser desorption / ionisation time-of 15 flight mass spectrometry (MALDI-TOF MS) using a Perseptive Biosystems Voyager DE STR. Spectra were obtained in reflectron mode using an accelerating voltage of 20 kV. Mass calibration was performed using trypsin autolysis peaks, 2211.11 Da and 842.51 Da as internal standards. Data generated from peptide mass fingerprinting (PMF) was used to confirm the identity of the protein. Searches (primarily of Homo sapien (Human) and 20 mammalian entries) were performed in databases such the SWISS-PROT and TrEMBL, via the program PeptIdent (www.expasy.ch/tools/peptident.html). Identification parametres included peptide mass tolerance of 0.1 Da, a maximum of one missed tryptic cleavage per peptide, and the methionine sulfoxide and cysteine-acrylamide modifications. Identifications were based on the number of matching peptide masses and the total 25 percentage of the amino acid sequence that those peptides covered, in comparison to other database entries. Generally, a peptide match with at least 30% total sequence coverage was required for confidence in identification, but very low and high mass proteins, and those resulting from protein fragmentation, may not always meet this criterion, therefore requiring further identification. 30 Where inconclusive or no protein identification could be obtained from MALDI-TOF PMF analysis, the remaining peptide mixture or the identical spot cut from a replicate gel was WO 2007/028212 PCT/AU2006/001325 -275 subjected to tryptic digest and analysed by electrospray ionization tandem MS (ESI MS/MS). For ESI-MS/MS, peptides were eluted from Poros R2 micro-columns in 1-2 ptl of 70% acetonitrile, 1% formic acid directly into borosilicate nanoelectrospray needles (Micromass, Manchester, UK). Tandem MS was performed using a Q-Tof hybrid 5 quadrupole/orthogonal-acceleration TOF mass spectrometer (Micromass). Nanoelectrospray needles containing the sample were mounted in the source and stable flow obtained using capillary voltages of 900-1200V. Precursor ion scans were performed to detect mass to charge ratio (in/z) values for peptides within the mixture. The m/z of each individual precursor ion was selected for fragmentation and collided with argon gas using 10 collision energies of 18-30eV. Fragment ions (corresponding to the loss of amino acids from the precursor peptide) were recorded and processed using MassLynx Version 3.4 (Micromass). Amino acid sequences were deduced by the mass differences between y- or b-ion 'ladder' series using the program MassSeq (Micromass) and confirmed by manual interpretation. Peptide sequences were then used to search the NCBI and TrEMBL 15 databases using the program BLASTP "short nearly exact matches". A minimum of two matching peptides were required to provide confidence in a given identification. The identity of the bands were confirmed to be noggin. 20 Further, a iDa shift in the masses of two tryptic peptides (EKDLNETLLR and DLNETLLR) indicated the asparagine residue (N) of the NET motif found in the theoretical amino acid sequence of human noggin was modified to aspartic acid (D), consistent with the known ability of PNGase F to induce an N to D residue modification upon removal of associated N-linked oligosaccharides. Hence, the site of N-glycosylation 25 of the noggin molecule of the present invention is N-62 (when numbered from the start of the signal sequence).
WO 2007/028212 PCT/AU2006/001325 - 276 EXAMPLE 4 Analysis of Amino Acid, Monosaccharide, Oligosaccharide, Phosphate, Sulfate and Isoform Composition of Noggin of the Present Invention 5 (a) Preparation of Samples for Amino Acid, Monosaccharide, Oligosaccharide, Phosphate, Sulfate and Isoform Analysis For characterisation of monosaccharide and oligosaccharide glycosylation and phosphate and sulfate post-translational modifications, the saccharides are first removed from the 10 polypeptide backbone by hydrolytic or enzymatic means. The sample buffer components are also removed and exchanged with water to avoid inhibition of the hydrolysis and enzymatic reactions before analysis began. A solution of purified noggin in PBS is dialysed extensively against 4 litres of deionised ultrafiltered water (18 MOhm) for four days with two changes per day using a regenerated cellulose dialysis membrane 15 (Spectrapore) with a nominal molecular weight cut-off (NMWC) of 5 KDa. After dialysis the solution is dried using a Savant Speed Vac (New York, USA). The dried down sample is then resuspended in 2 ml of deionised ultrafiltered water (18 MOhm) and divided into aliquots for the various analyses. 20 (b) Analysis of Amino Acid Composition by the Gas Phase Hydrolysis Method Amino acids in the samples are analysed using precolumn derivatisation with 6 aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The stable fluorescent amino acid derivatives are separated and quantified by reversed phise (C18) HPLC. The 25 procedure employed is based on the Waters AccQTag amino acid analysis methodology. Three samples of the noggin preparation are taken and dried in a Speed Vac . The dried samples are then hydrolysed for 24 hours at 110 C. After hydrolysis the samples are dried again before derivatisation as follows. The dried samples are re-dissolved in 10 tL of an 30 internal amino acid standard solution (c-aminobutyric acid, AABA), 35 pL of borate buffer is added followed by 15 pL of AQC derivatising reagent. The reaction mix is heated at 50'C for 12 minutes in a heating block. The derivatised amino acid sample is transferred WO 2007/028212 PCT/AU2006/001325 - 277 to the autosampler of a HPLC system consisting of a Waters Alliance 2695 Separation Module, a Waters 474 Fluorescence Detector and a Waters 2487 Dual X Absorbance Detector in series. The control and analysis software is Waters Empower Pro Module (Waters Corporation, Milford. MA, USA). The samples are passed over a Waters 5 AccQTag column (15cm x 3.9mm ID) using chromatographic parameters (i.e. suitable eluents and gradient flows) known in the art. (c) Analysis of Neutral and Amino Monosaccharide composition 10 Two samples of the noggin preparation are taken and treated in two different ways to liberate monosaccharides. Each treatment, as described below, is performed in triplicate. 1. Hydrolysed with 2 M trifluroacetic acid (TFA) heated to 1000 C for four hours to release neutral sugars (galactose, glucose, fucose and mannose). 15 2. Hydrolysed with 4 M HCl heated to 100* C for four hours to release amino sugars (N-acetyl-galactosamine, N-acetyl-glucosamine). All of the hydrolysates are lyophilised using a Speed Vac system, redissolved in 200 1d water containing 0.8 nmols of internal standard. For neutral and amino sugars the internal 20 standard is 2-deoxy-glucose. The samples are then centrifuged at 10,000 g for 30 minutes to remove protein debris. The supernatant is transferred to a fresh tube and analysed by high pH anion exchange chromatography using a Dionex LC 50 system with a GP50 pump and an ED50 pulsed amperometric detector (Dionex Ltd). Analysis of neutral and amino sugars is performed using a Dionex CarboPac PA-20 column. Elution is performed with an 25 isocratic hydroxide concentration of 10 mM over 20 minutes. This is achieved with the Dionex EG50 eluent generation system. (d) Analysis of Acidic Monosaccharide Composition 30 A sample of the noggin preparation is taken and treated in the following way to liberate sialic acid monosaccharides. The treatment is performed in triplicate. The sample is hydrolysed with 0.1 M TFA at 800 C for 40 minutes to release N-Acetyl and N-Glycolyl WO 2007/028212 PCT/AU2006/001325 - 278 neuraminic acid. The hydrolysates are lyophilised using a Speed Vac, redissolved in 200 pl water containing 0.8 nmols of internal standard. For sialic acid analysis the internal standard is lactobionic acid. Samples are then centrifuged at 10,000 g for 30 minutes. The supernatant is transferred to a fresh tube and analysed by high pH anion exchange 5 chromatography using a Dionex LC 50 system with a GP50 pump and an ED50 pulsed amperometric detector. Analysis of sialic acids is performed using a Dionex CarboPac PAl using using chromatographic parameters (i.e. suitable eluents and gradient flows) known in the art. 10 (e) Analysis of Oligosaccharide Composition For analysis of oligosaccharide composition two samples of the noggin preparation are taken in triplicate and treated in one of the following ways: 15 1. Release of N-linked oligosaccharides is achieved with the enzyme Peptide-N4-(N acetyl--D-glucosaminyl) Asparagine Amidise (PNGase). First, a 1
/
5 th volume of denaturation solution (2 % SDS (Sigma)/1 M p-mercaptoethanol (Sigma)) is added to the sample. The sample is heated to 100 C for 5 minutes. A 1/10 volume of 15 % Triton X100 (Sigma) is added to the sample. The sample is mixed gently and allowed to cool to 20 room temperature. 25 Units of PNGase (Sigma) is added and incubated overnight at 37*C. 2. Release of O-linked oligosaccharides is achieved by the process of p-elimination. First, a 1/2 volume of 4M sodium borohydride (freshly made) (Sigma) solution is added to the sample. A 1/2 volume of 0.4 M NaOH (BDH, HPLC grade) is added to the sample. The 25 sample is incubated at 50" C for 16 hours. The sample is cooled on ice and a M volume of 0.4 M acetic acid (Sigma) is added to the sample. Both the N-linked and O-linked samples are further processed to remove buffer components using a Carbo Pac graphitised carbon SPE column. The column equilibration 30 and elution conditions are is follows: WO 2007/028212 PCT/AU2006/001325 - 279 Firstly, the column is pre-equilibrated with 1 column volume of 80 % acetonitrile (Sigma) followed by two column volumes of H20. The sample is loaded under gravity flow and the column washed with two column volumes of H 2 0. To elute neutral oligosaccharides 2 ml of 50 % acetonitrile is applied to the column. To elute acidic oligosaccharides 2 ml of 50 5 % acetonitrile/0.1% formic acid is applied to the column. Any remaining oligosaccharides are eluted by the addition of 2 ml of 80 % acetonitrile/0.1 % formic acid. Individual fractions from the SPE columns containing the neutral or acidic N-linked oligosaccharides and the neutral or acidic O-linked oligosaccharides are dried down to completion using a Speed Vac. The samples are redissolved in 200 ptl water and analysed by high pH anion 10 exchange chromatography using a Dionex LC 20 system with a GP50 pump and an ED50 pulsed amperometric detector. Analysis of neutral and acidic oligosaccharides is performed using a CarboPac PA100 column and chromatographic parameters (i.e. suitable eluents and gradient flows) known in the art. 15 (f) Analysis of Sulfate and Phosphate Composition Sulfate/phosphate analysis is performed essentially by the method described by Harrison and Packer (Methods Mol Biol 125:211-216, 2000). A sample of the noggin preparation is taken for sulfate/phosphate analysis and hydrolysed in 4 M HCl at 100 'C for four hours. 20 The HCl is removed by drying the samples in a Speed Vac system. Samples are then redissolved into 200 pl H 2 0. A suitable volume of sample is injected onto a Dionex LC 50 system with a GP50 pump and a ED50 conductivity detector. Separation is performed by a Dionex lonPac IS11 Anion exchange column using chromatographic parameters (i.e. suitable eluents and gradient flows) known in the art. 25 (g) Further Separation of Protein Isoforms Further separation of noggin isoforms is performed using a pellicular anion exchange column. A suitable volume of sample, for example, 24 pl, is separated through a ProPac 30 SAX-10 column (Dionex Ltd) using a Dionex SUMMIT system with UV-Vis detector (Dionex Ltd). Separation is performed using suitable eluents and gradients known in the art. noggin isoforms are found to elute in a pattern of distinct peaks.
WO 2007/028212 PCT/AU2006/001325 -280.
EXAMPLE 5 Glyco Mass Fingerprinting 5 Noggin of the present invention was separated using 2D gel electrophoretic techniques as in Example 3 and blotted onto polyvinyl difluorethane (PVDF) membrane. The spots were stained using one of a standard array of protein stains (Colloidal Coomassie Blue, Sypro Ruby or Deep Purple), and the isoform relative amounts quantified using densitometry algorithms. Individual spots were excised and treated with an array of deglycosylating 10 enzymes and/or chemical means, as appropriate, to remove the oligosaccharides present according to methods described above in Example 4(e). Once the oligosaccharides were removed, they were separated and analysed on a liquid chromatography-electrospray mass spectrometry system (LC-MS) using a graphitised 15 carbon column and organic solvent (MeCN) gradient elution system. The generated peak profile represents a "fingerprint" of the N-linked and 0-linked oligosaccharides present on Noggin of the present invention (Figures 2(a), 3(a) and 2(b), 3(b), respectively). In addition, individual mass peaks were fragmented multiple times to give MS" spectra. 20 These fragments allowed a structural prediction of both the N-linked oligosaccharides (Figures 4(a), 4(b) and 4(c); Table 9) and O-linked oligosaccharides (Figures 4(d) and 4(e); Table 10) using a bioinformatics database program (GlycosidIQ; http://www.glycosuite.com/).
WO 2007/028212 PCT/AU2006/001325 -281 TABLE 9 Predicted structures of the N-glycans present in the Noggin of the present invention using GlycosidIQ MW Structure % Total 1972 15.8 Gal b--4 Glclaci-- 2 Hanal G2 han bi- 4 GlcNR fbi- 4 GlcNflc NeuRc a2-6 GalNnc bl-4 GlcHAnbi- 2 Hanal 2368 35.8 Fuc eufe a2- 3 Gal bi-4 GlcNRbi- 2 lana1 a 6 hanbi-4 GlcNflcbi-4 GlcNRc NeuRc a2- 3 Gal bi-4 GlcNHbi- 2 Hanal 2159 48.4 Fuc al. GaINRcbi-4 GlcNcbi- 2 hanaI a HNabi-4 G].cNflb-4 GlcNRc NeuRc a2--6 GaINRcbi-4 GlcNAcbi- 2 Hanal 5 TABLE 10 Predicted structures of the O-glycans present in the Noggin of the present invention using GlycosidIQ MW Structure % Total 965 90.3 Neu~o Neufca2- 3 Galbi 1330 9.7 Heuflca2- 6 Gal bi-4 GicHflc\ 3 NeuAc a2- 3 Galbi 10 WO 2007/028212 PCT/AU2006/001325 -282 EXAMPLE 6 Fluorophore Assisted Carbohydrate Electrophoresis Oligosaccharide profiles of the noggin of the present invention are derived using the 5 fluorophore assisted carbohydrate electrophoresis protocols (FACE protocols). The oligosaccharides from the noggin of the present invention are hydrolysed from the amino acid backbone using ammonium hydroxide and subsequently labelled using the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). Polyacrylamide gel electrophoresis is used to separate the species and standards used to identify an 10 oligosaccharide profile that is typical of the noggin of the present invention. Further, the oligosaccharides are identified using matrix assisted laser desorption and ionisation - time of flight mass spectrometry (MALDI-TOF) relying on the fluorophore and a specific matrix to ionise each sugar. The mass of each sugar is determined and potential structures identified using the GlycoSuite database. The potential sugar structures are further 15 characterised by tandem mass spectrometric techniques, which allows partial or complete characterisation of the oligosaccharides present and their relative amounts. Further, the process is repeated using the isoforms identified by 2D gel electrophoresis to generate a profile of the oligosaccharides present on each of the isoforms isolated. 20 EXAMPLE 7 QCM and SPR The binding characteristics and activity of the noggin of the present invention is determined using either quartz crystal microbalance (QCM) or surface plasmon resonance 25 (SPR). In both cases a suitable receptor for the molecule is bound to a wafer using the chemistry described by the manufacturer. The noggin of the present invention is dissolved into a suitable biological buffer and allowed to interact with the receptor on the chip by passing the buffer over it. Changes in the total protein mass on the surface of the wafer are measured either by change of oscillation frequency (in the case of QCM) or changes in the 30 light scattering qualities of the chip (in the case of SPR). The chip is then treated with the biological buffer alone to observe the release of the target molecule back into solution. The WO 2007/028212 PCT/AU2006/001325 -283 rate at which the receptors reach saturation and complete disassociation is then used to calculate the binding curve of the noggin of the present invention. EXAMPLE 8 5 Generation of a Transgenic Host Cell Line (a) Transgenic Host Cell Line with alpha-2,6-sialyltransferase The cDNA coding for alpha-2,6-sialyltransferase (alpha 2,6ST) is amplified by PCR from 10 poly(A)-primed cDNA. The PCR product is ligated into pIRESpuro4 or pCEP4, to generate an alpha 2,6ST plasmid. The cloned cDNA is sequenced and its identity verified by comparison with the published alpha-2,6ST cDNA sequence. DNA sequencing is performed using known methods. 15 Mammalian host cells, including cell clones of the same lineage that express high levels of noggin (cell line- noggin) are transfected with the alpha 2,6ST plasmid, which also carries an antibiotic resistance marker. Selection of stably transfected cells is performed by incubaton of the cells in the presence of the antibiotic; colonies of antibiotic-resistant cells that appear subsequent to transfection are pooled and examined for intracellular alpha 20 2,6ST activity. To isolate individual cell clones expressing alpha 2,6ST, cell pools are cloned by a limiting dilution process as described by Kronman (Gene 121: 295-304, 1992). Individual cell clones are chosen at random, cells expanded and clones tested for alpha 2,6ST activity. 25 Cell pellets are washed, resuspended in lysis buffer and left on ice prior to sonication. The cell lysate is centrifuged and the clear supernatant is assayed for protein concentration (via known methods) and sialyltransferase activity. Sialyltransferase activity is assayed by known methods, for example the method detailed by Datta et al. (JBiol Chem 270:1497 1500, 1995). 30 Expressed noggin is purified from high-expressing alpha 2,6ST cell line- noggin cells and subjected to in vitro and/or in vivo half-life bioassays (see Example 10). noggin from high- WO 2007/028212 PCT/AU2006/001325 -284 expressing alpha 2,6ST cell displays an increased in vitro and/or vivo half-life in comparison to rh noggin derived from the same parent cell line without any subsequent transgene manuipulation or rh noggin derived from other cell lines. 5 (b) Transgenic Host Cell Line with fucosyltransferase The cDNA coding for a fucosyltransferase (FT) such as FUTI, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT1 0, FUTI 1 is amplified by PCR from poly(A) primed cDNA. The PCR product is ligated into a suitable vector, for instance pIRESpuro4 10 or pCEP4, to generate an alpha 2,6ST plasmid. The cloned cDNA is sequenced and its identity verified by comparison with the published FT cDNA sequence. DNA sequencing is performed using known methods. Human host cells, including cell clones of the same lineage that express high levels of 15 noggin (cell line- noggin) are transfected with the FT plasmid, which also carries an antibiotic resistance marker. Selection of stably transfected cells is performed by incubation of the cells in the presence of the antibiotic; colonies of antibiotic-resistant cells that appear subsequent to transfection are pooled and examined for intracellular FT activity. To isolate individual cell clones expressing FT, cell pools are cloned by a limiting 20 dilution process as described by Kronman 1992 supra; individual cell clones are chosen at random, cells expanded and clones tested for FT activity. Cell pellets are washed, resuspended in lysis buffer and left on ice prior to sonication. The cell lysate is centrifuged and the clear supernatant is assayed for protein concentration (via 25 known methods) and FT activity. FT activity is assayed by known methods, for example the method detailed by Mas et a[. (Glycobiology 8(6):605-13, 1998). Expressed noggin is purified from high-expressing FT cell line-target molecule cells. A Lewis x-specific antibody, such as L5 and a sialyl Lewis x-specific antibody such as 30 KV193, HECA493, 2H5 or CSLEX are used to test the presence of Lewis x or sialyl Lewis x structures according to methods known in the art, for example, as detailed in Lucka et al. (Glycobiology 15(1):87, 2005). Alternatively, the presence of Lewis x or sialyl Lewis x WO 2007/028212 PCT/AU2006/001325 -285 structures may be detected by treating the sample with appropriate glycosidases and detecting the effect of the glycosidases on parameters such as mass using MS or retention time using HPLC. Glyco mass fingerprinting, as described in Example 5, may also be employed to predict the presence of Lewis x or sialyl Lewis x structures. 5 EXAMPLE 9 Differential Gene Expression Differences in gene expression can be analyzed using a target cell line of the noggin of the 10 present invention. The target cells are grown to the appropriate density and treated with a range of concentration of noggin of the present invention or buffer control for a number of hours, for instance, 72 hours. At various time points RNA is harvested, purified, and reverse transcribed according to 15 Affymetrix protocols. Labelled cRNA (e.g. biotin labelled) is then prepared and hybridised to expression arrays e.g. U133 GeneChips. Following washing and signal amplification, the GeneChips are scanned using a GeneChip scanner (Affymetrix) and the hybridisation intensities and fold change information at various time points is obtained using GeneChip software (Affymetrix). 20 The noggin of the present invention induces unique gene expression and results in different mRNA profiles upon comparison with profiles induced by cytokines or receptors produced from different sources e.g. E. coli, yeast or CHO cells. 25 EXAMPLE 10 Determining the Half-Life of the Noggin The half-life of the noggin of the present invention is determined in an in vitro system. Composition containing noggin of the present invention is mixed into human 30 serum/plasma and incubated at a particular temperature for a particular time (e.g. 37 degrees for 4 hours, 12 hours etc). The amount of noggin of the present invention remaining after this treatment is determined by ELISA methods or dot blot methods known WO 2007/028212 PCT/AU2006/001325 -286 in the art. The biological activity of the remaining noggin of the present invention is determined by performing a suitable bioassay chosen by a person skilled in the relevant art. The serum chosen may be from a variety of human blood groups (eg A, B, AB, 0 etc.). 5 The half-life of noggin of the present invention is also determined in an in vivo system. Composition containing noggin of the present invention is labelled by a radioactive tracer (or other means) and injected intravenously, subcutaneously, retro-orbitally, intramuscularly or intraperitonally into the species of choice for the study, for instance, mouse, rat, pig, primate or human. Blood samples are taken at time points after injection 10 and assayed for the presence of noggin of the present invention (either by ELISA methods, dot blot methods or by trichloroacetic acid (TCA)-precipitable label e.g. radioactive counts). A comparison composition consisting of rh noggin produced from other sources eg E. coli, yeast, or CHO cells can be run as a control. 15 EXAMPLE 11 (a) In Vivo Studies using the Noggin The individual subjects of the in vivo studies described herein are warm-blooded vertebrate 20 animals, which includes humans. The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. 25 Preferably to account for the psychological effects of receiving treatments, the trial is conducted in a double-blinded fashion. Volunteers are randomly assigned to placebo or noggin treatment groups. Furthermore, the relevant clinicans are blinded as to the treatment regime administered to a given subject to prevent from being biased in their post-treatment observations. Using this randomization approach, each volunteer has the 30 same chance of being given either the new treatment or the placebo.
WO 2007/028212 PCT/AU2006/001325 - 287 Volunteers receive either the noggin of the present invention or placebo for an appropriate period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements 5 include the levels of noggin in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease such as specific disease indicators or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements. 10 Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition. 15 Volunteers taking part in this study are adults aged 18 to 65 years and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and noggin treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with noggin of the present invention show positive trends in their 20 disease state or condition index at the conclusion of the study. (b) Treatment of an Acute Spinal Cord Injury using a purified population of Oligodendrocyte Progenitor Cells differentiated from hES by the use of Noggin 25 The individual subjects of the in vivo studies described herein are human and in particular, humans presenting with an acute demyelinating event as a result of spinal cord injury. The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. 30 In a particular embodiment, to account for the psychological effects of receiving the treatment, the trial is conducted in a double-blinded fashion. Volunteers are randomly assigned to oligodendrocyte transplantation or placebo treatment groups. Furthennore, the WO 2007/028212 PCT/AU2006/001325 -288 relevant clinicans, for example, neurologists, are blinded as to the treatment regime administered to a given subject to prevent from being biased in their post-treatment observations. Using this randomization approach, each volunteer has the same chance of being given the treatment or the placebo. 5 The site of acute demyelination within the spinal cord is determined by a suitable imaging technique, for example, the presence of hyperintensity on a T1W magnetic resonance image. 10 A highly purified population of cells with an oligodendrocyte lineage is prepared using the 04+ tag (see Example 18, below) and standard cell sorting techniques. Volunteers receive injections of a suitable number of 04+ cells or 04+ cell clumps, for example 125,000 cells into the site of acute demyelination or placebo for an appropriate period with biological parameters associated with an acute demyelination consistent with a spinal cord injury 15 being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include indices of the disease state or condition being treated such as the area of T1W hyperintensity on a magnetic resonance image, Wilcoxon's signed rank test as well as body weight, blood pressure, serum titers of antibodies which indicate allograft rejection and 20 ADME (absorption, distribution, metabolism and excretion) measurements. Information recorded for each patient includes age (years), gender, height (cm) and motivation rating (some/moderate/great) and previous family history of demyelinating events such as multiple sclerosis (yes/no). 25 Volunteers taking part in this study are adults aged 18 to 65 years and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for transplantation or placebo treatments. 30 In general, the volunteers that receive transplantation show positive trends in their disease state or condition index at the conclusion of the study compared with volunteers treated with placebo.
WO 2007/028212 PCT/AU2006/001325 -289 EXAMPLE 12 (a) In vitro comparison of the bioactivities of Noggin and Recombinant Human 5 Noggin expressed using non-human systems in the inhibition of BMP-4 Mediated Alkaline Phosphatase Production in MC3T3 Cells BMP-4 has been reported to increase the production of alkaline phosphatase (AP) in mouse MC3T3-E1 clonal osteogenic cells. Noggin, a specific inhibitor of BMP-4, binds to BMP-4 10 and thus competitively inhibits the binding of BMP-4 to cellular BMP-4 receptor sites. Thus, incubating BMP-4 with Noggin will inhibit BMP-4 mediated AP production in MC3T3 cells. MC3T3-E1 subclone 4 cells (ATC Number CRL-2593) were plated in 96 well plates in 15 triplicate at a concentration of 40,000 cells/well in assay medium (alpha-MEM, #01 0083DJ, Gibco; 1% FCS, #12003-500M, JRH Biosciences; 1% Pen/Strep, #P4458, Sigma; 0.05 g/L ascorbic acid, #A4034, Sigma; 3mM beta-glycerophosphate, #G9891, Sigma) and treated with BMP-4 (75 ng/ml; R&D Systems, Cat No. 314-BP-010). Noggin of the present invention was serially diluted (0-3000 ng/ml) and added to wells. Cells in control 20 wells were treated with noggin of the present invention (0-3000 ng/ml) alone in assay medium. Cells were incubated for 48 hours at 37"C and 5% CO 2 after which alkaline phosphatase production was measured using p-nitrophenyl phosphate disodium salt (1 mg/ml, # N9389, 25 Sigma). The amount of AP produced was determined by reading the absorbance of the resultant solution at 405nm by a spectrophotometer (E max precision microplate reader, Molecular Devices). The above assay was repeated using a recombinant human noggin expressed in E. coli 30 (Peprotech; Cat No. 120-10).
WO 2007/028212 PCT/AU2006/001325 -290 The ED50 for the noggin of the present invention was estimated using standard methods after curve fitting the absorbance and the noggin concentration values (Figure 5(a)). The ED50 for the noggin of the present invention was found to be 144-216 ng/ml, whereas 5 the recombinant human noggin expressed in E. coli was found to be ineffective at inhibiting BMP-4 mediated production of alkaline phosphatase in MC3T3-E1 pre-osteoblast cells (Figure 5(a)). (b) In vitro comparison of the bioactivities of Noggin and Recombinant Human 10 Noggin expressed using non-human systems in the BMP-4 mediated proliferation of CD34* human hematopoietic cells Sonic hedgehog (ShH) and BMP-4 have been reported to induce the differentiation and proliferation of primitive human hematopoietic cells. The actions of ShH are dependent on 15 downstream BMP-4 signals. Noggin, a specific inhibitor of BMP-4, binds to BMP-4 and thus competitively inhibits the binding of BMP-4 to cellular BMP-4 receptor sites. Thus, incubating either ShH or BMP-4 with Noggin will inhibit the proliferation of primitive human hematopoietic cells. 20 Primitive (CD34*) human hematopoietic cells (PoieticsTM; Cat No. 2C-101B; Cambrex) were plated in 48 well plates in duplicate at a concentration of 1000 cells/well in HPGM medium (Cambrex; Cat No. PT-3926) and treated with either BMP-4 (25 ng/nl; R&D Systems, Cat No. 314-BP-010) or ShH (100 ng/ml; R&D Systems, Cat No. 1314-SH-025). Noggin of the present invention (25 ng/ml) was added to some dishes. Control dishes were 25 treated with Noggin of the present invention (25 ng/ml) in HPGM medium. Cells were incubated for 4-7 days at 37*C and 5% CO 2 after which cell numbers were determined using a standard Trypan Blue staining procedure and cell counts using a haemocytometer. The above assay was repeated using a recombinant human noggin expressed in K coli 30 (Peprotech; Cat No. 120-10).
WO 2007/028212 PCT/AU2006/001325 -291 After 4 days incubation, the noggin of the present invention was found to be 16-37% more effective (Figure 5(b); p = 0.02, two-tailed Students t-test) than a recombinant human noggin molecule expressed in E. coli in the inhibition of a ShH-induced, BMP-4 mediated proliferation of CD34+ human hematopoietic cells. 5 (c) In vitro comparison of the bioactivities of Noggin and Recombinant Human Noggin expressed using non-human systems in a colony forming unit (CFU) assay Sonic hedgehog (Shh) and BMP-4 have been reported to induce the differentiation and 10 proliferation of primitive human hematopoietic cells. The actions of Shh are dependent on downstream BMP-4 signals. Noggin, a specific inhibitor of BMP-4, binds to BMP-4 and thus competitively inhibits the binding of BMP-4 to cellular BMP-4 receptor sites. Thus, incubating either Shh or BMP-4 with Noggin will inhibit the differentiation and proliferation primitive human hematopoietic cells, including the ability to form colonies. 15 Primitive (CD34+) human hematopoietic cells (PoieticsTM; Cat No. 2C-101B; Cambrex) are plated in 48 well plates in duplicate at a concentration of 1000 cells/well in a suitable medium, for example, HPGM medium (Cambrex; Cat No. PT-3926) or serum-free conditioned BSA, insulin, transferrin (BIT) media (Stem Cell Technologies) and treated 20 with either BMP-4 (25 ng/ml; R&D Systems, Cat No. 314-BP-010) or Shh (100 ng/ml; R&D Systems, Cat No. 1314-SH-025). Noggin of the present invention (25 ng/ml) is added to some dishes. Control dishes are treated with Noggin of the present invention (25 ng/ml) in HPGM medium. 25 Cells are incubated for 7 days at 37'C and 5% CO 2 after which cell numbers are determined using a standard Trypan Blue staining procedure and cell counts performed using a haemocytometer. Cells which did not undergo staining are re-suspended in a suitable medium, re-plated in a methylcellulose cocktail, for example, MethoCult H4434 (Stem Cell Technologies) at a range of concentrations, for example 100-1000 cells, and 30 incubated for 7-14 days at 37 0 C and 5% CO 2 in a humidified environment. Colonies are stained with crystal violet and counts performed using an inverted microscope and a WO 2007/028212 PCT/AU2006/001325 - 292 100mm culture dish marked with the scoring grid. A colony consists of approximately 40 cells or more. The above assay is repeated using a recombinant human noggin expressed in E. coli 5 (Peprotech; Cat No. 120-10). It is envisaged the significantly fewer colonies are produced when cells are exposed to noggin of the present invention compared with recombinant human noggin expressed in E. coli over a range of concentrations. Thus, the noggin of the present invention has a 10 significantly greater capacity to inhibit the BMP-4 mediated differentiation of primitive human hematopoietic cells into CFUs than a recombinant human noggin molecule expressed in E. coli. EXAMPLE 13 15 In vitro comparison of Immunoreactivity Profiles between Noggin and recombinant human and mouse noggin expressed using non-human systems Protein estimation of noggin of the present invention was determined using a standard 20 protein assay technique, for example, by the A280 absorbance method using the calculated extinction coefficient (s) and the measured molecular mass based on SDS-PAGE analysis. Mature human and mouse noggin differs by only one amino acid in a 205 amino acid length and, as such, a recombinant mouse noggin expressed in mouse NSO cells is likely to 25 exhibit a similar, if not identical, immunoreactivity profile to a recombinant human noggin expressed in mouse NSO cells. A quantitative immunoassay procedure developed "in-house" using components available from commercially available sources was used to detennine levels of noggin of the present 30 invention. Specifically, a rabbit polyclonal (PAb) anti- human noggin antibody (Santa Cruz Biotechnology, Inc.; Cat # FL-232) was used as a capture antibody; a goat anti-mouse noggin PAb tagged with biotin (R&D Systems; Cat # BAF719) was used as a detection WO 2007/028212 PCT/AU2006/001325 -293 antibody; and two different recombinant human and mouse noggins expressed in non human cells, for example, a recombinant human noggin expressed in E. coli (Peprotech; Cat # 120-10) and a recombinant mouse noggin expressed in mouse NSO cells (R&D Systems; Cat # 1967-NG) were used as protein standards. 5 Firstly, a saturating concentration of rabbit anti-human noggin binding against immobilized noggin of the present invention was determined by direct ELISA titration using methods known in the art. In this assay, the biotinylated goat anti-mouse noggin detection antibody was used at a concentration recommended by the manufacturer. The 10 saturating concentration of rabbit anti-human noggin thus determined was used for immobilization of capture antibody for use in the quantitative immunoassay. Secondly, protein concentrations of noggin of the present invention, standardised using the standard protein assay results, were assayed with the above-mentioned reagents using 15 ELISA methods known in the art. Alternatively, noggin of the present invention, standardised using the standard protein assay results, is diluted and tested in a suitable, commercially available noggin quantitative immunoassay procedure supplied with a non-human cell expressed noggin standard, for 20 example, an anti-noggin ELISA kit used in accordance with the manufacturer's instructions. The "in-house" ELISA kit results showed a markedly different concentration-OD curve of noggin of the present invention when compared with that of recombinant human noggin 25 expressed in E. coli and recombinant mouse noggin expressed in NSO cells (Figure 6). The results represent an underestimation of the noggin of the present invention concentration by the above-described ELISA assay, which employs human noggin expressed in E. coli cells and mouse noggin expressed in mouse NSO cells as standards and 30 antibodies against human noggin expressed in E. coli cells and mouse noggin expressed in mouse NSO cells (c.f. human noggin expressed in mouse NSO cells), suggesting inherent WO 2007/028212 PCT/AU2006/001325 - 294 differences in immunoreactivity between the noggin of the present invention and the noggin proteins expressed in non-human cell systems. EXAMPLE 14 5 Further Purification of Noggin and Peptide Mass Fingerprinting by ESI-MS/MS In addition to the purification protocol as described in Example 2, purification of the noggin of the present invention is further performed by RP-HPLC, using a commercially available column. Eluting proteins are monitored by the absorbance at 215 or 280 nm and 10 collected with correction being made for the delay due to tubing volume between the flow cell and the collection port. A gel piece containing the protein sample from a ID or 2D gel is digested in trypsin solution as described in Example 3. Alternatively, a solution containing the protein sample 15 is digested with trypsin in an ammonium bicarbonate buffer (10-25 mM, pH 7.5-9). The solution is incubated at 370 C overnight. The reaction is then stopped by adding acetic acid until the pH is in the range 4-5. The peptide samples are concentrated and desalted using C18 Zip-Tips (Millipore, Bedford, MA) or pre-fabricated micro-columns containing Poros R2 chromatography resin (Perspetive Biosystems, Framingham, MA) as described in 20 Example 3. The protein sample (2-5 .tl) is injected onto a micro C18 precolumn and washed with 0.1% formic acid at 30 pl/min to concentrate and desalt. After a 3 min wash the pre-column is switched into line with the analytical column containing C18 RP silica (Atlantis, 75tm x 25 100mm, Waters Corporation). Peptides are eluted from the column using a linear solvent gradient, with steps, from H 2 0:CH 3 CN (95:5; + 0.1% formic acid) to H 2 0:CH 3 CN (20:80, + 0.1% formic acid) at 200 nl/min over a 40 min period. The LC eluent is subject to positive ion nanoflow electrospray analysis on a Micromass QTOF Ultima mass spectrometer (Micromass, Manchester, UK). 30 Tandem MS is performed using a Q-Tof hybrid quadrupole / orthogonal-acceleration TOF mass spectrometer (Micromass). The QTOF is operated in a data dependent acquisition WO 2007/028212 PCT/AU2006/001325 -295 mode (DDA). A TOFMS survey scan is acquired (m/z 400-2000, 1.0s), with the three largest multiply charged ions (counts >15) in the survey scan sequentially subjected to MS/MS analysis. MS/MS spectra are accumulated for 8 s (m/z 50-2000). 5 The LC/MS/MS data are searched using Mascot (Matrix Science, London, UK) and Protein Lynx Global Server ("PLGS") (Micromass). The protein sample is anticipated to be human noggin. EXAMPLE 15 10 (a) Immunogenicity in non-human animals (i) Animal immunization with target protein 15 Separate groups of non-human animals, for example, mice are immunized either sub cutaneously, intramuscularly or intraperitoneally (IP) with 1-100ug of noggin of the present invention and rh noggin expressed in non-human cells, respectively. Animals receive a secondary immunization one month following immunization. Prior to immunization, protein is emulsified in an adjuvant, for example, complete Freud's 20 adjuvant for the primary immunization and incomplete Freud's adjuvant for the secondary immunization. (ii) Detection of antibodies directed to target protein 25 For the detection of antibody response, animals from each group are bled from the tail and sera pooled. Protein-specific antibodies are detected by a solid phase ELISA using 50ng/well of noggin of the present invention. Different immunoglobulin isotypes are detected by using labelled detection antibodies raised against IgG1, IgG2, IgG2b, IgG3, IgM, IgA, IgD. Alternatively, antibody response is measured against noggin of the present 30 invention blotted onto a membrane either as a dot blot or Western blot. Detection of different immunoglobulin isotypes are as described above. It is anticipated that the noggin WO 2007/028212 PCT/AU2006/001325 -296 of the present invention will elicit an antibody response that is distinct to that of rh noggin expressed in non-human cells. (iii) T cell proliferation assay 5 Immunised animals are euthanised and spleen cells prepared. A suitable number of spleen cells, for example, 5 x 105 cells, from animals immunized with noggin of the present invention are cultured with various concentrations of noggin of the present invention while and equivalent number of spleen cells from animals immunized with rh noggin expressed 10 in non-human cells are cultured with various concentrations of rh noggin expressed in non human cells. For T cell proliferation assays, spleen cells are cultured for 96 hours and treated with 1 micro Ci [ 3 H] thymidine (6-7 micro Ci/micromol) during the final 16 hours. The cells are harvested onto filter strips and [ 3 H] thymidine incorporation determined using standard methods. It is anticipated that the noggin of the present invention will elicit 15 a different proliferation response compared to rh noggin expressed in non-human cells. (iv) IFN gamma assay For the IFN gamma assay, culture supernatant from spleen cells incubated with either the 20 noggin of the present invention or rh noggin expressed in non-human cells are harvested at 96 hours and IFN gamma production is detected by a sandwich ELISA, for example, a R&D Systems anti-IFN gamma Quantikine* ELISA kit (Cat # DIF50) in accordance with the manufacturer's instructions. It is anticipated that IFN gamma production will be different in culture supernatant derived from cells incubated with noggin of the present 25 invention compared with culture supernatant derived from cells incubated with rh noggin expressed in non-human cells. (b) In vitro Human Immunogenicity assays 30 (i) Human T-Cell response assay Human dendritic cells and CD4+ T cells are prepared from human blood as described in Stickler et al. Toxicological Sciences 77:280-289, 2004. Co-cultures of dendritic cells and WO 2007/028212 PCT/AU2006/001325 -297 CD4* T cells are plated out in 96 well plates containing 2 x 104 dendritic cells and 2 x 105 CD4+ T cells. The noggin of the present invention and rh noggin expressed in non-human cells undergo enzymatic digestion into peptide fragments using a suitable enzyme determined by cleavage site prediction software, for example, Peptide Cutter 5 (http://au.expasy.org/tools/peptidecutter). The resulting peptide fragments are purified by a suitable technique, for example, liquid chromatography and added to the co-cultures to a final concentration of 5ug/ml. Cultures are incubated for 5 days and 0.5 uCi 3 H thymidine is then added to each culture. The cells are harvested onto filter strips and cell proliferation is determined by [ 3 H] thymidine incorporation. 10 It is anticipated that the peptides derived from noggin of the present invention will elicit a weaker proliferation response compared to peptides derived from the rh noggin expressed in non-human cells. 15 (ii) Human antibody response assay Human donors undergoing treatment with rh noggin expressed in non-human cells are bled and sera prepared. Human noggin-specific antibodies are detected by a solid phase ELISA against both 50ng/well of noggin of the present invention and rh noggin expressed in non 20 human cells. Different immunoglobulin isotypes are detected by using labelled detection antibodies raised against human IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD. Alternatively, antibody response is measured against noggin of the present invention and rh noggin expressed in non-human cells blotted onto a membrane either as a dot blot or 25 Western blot. Detection of different immunoglobulin isotypes are as described above. It is anticipated that the immunoglobulin present in the sera of people treated with rh noggin expressed in non-human cells will bind to rh noggin expressed in non-human cells while either binding weakly or not binding with noggin of the present invention. 30 WO 2007/028212 PCT/AU2006/001325 - 298 EXAMPLE 16 Comparison of Noggin and Noggin or chimeric Noggin expressed using non-hunan systems in the ability to maintain human embryonic stem cells in an undifferentiated phenotype 5 Human ES cells (hES), for example, HI cells (WiCell Research Institute, Madison, WI) are cultured in unconditioned medium (UM), for example, 80% DMEM/F 12, 20% KNOCKOUT serum replacement, supplemented with 1 mM L-glutamine and 1% non essential amino acids; (all form Invitrogen) on plates coated with a suitable preparation, for 10 example, Matrigel (BD Scientific, North Ryde, NSW, Australia) for 10-14 days. Alternate unconditioned media suitable for maintaining hES include pooled, heat-inactivated human serum selected for low titres of antibodies specific for N-glycolylneuraminic acid (NeuGc) (Martin et al. 2005 supra). Other media suitable for coating plates include human laminin or a combination of poly-L lysine and fibronectin. Human bFGF (40ng/ml; R&D Systems) 15 and noggin of the present invention (concentration range 50-1000ng/ml) is added to the culture medium. hES are dissociated with a suitable enzyme preparation, for example, trypsin/EDTA and resuspended in PBS with serum, for example, 10% goat serum for 15 minutes at 4"C. Cells 20 are then incubated for a suitable period, with an antibody directed against a marker of undifferentiated hES, for example, Tra-1-60 or Oct4, followed by a further incubation with a suitable secondary antibody tagged with a suitable fluorophore. Cells are analyzed by a flow cytometer and resulting data analysed with suitable analysis software. 25 hES treated with the combination of noggin of the present invention and human bFGF remains in an undifferentiated state as determined by the high percentage of cell surface markers, for example, Tra-1-60 or Oct4. The above culture assay is repeated using noggin or a chimeric noggin molecule expressed 30 in non-human cell systems, e.g. E. coli, yeast or CHO cells and the percentage of undifferentiated hES is found to be lower.
WO 2007/028212 PCT/AU2006/001325 - 299 EXAMPLE 17 Comparison of Noggin and Noggin or chimeric Noggin expressed using non-human systems in the ability to generate neural precursor cells from human embryonic stem 5 cells Human ES cells, for example, H7 or HES-2 cells are routinely maintained, for example, using the protocol of Example 16 (above) or, alternatively, cultured on feeder layer cells, for example, mouse embryo fibroblasts, using protocols known in the art. Differentiation 10 of hES is induced by a suitable technique known in the art, for example, by the simultaneous withdrawal of growth factors and the addition of 100mM all-trans retinoic acid (Roach et al. Experimental Cell Research 215:189-198, 1994). hES are subcultured by routine means, for example, the protocol used by Reubinoff et al. Nature Biotechnology 19:1134-1140,2001. 15 Noggin of the present invention (concentration range 50-10OOng/ml) is added to the culture medium at a suitable time and frequency following subculture, for example, at day one and is continued for 5-14 days. 20 The proportion of hES cultures converted to neural progenitors is assayed by first dissecting the colonies followed by culturing in a suitable neural progenitor medium, for example, that described by Reubinoff et al. 2001 supra, for a suitable period, for example, 1 week. The resulting embryoid bodies are treated using suitable methods, for example, that described by Reubinoff et al. 2001 supra, in which neurospheres are replated onto 25 laminin-coated culture dishes with neural progenitor medium lacking growth factors, allowed to attach and grow for 2 days and positively identified using an anti-nestin antibody in combination with standard immunohistochemical protocols. The proportion of embryoid bodies showing characteristics of neural progenitors, for 30 example, uniform positive immunoreactivity for nestin (Reubinoff et al. 2001 supra) treated with noggin of the present invention is significantly higher than in control cultures. The above culture assay is repeated using noggin or a chimeric noggin molecule expressed WO 2007/028212 PCT/AU2006/001325 - 300 in non-human cell systems, e.g. E. coli, yeast or CHO cells and the proportion of embryoid bodies showing characteristics of neural progenitors is found to be lower than that of cultures treated with noggin of the present invention. 5 EXAMPLE 18 Comparison of Noggin and Noggin or chimeric Noggin expressed using non-human systems in the ability to generate oligodendrocyte precursor cells from human embryonic stem cells 10 As described in Example 17, treatment of human embryonic stem cell (hES) cultures with noggin of the present invention is anticipated to yield a higher proportion of embryoid bodies showing characteristics of neural progenitors than that obtainable using noggin or a chimeric noggin molecule expressed in non-human cell systems, e.g. E. coli, yeast or CHO cells. 15 Embryoid bodies prepared using noggin of the present invention (as described in Example 17) are plated onto a suitably prepared surface, for example, dishes coated with fibronectin over polyornithine (Murray & Dubois-Dalcq, J Neurosci Res 50:146-156, 1997) then passaged and propagated with media containing (i) bFGF (10-40ng/ml), then (ii) bFGF 20 (10-40ng/ml) + EGF (10-40ng/ml), followed by bFGF (10-40ng/ml) + PDGF-AA (10-40 ng/ml) to promote the proliferation of glial precursor cells (Brtstle et al. Science 285:754 756, 1999). Cells are incubated at low densities in media containing noggin of the present invention (50-200 ng/ml) for a suitable period of time, for example, 10-14 days, to enhance the elaboration of oligodendrocytes (Mehler et al. Dev Neurosci 22:74-85, 2000). Cells 25 then undergo dissociation to single cells or cell clumps with a suitable enzyme preparation, for example, trypsin/EDTA and then resuspended in PBS with autologous human serum, for example, 10% human serum for 15 minutes at 4'C. Cells or cell clumps are then incubated for a suitable period, with an antibody directed against a marker of oligodendrocyte lineages, for example, 04 (Sommer & Schachner Dev Biol 83:311-327, 30 1981) followed by a further incubation with a suitable secondary antibody tagged with a suitable fluorophore. Cells are analyzed by a flow cytometer and resulting data analysed with suitable analysis software.
WO 2007/028212 PCT/AU2006/001325 - 301 The above culture assay is repeated using noggin or a chimeric noggin molecule expressed in non-human cell systems, e.g. E. coli, yeast or CHO cells. 5 Embryoid bodies treated with noggin of the present invention yield a higher population of 04+ cells than that exhibited by embryoid bodies treated with noggin or a chimeric noggin molecule expressed in non-human cell systems, e.g. E. coli, yeast or CHO cells. EXAMPLE 19 10 Comparison of Noggin and Noggin or chimeric Noggin expressed using non-human systems in the ability to generate cardiomyocytes from human embryonic stem cells Human ES cells, for example, HI, H7, H9.2, H14 or HES-2 cells are routinely maintained, for example, using the protocol of Example 16 (above) or, alternatively, cultured on feeder 15 layer cells, for example, mouse embryo fibroblasts, using protocols known in the art. Differentiation of hES cells is induced by suitable techniques known in the art, for example, by dissociation with a suitable enzyme preparation, for example, trypsin/EDTA, followed by preparation of suspension cultures for a suitable time period, for example 10 14 days. Recombinant human noggin (concentration range 50-250 ng/ml) is added to the 20 cultures at a suitable time and frequency in relation to the onset of embryonic body formation, for example, at 3 days prior to, and on the day of, embryonic body formation. At a suitable time point, for example, 10-14 days, total RNA is harvested, purified, and reverse transcribed (RT) according to standard protocols, for example, the use of an 25 RNeasy kit (Qiagen) followed by RT with the Improm-II system (Promega). Quantitative PCR is performed using a suitable mastermix, for example, the SYBR green Q-PCR mastermix (Stratagene) on a real time PCR system under appropriate conditions determined by persons skilled in the art. An endogenous reference transcript, for example, GAPDH, is used to calculate relative expression levels of relevant cardiac transcription 30 factor genes, for example, Nkx2.5, GATA4 and Tbx5, and cardiac-specific protein genes, for example, ac-MHC and p-MHC.
WO 2007/028212 PCT/AU2006/001325 - 302 Noggin or chimeric noggin molecule of the present invention will induce a unique gene expression pattern in mRNA profiles upon comparison with gene expression profiles induced by noggin or chimeric noggin molecule produced from different non-human sources e.g. E. coli, yeast, mouse or CHO cells, an expression pattern consistent with a 5 high incidence of cardiomyocytes. Cardiomyocytes may be used for therapeutic purposes, such as repairing myocardial infarction injury. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood 10 that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. 15 WO 2007/028212 PCT/AU2006/001325 - 303 BIBLIOGRAPHY Ackland et al. Chromatogr 540:187-198, 1991 Aloj et al. JBiolChein 247:1146-1151, 1971 Altschul et al. NuclAcids Res 25:389, 1997 Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988) Aronsson et al. FEBS Lett 411:359-364, 1997 Atherton and Shephard Synthetic Vaccines 9: Blackwell Scientific Publications Ausubel et al. In: Current Protocols in Molecular Biology John Wiley & Sons Inc. 1994 1998) Baneyx Current Opinion in Biotechnology 10:411-421, 1999 Bernstein Methods Mol Biol 237:195-204, 2004 Bird Science 242:423, 1988 Blenis and Resh Curr Opin Cell Biol 5(6):984-9, 1993 Bonner and Laskey Eur JBiochen 46:83, 1974 Brnstle et al. Science 285:754-756, 1999 Caprioli et al. Biochem Biophys Res Commun 146:291-299, 1987 Carr et al. Anal Biochem 175: 492-499, 1988 WO 2007/028212 PCT/AU2006/001325 - 304 Carr et al. Anal Chem 63:2802-2824, 1991 Carr et al. JBiol Chem 264(35):21286-21295, 1989 Clackson et al. Nature 352:624-628, 1991 Clarke Curr Opin Cell Biol 5:977 983, 1993 Datta et al. JBiolChem 270:1497-1500, 1995 Edman Mol Biol Biochem Biophys 8:211-55, 1970 Erickson et al. Science 249:527-533, 1990 Farruggia et al. Int JBiol Macromol 20:43-51, 1997 Figeys and Aebersold, Electrophoresis 19:885-892, 1998 Franks et al. Characterization ofproteins, Humana Press, Clifton, NJ, 1988 Fukuhara et al. JBiol Chem 260:10487-10494, 1985 Gelb et al. Curr Opin Chem Biol 2(1):40-8, 1998 Harrison and Packer Methods Mol Biol 125:211-216, 2000 Hearn et al. Methods in Enzymol 104:190-212, 1984 Herzberg et al. Infrared and Raman Spectra of Polyatomic Molecules, Van Nostrand Reinhold, New York, NY, 1945 Hodgson BiolTechnology 9:19-21, 1991 Hoffman et al. Nat Biotechnol 23(6):699-708, 2005 WO 2007/028212 PCT/AU2006/001325 -305 Holzwarth et al. JAm Chem Soc 178:350, 1965 Honroe et al. Biochem J258:99-204, 1989 Huston et al. Proc Natl Acad Sci USA 85:5879, 1988 JBiochem 336:647-658, 1998 JBiochem 363:619-631, 2002 James and Bottomley Arch Biochem Biophy 356:296-300, 1998 Jones et al. Nature 321:522-525, 1986 Kennet et al. Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, New York, 1980 Kivirikko et al. FASEB Journal 3:1609-1617, 1989 Kohler et al. Nature 256:495, 1975 Kortt et al. Protein Engineering 10:423, 1997 Krimm and Bandekar Adv protein chem 38:181-364, 1986 Kronman Gene 121:295-304, 1992 Kurochkin et al. JMol Biol 248:414-430, 1995 Kwon and Yu Biophim Biophys Acta 1335:265-272, 1997 Larrick et al. Bio/Technology 7:934, 1989 Li et al. Biochemistry 34:5762-5772, 1995 WO 2007/028212 PCT/AU2006/001325 -306 Liu et al. JImmunol 158:604-613, 1994 Lucka et al. Glycobiology 15(1):87, 2005 Marks et al. JMol Biol 222:581-597, 1991 Marmur and Doty JMol Biol 5:109, 1962 Martin et al. Nat Med 11(2):228-32, 2005 Mas et al. Glycobiology 8(6):605-13, 1998 McGettrick et al. Methods Mol Biol 244:151-7 2004 Mehler et al. Dev Neurosci 22:74-85, 2000 Mire-Sluis et al. J Immunol Methods 289(1-2):1-16, 2004 Moore JBiol Chem 278(27):24243-24246, 2003 Morrison et al. Proc Natl Acad Sci USA 81:6851-6855, 1984 Murray & Dubois-Dalcq, JNeurosci Res 50:146-156, 1997 Nguyen et al. J Chromatogr A. 705:21-45, 1995 Packer et al. Glycoconj J5(8):737-47, 1998 Phillies Anal Chem 62:1049A-1057A, 1990 Pikal et al. Pharm Res 8:427-436, 1991 Presta, Curr Op Struct Biol 2:593-596, 1992 WO 2007/028212 PCT/AU2006/001325 - 307 Rando Biochim Biophys Acta 1300(1):5-16, 1996 Reichmann et al. Nature 332:323-329, 1988 Reubinoff et al. Nature Biotechnology 19:1134-1140, 2001 Roach et al. Experimental Cell Research 215:189-198, 1994 Sambrook et al. Molecular Cloning - A Laboratory Manual, Cold Spring Harbour, New York, USA, 1990 Schmid et al. Protein structure, a practical approach, Creighton Ed., IRI Press, Oxford, England, 1989 Sommer & Schachner Dev Biol 83:311-327, 1981 Stickler et al. Toxicological Sciences 77:280-289, 2004 Triguero et al. J ofNeurochemistry 54:1882-1888 1990 Ward et al. Nature 334:544, 1989 Wells Methods Enzymol 202:2699-2705, 1991 Wilkinson Annu Rev Nutr 15:161-89, 1995 Winter and Harris TIPS 14:139, 1993 Yoshioka et al. Pharn Res 10:103-108, 1993

Claims (20)

1. An isolated protein comprising a profile of measurable physiochemical parameters, wherein said profile is indicative of, associated with or forms the basis of one or more distinctive pharmacological traits.
2. The isolated protein of Claim 1, wherein said isolated protein comprises a physiochemical profile comprising a number of measurable physiochemical parameters, {[Pxi, [Px] 2 ,... [Pxa, }, wherein P, represents a measurable physiochemical parameter and "n" is an integer >1, wherein each of [Px] 1 to [P]n is a different measurable physiochemical parameter, wherein the value of any one of the measurable physiochemical characteristics or an array of values of more than one measurable physiochemical characteristics is indicative of, associated with, or forms the basis of, a distinctive pharmacological trait, Ty, or an array of distinctive physiochemical traits {[Ty]1, [Ty] 2 , .... [Ty]m} wherein Ty represents a distinctive pharmacological trait and m is an integer 1 and each of [Ty] 1 to [Ty]m is a different pharmacological trait.
3. The isolated protein of Claim 1 or 2 wherein the isolated protein is noggin.
4. The isolated protein of Claim 3, wherein said protein comprises the following measurable physiochemical parameters set forth in Table 2.
5. The isolated protein of Claim 3 or 4 wherein said protein comprises the distinctive pharmacological trait or traits set forth in Table 3.
6. A chimeric molecule comprising the isolated protein of any one of Claims 1 to 5, or fragment thereof, fused to one or more peptide, polypeptide or protein moieties.
7. The chimeric molecule of Claim 6 wherein the peptide, polypeptide or protein moiety comprises the constant (Fc) or framework region of human immunoglobulin. WO 2007/028212 PCT/AU2006/001325 - 309
8. The chimeric molecule of Claim 6 or 7 wherein the chimeric molecule is noggin Fc.
9. A pharmaceutical composition comprising the isolated protein or chimeric molecule of any one of Claims I to 8.
10. A method of treating or preventing a condition in a mammalian subject, wherein said condition can be ameliorated by increasing the amount or activity of a protein, said method comprising administering to said mammalian subject an effective amount of an isolated protein according to any one of Claims I to 5, a chimeric molecule according to any one of Claims 6 to 8 or the pharmaceutical composition of Claim 9.
11. The method of Claim 10 wherein the isolated protein is the isolated protein of Claim 5.
12. The method of Claim 10 wherein the chimeric molecule is the chimeric molecule of Claim 8.
13. A nucleotide sequence selected from the list consisting of SEQ ID NOs: 23, 25, 27, 29, 31, or a nucleotide sequence having at least about 90% identity to any one of the above-listed sequence or a nucleotide sequence capable of hybridizing to any one of the above sequences or their complementary forms under low stringency conditions.
14. An isolated noggin or chimeric noggin molecule encoded by a nucleotide sequence selected from the list consisting of SEQ ID NOs: 23, 25, 27, 29, 31, or a nucleotide sequence having at least about 90% identity to any one of the above-listed sequence or a nucleotide sequence capable of hybridizing to any one of the above sequences or their complementary forms under high stringency conditions.
15. An isolated nucleic acid molecule encoding noggin or chimeric noggin molecule or a functional part thereof comprising a sequence of nucleotides having at least 90% similarity SEQ ID NOs: 23, 25, 27, 29, 31 or after optimal alignment and/or being capable WO 2007/028212 PCT/AU2006/001325 -310 of hybridizing to one or more of SEQ ID NOs: 23, 25, 27, 29, 31 or their complementary forms under high stringency conditions.
16. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding a noggin or chimeric noggin molecule having an amino acid sequence substantially as set forth in one or more of SEQ ID NOs: 24, 26, 28, 30, 32, or an amino acid sequence having at least about 90% similarity to one or more of SEQ ID NOs: 24, 26, 28, 30, 32, after optimal alignment.
17. A kit for determining the level of human cell expressed human noggin or chimeric noggin present in a biological preparation, said kit comprising (a) a solid phase support matrix; (b) one or more antibodies directed against human noggin or chimeric noggin molecule; (c) a blocking solution; (d) one or more stock solutions of substrate; (e) a solution of substrate buffer; (f) a standard human noggin sample; and (g) instructions for use.
18. The kit of Claim 17, wherein the standard human noggin sample is a preparation of the isolated protein of claim 5 or the chimeric molecule of claim 8.
19. The kit of Claim 17 or 18, wherein the or each antibody is derived from an immunization of a mammal with a preparation comprising the isolated protein of claim 5 or the chimeric molecule of claim 8.
20. The kit of any of Claims 17 to 19, wherein the human cell expressed human noggin is naturally occurring human noggin.
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