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Enzyme-Replacement Therapy in Lysosomal Storage

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Enzyme-Replacement Therapy in Lysosomal Storage ENZYME.REPLACEMENT THERAPY IN LYSOSOMAL STORAGE DISEASES Julie Bielicki BSc Thesis submitted for the degree of Doctor of Philosophy 1n The University of Adelaide (F aculty of Medicine) Lysosomal Diseases Research Unit Department of Chemical PathologY 'Women's and Children's Hospital South Australia and Department of Paediatrics Faculty of Medicine University of Adelaide South Australia 2003 TABLE OF CONTENTS ABBREVIATIONS x THESIS ABSTRACT xv DECLARATION XViii ACKNO\MLEDGEMENTS xix Chanter 1: Introduction and review 1.1 Preface 1 1.2 Lysosomes 2 1.3 Lysosomal biogenesis J 1.4 Lysosomal enzymes 5 1.5 M6PR-mediated transport of lysosomal enzymes 6 1.6 M6PR-independent transport of lysosomal enzymes 8 1.7 Glycosaminoglycans and proteoglycans 8 1.7.1 Structure and function 8 1.7.2 Biosynthesis 11 1.7.3 Degradation t2 1.8 Glycoproteins T4 1.8.1 Structure and function I4 1.8.2 Biosynthesis 15 1.8.3 Degradation 15 1.9 Lysosomal storage diseases 18 1.10 The mucopolysaccharidoses t9 1.11 The oligosaccharidoses 2l 1.12 Human MPS VI 22 Ll2.l Historical 22 1'12.2 Genetics 22 1.12.3 Clinical 23 1.I2.4 Biochemistry 24 1.12.4.I Storage and PathologY 24 1.12.4.2 Enzymology 25 1.12.5 Diagnosis 25 1l 1.1,2.5.1 Urine analysis 26 1.12.5.2 45 activity 26 1.13 Bone synthesis and growth 27 1.13.1 Synthesis 27 1l3 .2 Elongation/growth 28 1.14 Human fucosidosis 29 1.14.1 Historical 29 |.14.2 Genetics 29 1.14.3 Clinical 30 1.14.4 Pathology 30 1.14.5 Diagnosis 31 1.15 Pathogenesis of LSD 31 1.16 Therapies for LSD JJ 1.16.1 Medical and surgical JJ 1.16.2 Organ and tissue transplantation 34 1. 1 6.3 Bone marrow transplantation 34 l .l 6.4 Enzyme-replac ement therapy 36 1.16.5 Somatic cell gene therapy 37 l. 1 6.6 Substrate deprivation therapy 39 116.7 Neural and embryonic stem cell therapy 40 1.17 Animal models of LSD 4t 1.18 Feline MPS VI 43 1.19 Therapies for feline MPS vI and other animal models of LSD 45 1 .19.1 Bone marrow transplantation 45 I .19 .2 Enzyrne-replac ement therapy 45 1.20 Canine fucosidosis 47 1.21 Therapies for canine fucosidosis and other animal models of LSD 52 1.22 The blood-brain b arrier 54 1.23 Mechanisms for transport into the brain 57 1.24 Transport of conjugates and modified proteins into the brain 6l 1 .24.1 Via adsorptive-mediated transcytosis 6l 1 .24.2 Vi a r eceptor-mediated transcytosis 63 1.25 Aims of the project 67 l1l Chapte r 2z Materials and methods 2.1Materials 68 2.1.I Tissue culture 68 2.1.2 Antibiotics 68 2.1.3 Cell lines 68 2.1.4 Gel chromatography media 69 2.1.5 Heterobifunctional crosslinkers 69 2.1.6 Enz¡rmes 69 2.1.7 Antibodies 69 2.1.8 Radiochemicals 70 2.1.9 Substrates for enzyrne reactions 70 2.I.rc Electrophoresis reagents 70 2.l.ll Animal colonies 7I 2.1.12 Buffers and solutions 7l 2.I.13 Chemicals 72 2.7 .1 4 Miscellaneous materi als 75 2.2 Methods 76 2.2.1 Tissue culture 76 2.2.1.1CHO-KI cells 76 2.2.1.2 MDCK cells 76 2.2.1.3 Human skin fibroblasts 76 2.2.1.4 Feline skin fibroblasts 77 2.2.1.5 H4IIE rat hepatoma cells 77 2.2.2 Large-scale production of rfilS for clinical trial 77 2.2.3 Purification of r?tS by immunoaffinity chromatography 78 2.2.4 Coupling of F58.3 monoclonal antibody to Affi-Gel 10 78 2.2.5 Enzymeassays 79 2.2.5.1Assay of 45 using 4MUS substrate 79 2.2.5.2 Assay of 45 using trisaccharide substrate 79 2.2.5.3 Assay of FUC using 4MUF 80 2.2.5.4 Assay of p-hexosaminidase 80 1V 2.2.6 Enzyme characterisation 80 2.2.6.1 Determination of native molecular mass 80 2.2.6.2 Determination of subunit molecular mass 81 2.2.6.3 Determination of degree of glycosylation 81 2.2.6.4 Determination of kinetic parameters 81 2.2.6.5 pI determination 82 2.2.7 Correction of storage in feline MPS vI skin fibroblasts with rfilS 83 2.2.8 Experimental animals for feline MPS VI ERT trial 84 2.2.9 ERT protocol for MPS VI cats 84 2.2.10 Determination of antibody titre in MPS VI cats undergoing ERT using Elisa 86 2.2.1I Western blot analysis of plasma from rf,lS-treated MPS VI cats 86 2.2.lzlmmunobinding assay for 45 87 2.2.13 Sandwich Elisa of monoclonal antibody binding to 45 88 2.2.14 Urinary GAG analysis 88 2.2.I4.1Alcian Blue spectrophotometric detection of total GAG 88 2.2. | 4.2 High-resolution electrophoresis of GAG 89 2.2.14.3 Uronic acid quantification of GAG 89 2.2.14.4 Gradient-PAGE analysis of GAG 90 2.2.15 Post mortem examination of MPS VI cats on ERT 90 2.2.16 Bone histomorphometric analysis of MPS VI cats on ERT 9l 2.2.I7 Residual45 activity in MPS VI cat liver 92 2.2.18 Assessment of hepatomegaly in treated MPS VI cats 92 3H-rf4S 2.2.19 Distribution of in kittens 92 2.2.20 Construction of cFUC expression vector 93 2.2.21Transfection of cells with pEFNeocFUC 94 2.2.21. I Electroporation 94 2.2.2t.2 DOTAP 94 2.2.22 Large-scale production of rcFUC 94 2.2.23 Purification of rcFUC by affinity chromatography 95 2.2.24 Purification of rat liver FUC 95 2.2.25 Correction of storage in human fucosidosis cells with rcFUC 96 2.2.26 Radio I ab elling o f recombinant enzyrnes 96 2.2.27 Growth of OX26 hybridoma cells 97 V 2.2.28 Purification of OX26 97 2.2.29 Production of spleen feeder cells 97 2.2.30 Production of monoclonal antibodies to rcFUC 98 2.2.3I Production of rabbit anti-rcFUC antibody 99 2.2.32 Immunohistochemical analysis of cFUC expression 100 2.2.33 Preparation of insulin peptide fragment, F007 100 2.2.34 Pyridyldithiopropionate modification of insulin and rcFUC 101 2.2.35 Quantification of pyridyldithiopropionate modification of insulin and rcFUC 101 2.2.36 Conjugation of insulin to rcFUC r02 2.2.37 Thiolation of rcFUC with SATA 102 2.2.38 Quantification of modification of rcFUC with SATA t02 2.2.39 Conjugation of SATA-defüvatized rcFUC with SPDP-denvatized insulin 103 2.2.40 Pyridyldithiopropionate modification of OX26 through amine gloups 103 2.2.4I Pyridyldithiopropionate modification of OX26 through carbohydrate groups 103 2.2.42 Conjugation of rcFUC to OX26 104 2.2.43 FACS analysis r04 2.2.44 Binding specificity of OX26 andOX26-FUC conjugates to Tf receptors on H4IIE cells 105 2.2.45 Cationization of rcFUC with putrescine 105 2.2.46 In vivo distribution studies and plasma half-life of rcFUC in rats 106 2.2.47 Capillary depletion r07 2.2.48 Europium-labelling of OX26 108 2.2.4g Rat tissue distribution of OX26-Eu*3 109 2.2.50Isolation and enzyme assays of canine blood leucocytes 109 2.2.51Isolation of canine fucosidosis brain storage products 110 2.2.52 Thin layer chromatography of fucosidosis dog brain products 111 2.2.53 Tissue distribution of rcFUC in fucosidosis dogs 111 2.2.54 Long-term ERT in a fucosidosis dog rt2 2.2.55 Determination of antibody titre in a fucosidosis dog on long-term ERT 113 2.2.56 Urinary analysis of L-fucose 113 V1 Chanter 3: Muconolvsaccharidosis tvne VI 3.1 Purification and characterisation of rf4S tl4 3.1 Introduction tt4 3.1 Results 115 3.1.1 Large-scale production of rf4S 115 3.1.2 Purification of rf?lS 116 3.1.3 Characterisation of r?lS tt7 3.1.3.1 Subunit Mr tt7 3.1.3.2 Native Mr 119 3.1.3.3 Kinetics t2l 3.7.3.4 Isoelectric focussing t22 3.1.3.5 Estimation of degree of glycosylation t22 3.1.4 Conection of GAG storage in feline MPS VI fibroblasts with rfztS 125 3.1 Discussion t27 3.2 ERT in MPS VI cats using rf4S 130 3.2Introduction 130 3.2 Results 130 3.2.1 Disease assessment in MPS VI cats undergoing ERT 130 3.2.1.1 Physical examination 130 3 .2.I .2 Neurological examination t34 3.2.1 .3 Radiological examination t34 3.2.2 Macroscopic pathology 136 3.2.3 Light and electron microscopy 138 3.2.4 Bone histomorphometry t43 3.2.5 lJnnary GAG analysis t46 3.2.5.1 Alcian Blue method r46 3.2.5.2 High-resolution electrophoresis 148 3.2.5.3 Gradient-PAGE 151 3.2.6 Antlbody response 156 3.2.7 Residual enzyrne activity in feline liver 156 v11 3.2.8 Assessment of hepatomegaly in MPS VI cats t57 3H-rf4S 3.2.9 Tissue distribution of in normal cats 160 3.2.I0 Plasma circulating half-life of r?lS in normal cats t62 3.2 Discussion r63 3.2 Conclusions r67 Chapter 4: Fucosidoiis 4.1 FUC: expression, purification and characterisation 170 4.1 Introduction 170 4.1 Results 771 4.1.1 Expression of rcFUC t7r 4.1.2 Large-scale production of rcFUC 172 4.1.3 Purification of rcFUC 173 4.1.4 Characterisation of rcFUC t76 4.1.4.1Subunit Mr 776 4.1.4.2 Native Mr t77 4.1.4.3 Kinetics 179 4.1.4.4rBF 183 4.1.4.5 Degree of glycosylation 183 4.1.5 Conection of fucosidosis SF by CHO/roFUC and MDCK/rcFUC r87 4.1.6 Treatment of dog brain products with rcFUC 187 4.1 Discussion 189 4.2 Chemical modification of rcFUC 194 4.2.I Preparation of insulin fragment F007 194 4.2.2 Pyridyldithiopropionate modification of insulin and rcFUC r97 4.2.3 Thiolation of rcFUC with SATA 201 4 .2.4 Conju gation o f S ATA-d eriv alized rcFUC with SPDP - derivatized insulin 20r 4.2.5 SPDP modification of OX26 through amine and carbohydrate groups 202 4.2.
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