US20070081984A1 - Compositions and methods for treating hypophosphatasia - Google Patents
Compositions and methods for treating hypophosphatasia Download PDFInfo
- Publication number
- US20070081984A1 US20070081984A1 US11/484,870 US48487006A US2007081984A1 US 20070081984 A1 US20070081984 A1 US 20070081984A1 US 48487006 A US48487006 A US 48487006A US 2007081984 A1 US2007081984 A1 US 2007081984A1
- Authority
- US
- United States
- Prior art keywords
- enzyme
- tnsalp
- enzymes
- anchorless
- rhtnsalp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 21
- 206010049933 Hypophosphatasia Diseases 0.000 title abstract description 38
- 238000000034 method Methods 0.000 title abstract description 18
- 150000001413 amino acids Chemical class 0.000 claims abstract description 7
- 239000002253 acid Substances 0.000 claims abstract 2
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 28
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 27
- 230000002378 acidificating effect Effects 0.000 claims description 17
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 230000002950 deficient Effects 0.000 claims description 14
- 235000018102 proteins Nutrition 0.000 claims description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 10
- 238000000338 in vitro Methods 0.000 claims description 9
- 201000010099 disease Diseases 0.000 claims description 7
- 238000001727 in vivo Methods 0.000 claims description 6
- 235000001014 amino acid Nutrition 0.000 claims description 5
- 235000003704 aspartic acid Nutrition 0.000 claims description 4
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 150000007523 nucleic acids Chemical group 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 claims 1
- 102000039446 nucleic acids Human genes 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 183
- 108090000790 Enzymes Proteins 0.000 abstract description 183
- 102100025683 Alkaline phosphatase, tissue-nonspecific isozyme Human genes 0.000 abstract description 65
- 238000002641 enzyme replacement therapy Methods 0.000 abstract description 43
- 229930004094 glycosylphosphatidylinositol Natural products 0.000 abstract description 33
- 239000012528 membrane Substances 0.000 abstract description 23
- 210000000988 bone and bone Anatomy 0.000 abstract description 19
- 238000011282 treatment Methods 0.000 abstract description 13
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 8
- 101710161969 Alkaline phosphatase, tissue-nonspecific isozyme Proteins 0.000 abstract description 2
- 101000574445 Homo sapiens Alkaline phosphatase, tissue-nonspecific isozyme Proteins 0.000 description 77
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 45
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 45
- 210000004027 cell Anatomy 0.000 description 28
- 230000000694 effects Effects 0.000 description 24
- 241000699670 Mus sp. Species 0.000 description 21
- 230000033558 biomineral tissue development Effects 0.000 description 21
- 210000001519 tissue Anatomy 0.000 description 17
- 238000001802 infusion Methods 0.000 description 15
- 102000045328 human ALPL Human genes 0.000 description 14
- 108010076504 Protein Sorting Signals Proteins 0.000 description 13
- 239000002609 medium Substances 0.000 description 13
- 108010038807 Oligopeptides Proteins 0.000 description 12
- 102000015636 Oligopeptides Human genes 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 238000001042 affinity chromatography Methods 0.000 description 12
- 239000002299 complementary DNA Substances 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 102000005348 Neuraminidase Human genes 0.000 description 11
- 108010006232 Neuraminidase Proteins 0.000 description 11
- DHCLVCXQIBBOPH-UHFFFAOYSA-N Glycerol 2-phosphate Chemical compound OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 10
- 238000004873 anchoring Methods 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 10
- 239000007983 Tris buffer Substances 0.000 description 8
- 210000000170 cell membrane Anatomy 0.000 description 8
- 230000007812 deficiency Effects 0.000 description 8
- 210000004185 liver Anatomy 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 8
- 210000003462 vein Anatomy 0.000 description 8
- 108090001090 Lectins Proteins 0.000 description 7
- 102000004856 Lectins Human genes 0.000 description 7
- 229960005261 aspartic acid Drugs 0.000 description 7
- 210000002798 bone marrow cell Anatomy 0.000 description 7
- 239000013078 crystal Substances 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 239000002523 lectin Substances 0.000 description 7
- 239000002243 precursor Substances 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 6
- 238000009826 distribution Methods 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 230000000717 retained effect Effects 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 6
- 206010010904 Convulsion Diseases 0.000 description 5
- 241000699802 Cricetulus griseus Species 0.000 description 5
- 239000012506 Sephacryl® Substances 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 230000018678 bone mineralization Effects 0.000 description 5
- 150000001720 carbohydrates Chemical class 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 229910052500 inorganic mineral Inorganic materials 0.000 description 5
- 235000010755 mineral Nutrition 0.000 description 5
- 239000011707 mineral Substances 0.000 description 5
- 125000005629 sialic acid group Chemical group 0.000 description 5
- 230000003442 weekly effect Effects 0.000 description 5
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 4
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 4
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 210000004349 growth plate Anatomy 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 229920001542 oligosaccharide Polymers 0.000 description 4
- 201000003045 paroxysmal nocturnal hemoglobinuria Diseases 0.000 description 4
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 4
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 4
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 4
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 208000015439 Lysosomal storage disease Diseases 0.000 description 3
- 108010031099 Mannose Receptor Proteins 0.000 description 3
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 3
- SUHOOTKUPISOBE-UHFFFAOYSA-N O-phosphoethanolamine Chemical compound NCCOP(O)(O)=O SUHOOTKUPISOBE-UHFFFAOYSA-N 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 230000002411 adverse Effects 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 description 3
- 235000011010 calcium phosphates Nutrition 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 3
- 235000011180 diphosphates Nutrition 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 238000011813 knockout mouse model Methods 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- 210000000963 osteoblast Anatomy 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 208000007442 rickets Diseases 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000010474 transient expression Effects 0.000 description 3
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 3
- 210000004291 uterus Anatomy 0.000 description 3
- 239000011592 zinc chloride Substances 0.000 description 3
- 235000005074 zinc chloride Nutrition 0.000 description 3
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 2
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 2
- ZAINTDRBUHCDPZ-UHFFFAOYSA-M Alexa Fluor 546 Chemical compound [H+].[Na+].CC1CC(C)(C)NC(C(=C2OC3=C(C4=NC(C)(C)CC(C)C4=CC3=3)S([O-])(=O)=O)S([O-])(=O)=O)=C1C=C2C=3C(C(=C(Cl)C=1Cl)C(O)=O)=C(Cl)C=1SCC(=O)NCCCCCC(=O)ON1C(=O)CCC1=O ZAINTDRBUHCDPZ-UHFFFAOYSA-M 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 208000004434 Calcinosis Diseases 0.000 description 2
- 229920002271 DEAE-Sepharose Polymers 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 208000015872 Gaucher disease Diseases 0.000 description 2
- 102000009617 Inorganic Pyrophosphatase Human genes 0.000 description 2
- 108010009595 Inorganic Pyrophosphatase Proteins 0.000 description 2
- 108010044467 Isoenzymes Proteins 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 2
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 229920000805 Polyaspartic acid Polymers 0.000 description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- 208000008784 apnea Diseases 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000005221 enamel hypoplasia Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 208000005368 osteomalacia Diseases 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000009984 peri-natal effect Effects 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 108010064470 polyaspartate Proteins 0.000 description 2
- 230000001323 posttranslational effect Effects 0.000 description 2
- 230000002028 premature Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 229960002385 streptomycin sulfate Drugs 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000002626 targeted therapy Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 235000019158 vitamin B6 Nutrition 0.000 description 2
- 239000011726 vitamin B6 Substances 0.000 description 2
- 229940011671 vitamin b6 Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- JKYKXTRKURYNGW-UHFFFAOYSA-N 3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C(O)=C(O)C(S(O)(=O)=O)=C2 JKYKXTRKURYNGW-UHFFFAOYSA-N 0.000 description 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-M 4-nitrophenolate Chemical compound [O-]C1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-M 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 208000002109 Argyria Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 244000045232 Canavalia ensiformis Species 0.000 description 1
- 235000010520 Canavalia ensiformis Nutrition 0.000 description 1
- 102100037182 Cation-independent mannose-6-phosphate receptor Human genes 0.000 description 1
- 101710145225 Cation-independent mannose-6-phosphate receptor Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108010062580 Concanavalin A Proteins 0.000 description 1
- 208000032170 Congenital Abnormalities Diseases 0.000 description 1
- 208000009283 Craniosynostoses Diseases 0.000 description 1
- 206010049889 Craniosynostosis Diseases 0.000 description 1
- 208000012239 Developmental disease Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 102000002702 GPI-Linked Proteins Human genes 0.000 description 1
- 108010043685 GPI-Linked Proteins Proteins 0.000 description 1
- 208000037147 Hypercalcaemia Diseases 0.000 description 1
- 206010020590 Hypercalciuria Diseases 0.000 description 1
- 108010031792 IGF Type 2 Receptor Proteins 0.000 description 1
- 208000015580 Increased body weight Diseases 0.000 description 1
- 201000008114 Infantile hypophosphatasia Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- QUOGESRFPZDMMT-UHFFFAOYSA-N L-Homoarginine Natural products OC(=O)C(N)CCCCNC(N)=N QUOGESRFPZDMMT-UHFFFAOYSA-N 0.000 description 1
- 125000000570 L-alpha-aspartyl group Chemical group [H]OC(=O)C([H])([H])[C@]([H])(N([H])[H])C(*)=O 0.000 description 1
- QUOGESRFPZDMMT-YFKPBYRVSA-N L-homoarginine Chemical compound OC(=O)[C@@H](N)CCCCNC(N)=N QUOGESRFPZDMMT-YFKPBYRVSA-N 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 102000019218 Mannose-6-phosphate receptors Human genes 0.000 description 1
- 208000030136 Marchiafava-Bignami Disease Diseases 0.000 description 1
- 208000029725 Metabolic bone disease Diseases 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 208000001697 Odontohypophosphatasia Diseases 0.000 description 1
- 241001524178 Paenarthrobacter ureafaciens Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010034464 Periarthritis Diseases 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 208000024777 Prion disease Diseases 0.000 description 1
- 206010037407 Pulmonary hypoplasia Diseases 0.000 description 1
- 101800001295 Putative ATP-dependent helicase Proteins 0.000 description 1
- 101800001006 Putative helicase Proteins 0.000 description 1
- 102000009609 Pyrophosphatases Human genes 0.000 description 1
- 108010009413 Pyrophosphatases Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 208000004756 Respiratory Insufficiency Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 208000013201 Stress fracture Diseases 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 108010046516 Wheat Germ Agglutinins Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 108010011081 adenosine monophosphatase Proteins 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 201000008101 adult hypophosphatasia Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 230000002308 calcification Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 201000008113 childhood hypophosphatasia Diseases 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 208000030499 combat disease Diseases 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000004489 deciduous teeth Anatomy 0.000 description 1
- 238000011257 definitive treatment Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000005115 demineralization Methods 0.000 description 1
- 230000002328 demineralizing effect Effects 0.000 description 1
- 210000004513 dentition Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- OLSDWRNWUGHKSY-UHFFFAOYSA-J dicalcium;phosphonato phosphate;dihydrate Chemical compound O.O.[Ca+2].[Ca+2].[O-]P([O-])(=O)OP([O-])([O-])=O OLSDWRNWUGHKSY-UHFFFAOYSA-J 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000000483 effect on mineralization Effects 0.000 description 1
- 230000001037 epileptic effect Effects 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 230000000148 hypercalcaemia Effects 0.000 description 1
- 208000030915 hypercalcemia disease Diseases 0.000 description 1
- 208000016245 inborn errors of metabolism Diseases 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 208000015978 inherited metabolic disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- 230000037041 intracellular level Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 210000002414 leg Anatomy 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000001872 metatarsal bone Anatomy 0.000 description 1
- HOVAGTYPODGVJG-VEIUFWFVSA-N methyl alpha-D-mannoside Chemical compound CO[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]1O HOVAGTYPODGVJG-VEIUFWFVSA-N 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 1
- 150000004712 monophosphates Chemical class 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 239000006225 natural substrate Substances 0.000 description 1
- 201000000173 nephrocalcinosis Diseases 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000000505 pernicious effect Effects 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 230000009596 postnatal growth Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- FCHXJFJNDJXENQ-UHFFFAOYSA-N pyridoxal hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(C=O)=C1O FCHXJFJNDJXENQ-UHFFFAOYSA-N 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 201000004193 respiratory failure Diseases 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 208000002254 stillbirth Diseases 0.000 description 1
- 231100000537 stillbirth Toxicity 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 230000036346 tooth eruption Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03001—Alkaline phosphatase (3.1.3.1)
Definitions
- This invention relates generally to compositions and methods of enzyme replacement therapy (ERT). More specifically, the invention is directed to compositions and methods for treatment of enzyme deficient disease such as hypophosphatasia using a genetically modified polynucleotide to produce in an active secretory form of alkaline phosphatase.
- ERT enzyme replacement therapy
- Alkaline phosphatase is a ubiquitous plasma membrane-bound enzyme.
- hypophosphatasia is an inherited metabolic disorder of defective bone mineralization caused by deficiency of a form of ALP know as tissue-nonspecific alkaline phosphatase (TNSALP). Clinical severity is remarkably variable, ranging from death in utero to merely premature loss of dentition in adult life [1, 2]. Despite the presence of TNSALP in bone, kidney, liver, and adrenal tissue in healthy individuals, clinical manifestations in patients with hypophosphatasia are limited to defective skeletal mineralization that manifests as rickets in infants and children and osteomalacia in adults [2]. In the most pernicious form of hypophosphatasia, the perinatal lethal variant, profound skeletal hypomineralization results in caput membranaceum with shortened and deformed limbs noted. Some affected neonates survive for several days or weeks. They often succumb to respiratory failure brought on by pulmonary hypoplasia and structural failure of the weakened skeleton from demineralization [3].
- TNSALP tissue-nonspecific alkaline phosphatase
- Osteoblasts modulate the composition of the bone matrix, where they deposit mineral in the form of hydroxyapatite.
- Specialized buds from the osteoblasts' plasma membrane are called matrix vesicles (MVs).
- MVs matrix vesicles
- the initiation of matrix calcification by osteoblasts and chondrocytes appears to be mediated by release of MVs, which serve as a sheltered environment for hydroxyapatite crystal formation [4-7].
- MVs are alkaline phosphatase enriched, extracellular, membrane-invested bodies. Inside MVs the first crystals of hydroxyapatite bone mineral are generated.
- TNSALP hydrolyzes inorganic pyrophosphate (PP i ) to monophosphate (inorganic phosphate; P i ), which is important for growth of the hydroxyapatite crystal [4, 5, 8-10].
- ALP functions as an inorganic pyrophosphatase (PP i -ase) [14, 15].
- PP i itself impairs the growth of hydroxyapatite crystals as an inhibitor of mineralization [8, 11-13].
- Insufficient TNSALP activity fails to hydrolyze PP i and the resulting build-up of unhydrolyzed PP i in the perivesicular matrix inhibits the proliferation of pre-formed hydroxyapatite crystals beyond the protective confines of MV membranes.
- PP i The level of plasma PP i increases in hypophosphatasia [16-18].
- the other phosphatases AMPase and inorganic pyrophosphatase
- AMPase and inorganic pyrophosphatase can hydrolyze PP i , supplying P i for incorporation into initial mineral within MVs [19] but still be insufficient to remove excess PP i at the perimeter of MVs.
- initial mineral could form within MVs, while its propagation into perivesicular matrix would be inhibited by a local build-up of PP i [20, 21].
- Enzyme replacement therapy has proven effective in preventing or reversing lysosomal storage in patients and animal models with lysosomal storage diseases (LSDs) [22-28].
- LSDs lysosomal storage diseases
- Tremendous progress in the development of ERT has been made in the last three decades.
- Cellular uptake of enzyme from the blood following intravenous administration requires specific oligosaccharides on the enzyme itself corresponding to oligosaccharide receptors on the target cells. Examples include the binding of high-mannose oligosaccharides of the enzyme to the mannose receptor (MR) and binding of phosphorylated high-mannose oligosaccharides of the enzyme to the cation-independent mannose 6-phosphate receptor (M6PR).
- MR mannose receptor
- M6PR cation-independent mannose 6-phosphate receptor
- the cell-specific delivery system was also designed to enhance the clinical effectiveness of ERT.
- delivery of the enzyme to the affected cells was achieved by modifying the N-linked carbohydrate on the enzyme.
- hypophosphatasia caused by a deficiency of TNSALP seems to be a difficult disorder treated by ERT because TNSALP is a membrane-bound enzyme and is believed to require attachment at the cell surface to be functional.
- TNSALP is a membrane-bound enzyme and is believed to require attachment at the cell surface to be functional.
- the results of multiple intravenous infusions of plasma ALP or purified liver ALP in patients with hypophosphatasia have been disappointing [34-38].
- Administration of exogenous pyridoxal HCl delayed the onset of epileptic attacks and increased the life span of TNSALP ⁇ / ⁇ mice. Although the oldest survivor was 22 days old, all the homozygotes, however, died near weaning time, irrespective of their treatment regime [39].
- the inventors have genetically engineered a Chinese Hamster Ovarian (CHO) cell line to produce a C-terminus-anchorless TNSALP enzyme, in secreted form, [40] and showed clinical effectiveness of ERT on hypophosphatasia mice. These results indicate that the C-terminus-anchorless membrane enzyme possesses the characteristics necessary for use in ERT where the membrane-binding form is ineffective. Deletion of the C-terminus membrane anchor will be applicable to other membrane-binding proteins whose deficiency leads to other human disorders including but not limited to paroxysmal nocturnal haemoglobinuria (PNH).
- PNH paroxysmal nocturnal haemoglobinuria
- Targeted therapies have the advantage of reducing adverse effects on non-target organs as well as reducing the minimum effective systemic dose.
- Kasugai et al [41] has demonstrated that a small peptide consisting of a stretch of acidic amino acids (L-Aspartic acid or L-Glutamic acid) was selectively delivered to and retained in bone after a systemic administration.
- a small molecule, an estrogen, conjugated with an acidic-oligopeptide has been selectively targeted to bone, leading to dramatic improvement of the bone mineral density in ovariectomized mice with no or few adverse effects to liver and uterus [42].
- a bone-targeting system with an acidic oligopeptide could be applied to a large molecule such as an enzyme in a manner such that the enzyme is functional and efficiently produced remains unsolved.
- the inventors have sought to address the issue of enzyme replacement therapy using membrane bound enzymes genetically modified to be synthesized in an active secretory form.
- the inventors have applied this method to TNSALP as a treatment for hypophosphatasia.
- This method of releasing membrane bound enzymes in a functional form will offer new avenues for therapeutic strategies to combat disease of enzyme deficiency.
- the inventors have made the surprising discovery that removal of the nucleotide sequence encoding the C-terminus glycosylphosphatidylinositol (GPI) anchoring signal peptide of a membrane bound enzyme and expressing that nucleotide sequence in a host cell, will result in the synthesis and extracellular release of an active enzyme in a soluble form. Furthermore, a membrane bound enzyme such as tissue-nonspecific alkaline phosphatase (TNSALP) in an anchorless form is useful in enzyme replacement therapy for treatment of hypophosphatasia.
- TNSALP tissue-nonspecific alkaline phosphatase
- hypophosphatasia caused by deficient activity of TNSALP results in defective bone mineralization. Plasma infusions of TNSALP have not achieved clinical improvement. No definitive treatment is presently available. Enzyme replacement therapy for hypophosphatasia was not thought to be feasible since TNSALP exists as a membrane-bound enzyme and functions physiologically when the enzyme is present at the cell membrane.
- a tissue TNSALP knock-out mouse provides a model of infantile hypophosphatasia displaying impaired bone mineralization, epileptic seizures, apnoea, and abnormal apoptosis in the thymus, abnormal lumbar nerve roots, and postnatal death before the weaning.
- TNSALP telomere sequence
- anchorless rhTNSALP anchorless recombinant human TNSALP
- Targeted therapies are often advantageous because they can reduce overall total effective dose and in turn adverse consequences to patients.
- the inventors tagged anchorless rhTNSALP enzymes with an acidic oligopeptide, of six or eight residues of L-Aspartic acid, to provide high affinity binding to hydroxyapatite which is abundant in bone.
- the inventors characterized the biochemical properties of the purified tagged enzymes in comparison with the untagged enzyme to evaluate the feasibility of bone-directional delivery.
- CHO cell lines were established producing the tagged anchorless rhTNSALP enzymes as a secreted form. It was found that specific activities of the purified enzymes tagged with the acidic oligopeptide were almost the same as the untagged enzyme.
- an object of this invention is a method of modifying a membrane bound protein by eliminating the GPI anchor such the protein is not bound to the cell membrane and may exist extracellularly in a soluble active form.
- the object of this invention is a TNSALP, modified so that it does not comprise a GPI anchor, and that this anchorless TNSALP is not bound to the cell membrane and may exist extracellularly in a soluble active form such that it may be used therapeutically in enzyme replacement therapy for ALP deficient diseases such as hypophosphatasia.
- the object of this invention is a TNSALP, modified such that the TNSALP does not comprise a GPI anchor and this anchorless TNSALP is not bound to the cell membrane and may exist extracellularly in a soluble active form, and further comprises an acidic oligopeptide sequence, such as poly-aspartic acid, providing a high affinity for bone tissue so that it may be used therapeutically in ERT for ALP deficient diseases such as hypophosphatasia.
- an acidic oligopeptide sequence such as poly-aspartic acid
- the invention is drawn to a method of manufacturing an ALP ERT factor, comprising the steps of a) deleting the GPI anchor signal peptide encoding sequence form a nucleotide, b) transfecting a cell with said modified nucleotide, c) culturing the cell, and d) purifying the ALP ERT factor form the culture media.
- the invention is drawn to a method of treating a patient with hypophosphatasia using ALP ERT factors.
- the instant ALP ERT factors may be administered to patients in vivo, in a pharmaceutically acceptable formulation as a therapy for the treatment of hypophosphatasia, or encoded a nucleotide sequence to be expressed in cells within a patient to supply the aforementioned factors.
- FIG. 1 Construct of anchorless TNSALP.
- the glycosylphosphatidylinositol (GPI) anchoring signal peptide sequence of TNSALP was deleted from (A) the full-length of TNSALP cDNA to produce (B) the secreted form of the enzyme.
- GPI glycosylphosphatidylinositol
- FIG. 2 SDS-PAGE of ALP ERT factors from condition medium.
- the purified enzymes (0.2 ⁇ g) were subjected to SDS-PAGE under reducing condition and stained with silver. A Single band appeared in all the three enzymes.
- the molecular mass of the untagged anchorless rhTNSALP (lane 1) was approximately 80 kDa, while those of CD6- and CD8-TNSALP were larger (lanes 2 and 3, respectively).
- FIG. 3 Concentration-dependent binding curves of anchorless TNSALP and tagged anchorless TNSALP to hydroxyapatite.
- Purified enzymes were mixed with a hydroxyapatite suspension at a final concentration of 1.0, 2.5, 5.0, and 10.0 ⁇ g/ml. The mixture was mixed at 37° C. for 1 h, and centrifuged at 14,000 ⁇ rpm for 10 min to separate bound and unbound enzymes. To determine the amount of the unbound enzyme, the enzyme activity in supernatant was measured. The amount of bound enzyme was determined by measuring both total and unbound enzymes. Affinity for hydroxyapatite for oligo Aspartic acid tagged enzymes was 10-fold higher than that for the untagged enzyme. Also binding to hydroxyapatite was seen at lower concentrations of Aspartic acid tagged enzyme.
- FIG. 4 ConA affinity chromatography of three ALP ERT factors.
- Anchorless rhTNSALP (A), CD6-TNSALP (B), and CD8-TNSALP (C) were applied to a ConA affinity column. After washing the column, two fractions were eluted by two different concentrations, 0.01 M (arrow; a) and 0.5 M (arrow; b) of ⁇ MM. There was no difference in the elution profile among the three enzymes.
- FIG. 5 WGA affinity chromatography of ALP ERT factors.
- ALP ERT factors before (A-C) and after (D-F) the neuraminidase digestion were applied to the WGA affinity chromatography.
- the anchorless rhTNSALP (A and D), CD6-TNSALP (B and E), and CD8-TNSALP (C and F) enzymes were applied to the WGA column. After washing the column, two fractions were eluted by the two different concentrations, 0.1 M (arrow; a) and 0.5 M (arrow; b) of GlcNAc.
- FIG. 6 SDS-PAGE of ALP ERT factors before and after neuraminidase digestion.
- the enzymes (0.3 ⁇ g) were subjected to SDS-PAGE under reducing condition and stained with silver. A single band was observed at all the lanes. After the treatment with neuraminidase, the molecular mass of the three enzymes decreased in a similar proportion.
- FIG. 7 Biodistribution of fluorescence-conjugated ALP ERT factors to bone.
- Fluorescence-labeled ALP ERT factors (A) anchorless rhTNSALP, (B) CD6-TNSALP, and (C) CD8-TNSALP, were infused to mice from tail vein at the dose of 1 mg/kg of body weight.
- the legs were dissected and sectioned. The sections of legs were observed under a fluorescent microscopy to evaluate the enzyme distribution at the epiphyseal region.
- ALP ERT factors were distributed to the mineralized region (m), but not to the, growth plate (gp).
- FIG. 8 Relative area of fluorescence around growth plate after a single infusion of fluorescence-ALP ERT factors. The average of the relative areas of fluorescence from three fields of the fluorescent images at epiphyseal region was quantitated.
- FIG. 9 In vitro mineralization experiment with anchorless rhTNSALP enzyme.
- the bone marrow cells derived from a hypophosphatasia patient were seeded in 12-well plate at a density of 10,000 cells/cm2, and differentiated under existing 2.5 mM Pi or 2.5 mM ⁇ -glycerophosphate as a phosphate source.
- the effect on mineralization of anchorless rhTNSALP enzyme was evaluated in the presence of PPi.
- the calcium deposits were visualized 12 days after the initiation of differentiation of bone marrow cells.
- FIG. 10 Clinical phenotype of TNSALP ( ⁇ / ⁇ ) mouse treated by anchorless rhTNSALP.
- the upper mouse is a wild-type from the same littermate while the lower mouse is treated with anchorless rhTNSALP for 6 weeks.
- the stature and appearance of treated mouse is nearly the same as the wild-type control mouse.
- FIG. 11 Growth curve of mice injected with anchorless rhTNSALP of 5 mg/kg.
- TNSALP ( ⁇ / ⁇ ) mouse which received enzyme on the day after birth, followed by further weekly injection up to 10 weeks. At 0, 1, 2, 3, 4 weeks, the enzyme was injected by intraperitoneal. After 5 weeks through 10 weeks, enzyme was injected through tail vein weekly (black diamond line).
- the wild-type littermates of the treated TNSALP ⁇ / ⁇ )(open circles).
- the untreated TNSALP ⁇ / ⁇ ). The untreated mice died before the weaning (x ⁇ x).
- TNSALP is bound to plasma membranes by a GPI anchor, which is added after removal of a C-terminus peptide during post-translational processing.
- TNSALP functions as an ectoenzyme.
- the inventors have removed the nucleotide sequence encoding the GPI anchor signal from human TNSALP cDNA in order to express and secrete an anchorless form of TNSALP into the culture medium of overexpressing CHO—K1 cells.
- hypophosphatasia is a metabolic bone disease that establishes an important role for alkaline phosphatase (ALP) in skeletal mineralization.
- Subnormal serum ALP activity (hypophosphatasemia) constitutes the biochemical hallmark and reflects a generalized deficiency of activity of the tissue-nonspecific (liver/bone/kidney) ALP isoenzyme (TNSALP). Activities of the three tissue-specific ALP isoenzymes in humans—intestinal, placental, and germ-cell (placental-like) ALP—are not diminished.
- TNSALP is a zinc metalloglycoprotein that is catalytically active as a multimer of identical subunits. It is bound to plasma membranes by GPI linkage.
- Hypophosphatasia is characterized clinically by defective skeletal mineralization that manifests as rickets in infants and children and osteomalacia in adults.
- Clinical expressivity is, however, extremely variable. Stillbirth can occur from in utero onset in the perinatal (“lethal”) form, which is apparent in newborns and associated with the most severe skeletal hypomineralization and deformity.
- the infantile form presents as a developmental disorder by age 6 months. It may cause craniosynostosis and nephrocalcinosis from hypercalcemia and hypercalciuria and is often fatal. Premature loss of deciduous teeth and rickets are the cardinal clinical features of childhood hypophosphatasia.
- Odontohypophosphatasia refers to especially mildly affected individuals who have dental, but no skeletal, manifestations.
- PLP phosphoethanolamine
- PPi PPi
- PLP pyridoxal 5′-phosphate
- Extracellular accumulation of PPi which at low concentrations promotes calcium phosphate deposition but at high concentrations acts as an inhibitor of hydroxyapatite crystal growth, appears to account for the associated CPPD deposition and perhaps calcific periarthritis, as well as the defective mineralization of bones and teeth.
- Enzyme replacement by IV infusion of ALP from various tissue sources has generally not been of significant clinical benefit [34-38]. Therefore, it has long been thought that since TNSALP is a membrane-bound protein, via GPI linkage, TNSALP needs to be attached to the membrane to provide a physiological function.
- the inventors have invented an acidic-oligopeptide-tagged bone-directional anchorless rhTNSALPs for use in ERT, and have characterized these enzymes for their bone-targeting properties.
- the inventors tagged the anchorless rhTNSALP enzymes with an acidic oligopeptide (a six or eight stretch of L-Aspartic acid), to provide a high affinity for hydroxyapatite, which is abundant in bone.
- the inventors characterized the biochemical properties of the purified tagged enzymes in comparison with the untagged enzyme to evaluate the feasibility of the bone-directional delivery.
- CHO cell lines producing tagged (six or eight residues of L-Aspartic acid) and untagged anchorless rhTNSALP enzymes were established.
- the specific activity of purified enzymes tagged with the acidic oligopeptides was almost identical with the untagged enzyme.
- In vitro affinity assays showed that the tagged anchorless rhTNSALPs had a 10-fold higher affinity for hydroxyapatite than the untagged anchorless rhTNSALP.
- Lectin affinity chromatography showed little difference in carbohydrate structure among the tagged and untagged enzymes except for fewer sialic acid residues on the tagged enzymes.
- the invention is drawn to (1) a method of producing an anchorless membrane bound protein in a soluble active form, by deleting the GPI anchoring signal peptide, (2) composition and manufacture of an anchorless human recombinant TNSALP (anchorless rhTNSALP) for treatment of hypophosphatasia by deleting the GPI anchoring signal peptide nucleic acid sequence from cDNA and transfecting a host cell for high yield expression and release of the enzyme, (3) a method and composition for an acidic oligopeptide tagged variant of anchorless rhTNSALP for targeted delivery to bone, and (4) methods of using anchorless rhTNSALP and oligopeptide tagged variants of anchorless rhTNSALP to treat hypophosphatasia in a patient.
- anchorless recombinant human TNSALP or “anchorless rhTNSALP” refers to a TNSALP which has been modified by deletion of the GPI anchor.
- TNSALP generally referees to tissue non-specific alkaline phosphatase. As used in FIGS. 1, 3 , 6 , and 8 as well as the provisional application to which this application claims priority, TNSALP or rhTNSALP, where it is applicability described, is equivalent to, anchorless human recombinant TNSALP or anchorless rhTNSALP.
- CD6-TNSALP and “CD8-TNSALP” or “CD6” and “CD8” refer to “anchorless recombinant human TNSALP or anchorless rhTNSALP which have been tagged with 6 or 8 L-aspartic acid residues respectively.
- tagged or “oligopeptide tagged” means the act of adding to, in this case, referring to the adding of six or eight aspartic acids residues to anchorless rhTNSALP through genetic engineering or other chemical means.
- ALP refers to the family of alkaline phosphatase enzymes generally.
- ERT refers to enzyme replacement therapy for treatment of disease.
- a disease caused by enzyme deficiency treated through replacement of the deficient enzyme As used here it refers to replacement of the deficient enzyme, by way of explanation but not of limitation, inter venous infusion or administration of a corrective gene or cell containing a corrective gene to produce the deficient enzyme in a patient.
- ALP ERT factors refers generally to alkaline phosphatase enzymes useful in enzyme replacement therapy. More specifically this term is meant to include all compositions of anchorless rhTNSALP, CD6-TNSALP and CD8-TNSALP disclosed herein.
- GPI anchor is meant to refer to glycosylphosphatidylinositol attached at or near the C-terminus of a membrane bound protein, thereby binding the membrane bound protein to the membrane via its lipidphilic affinity with the membrane.
- GPI anchor signal peptide is meant to refer to the C-terminus amino acid sequence recognized during post-translational processing as a single for adding GPI and thereby anchoring the protein.
- GPSI anchor single peptide sequence refers to a nucleotide sequence encoding the GPI anchor signal peptide.
- active means a functional state of a molecule where it performs as it would in vivo, including reactions the enzymes is know to facilitate or binding or blocking functions receptors may be know to possess. Active also includes any pro-active state, pro-enzymes which normally exist in a precursor from; that is not capable of carrying out their known function until activated by another factor or co-factor.
- Sequence identity or percent identity is intended to mean the percentage of same residues between two sequences.
- the two reference sequences used are the entire peptide sequence of human tissue non-specific alkaline phosphatase precursor (residues 1-524), or the GPI anchor single peptide of human tissue non-specific alkaline phosphatase precursor (residues 506-524).
- the two sequences being compared are aligned using the Clustal method (Higgins et al, Cabios 8:189-191, 1992) of multiple sequence alignment in the Lasergene biocomputing software (DNASTAR, INC, Madison, Wis.).
- multiple alignments are carried out in a progressive manner, in which larger and larger alignment groups are assembled using similarity scores calculated from a series of pairwise alignments.
- Optimal sequence alignments are obtained by finding the maximum alignment score, which is the average of all scores between the separate residues in the alignment, determined from a residue weight table representing the probability of a given amino acid change occurring in two related proteins over a given evolutionary interval. Penalties for opening and lengthening gaps in the alignment contribute to the score.
- GPI anchor single peptide Shows are calculations of identity for comparisons of GPI anchor single peptide sequences from various mammalian species relative to the GPI binding signal peptide of human tissue non-specific alkaline phosphatase precursor.
- the GPI anchoring signal peptide 19 amino acid (SEQ:1 residues 506-524) sequence was removed from the C-terminal of the human TNSALP cDNA (SEQ:1) to release the enzyme in the media of CHO—K1 cells. ( FIG. 1 ).
- the resultant anchorless rhTNSALP enzyme (>95%) was mainly secreted to culture medium in a transient expression study (data is not shown).
- Acidic oligopeptide-tagged enzymes CD6-TNSALP and CD8-TNSALP, which also lack the GPI anchoring signal peptide, were secreted in to the culture medium as well.
- the purification of these enzymes was performed by a two-step column chromatography method, using DEAE-Sepharose and Sephacryl S-400, as summarized in Table 3.
- the overall purification yields of anchorless rhTNSALP, CD6-TNSALP, and CD8-TNSALP were 32%, 62%, and 56% of the total enzymes in the culture media, respectively, and the specific activities of each enzyme were 2744, 2411, and 2374 units/mg, respectively.
- the lower purification yield of anchorless rhTNSALP than those of the tagged enzymes was apparently due to a broader peak eluted from the DEAE column.
- K b binding constant and B max maximum binding rates were determined form double- reciprocal plots.
- K b B max (ug/100 ug (ug ⁇ 1 ml) hydroxyapatite) rhTNSALP 1.7 ⁇ 1.0 0.5 ⁇ 0.2 CD6-TNSALP 36.7 ⁇ 7.9 1.6 ⁇ 0.3 CD8-TNSALP 44.6 ⁇ 4.6 1.9 ⁇ 0.7 Elution Profiles of Enzymes by Lectin Affinity Chromatography
- ConA affinity chromatography Three enzymes, rhTNSALP, CD6-, and CD8-TNSALP, were subjected to ConA affinity chromatography. ( FIG. 4 ). ConA affinity chromatography indicated there was little unbound enzyme, whereas weakly-bound and strongly-bound enzymes were detected. Overall the elution profiles of these enzymes did not differ when two different concentrations of competitive sugars were added. Since ConA has a high reactivity to the mannosyl residues, the inventors concluded that these enzymes did not differ with respect to mannosyl residue composition. In contrast, the WGA elution profiles between the tagged and untagged enzymes were remarkably different in the ratio of strongly-bound enzyme and weakly-bound enzyme (FIGS. 5 A-C).
- Table 5 shows the percentages of the relative enzyme activity of three fractions on the WGA column. Approximately 30% of the tagged enzymes were weakly bound and 70% was strongly bound to the WGA column, while 66% of the untagged enzyme was weakly bound and 34% was strongly bound to the WGA column. The content of the weakly-bound enzyme was larger in the order of rhTNSALP>CD6-TNSALP>CD8-TNSALP.
- FIG. 7 shows the histological pictures of biodistribution of three enzymes at the epiphyseal region at 6, 24, 72, and 168 h after a single intravenous infusion.
- FIG. 8 shows the average of the relative area of fluorescence. Three enzymes were distributed to the mineralized region, but not to the growth plate. At 6 h, the relative areas of fluorescence at the tagged enzymes were four-fold larger than the area at the untagged enzyme.
- TNSALP gene knock-out mouse strains as models for hypophosphatasia had ⁇ 1% of wild-type plasma TNSALP activity. These TNSALP ⁇ / ⁇ mice were growth impaired, develop epileptic seizures and apnea, and died before weaning as described previously [39,47,48]. Postnatal growth of TNSALP ⁇ / ⁇ mice treated with anchorless rhTNSALP at 5 mg/kg of body weight and their littermate controls are shown in FIG. 10 . The average life span of untreated TNSALP ⁇ / ⁇ mice without anchorless rhTNSALP enzyme administration was 10 days [39,47, 48]. In treated mice, injected with anchorless rhTNSALP, no epileptic seizures appeared until at least 2 months old, in addition the mice lived approximately 4 and 7 times as long.
- FIG. 11 Growth curves of TNSALP ⁇ / ⁇ mice and littermate controls without treatment are shown in FIG. 11 for comparison.
- One mouse treated with IP infusion for 4 weeks did not grow well ( FIG. 11A ). However after IV infusion began, the mouse increased its body weight substantially.
- a second mouse treated with IV infusion at birth grew well at subnormal levels ( FIG. 11B ). Both of these mice exhibited no abnormal activity and seizures.
- TNSALPs human recombinant acidic oligopeptide-tagged and untagged TNSALPs (GenBank: NM — 000478.2)—The GPI anchoring signal peptide sequence of TNSALP (5′-CTTGCTGCAGGCCCCCTGCTGCTCGCTCTGGCCCTCTACCCCCTGAGCGTCCTGT TC-3′: c.1516C to c.1572C: Leu506 to Phe524) was deleted from the full-length of TNSALP cDNA to produce the enzymes as a secreted form.
- the three enzymes used for the further experiments were named as anchorless rhTNSALP (human TNSALP anchorless at the C-terminal), CD6-TNSALP (human TNSALP anchorless at the C-terminal tagged with a stretch of six L-Asp), and CD8-TNSALP (human TNSALP anchorless at the C-terminal tagged with a stretch of eight L-Asp), respectively.
- reverse transcriptase reaction was performed by using total RNA isolated from healthy human peripheral blood.
- PCR reactions were carried out with the following primers: TNSALP, forward 5′-GAATTCACCCACGTCGATTGCATCTCTGGGCTCCAG-3′ and reverse 5′-ctcgagTCAGCTGCCTGCCGAGCTGGCAGGAGCAC-3′: CD6-TNSALP, forward 5′-GAATTCACCCACGTCGATTGCATCTCTGGGCTCCAG-3′ and reverse 5′-tcaatcatcgtcgtcatcgtcggcctctgcttcaccggtGCTGCCTGCCGAGCTGGCAGGAGCACAGTG-3′: CD8-TNSALP, forward 5′-GAATTCACCCACGTCGATTGCATCTCTGGGCTCCAG-3′ and reverse 5′-tcagtc
- the nucleotide sequences compatible with six or eight of L-Asp were added to the reverse primers used here.
- the amplified cDNA were cloned and sequenced.
- the cDNA were then transferred into EcoRI cloning sites of mammalian expression vector pCXN, kindly provided by Miyazaki J., Osaka University, Suita, Japan (40).
- the anchorless rhTNSALP, CD6-TNSALP, and CD8-TNSALP cDNAs subcloned in pCXN were then transfected into Chinese hamster ovary (CHO—K1) cells with lipofectamine according to manufacture's instruction (Invitrogen). Selection of colonies was carried out in growth medium with Dulbecco's Modified Eagle Medium supplemented with 15% fetal bovine serum (FBS), plus 600 ⁇ g/ml G418 (Sigma-Aldrich) for 10-12 days. Individual clones were picked, grown to confluency, and analyzed for enzyme expression by measuring secreted enzyme activity in the medium as described below.
- FBS fetal bovine serum
- G418 Sigma-Aldrich
- the highest-producing clone was grown in collection medium with Ex-Cell tm 325 PF CHO Protein-free medium JRH Biosciences) and 15% FBS. When the cells reached confluency, the cells were rinsed with PBS and fed with collection media without FBS to collect enzyme for purification.
- a 50 ⁇ l of volume of sample was combined with 250 ⁇ l of 10 mM -nitrophenyl phosphate (pNPP) (Sigma-Aldrich, Mo.) as a substrate in 1 M diethanolamine, pH 9.8, containing 1 mM magnesium chloride and 0.02 mM zinc chloride, and incubated at 37° C.
- the time-dependent increase in absorbance at 405 nm was measured on a plate spectrophotometer (EL800, Bio-Tek Instrument, Inc., VT).
- One unit of activity was defined as the quantity of enzyme that catalyzed the hydrolysis of 1 ⁇ mol substrate in 1 min.
- the anchorless rhTNSALP enzyme was purified by a two-step column procedure.
- Tris buffer was 25 mM Tris-HCl, pH 8.0, containing 0.1 mM magnesium chloride and 0.01 mM zinc chloride. Unless stated otherwise, all steps were performed at 4° C.
- Step 1 The medium containing enzyme was filtered through a 0.2 ⁇ m filter, and then dialyzed against Tris buffer using Amicon stirred-cell ultrafiltration unit with Millipore ultrafiltration membrane YM-30.
- Step 2 The dialyzed medium was applied to a column of DEAE Sepharose (Sigma-Aldrich, MO) equilibrated with Tris buffer. The column was first washed with Tris buffer, and then the enzyme was eluted with 0-0.4 M NaCl in a linear gradient.
- DEAE Sepharose Sigma-Aldrich, MO
- Step 3 The active eluted fractions were pooled and dialyzed against Tris buffer containing 0.1 M NaCl by using Centricon centrifugal filter device with Millipore ultrafiltration YM-10 filter. The dialyzed fractions were then concentrated for step 4.
- Step 4 The concentrated enzyme was applied to a column of Sephacryl S-400-HR (Sigma-Aldrich, MO) equilibrated with Tris buffer containing 0.1 M NaCl. The enzyme was eluted with Tris buffer containing 0.1 M NaCl.
- Step 5 The active eluted fractions were pooled and dialyzed against Tris buffer containing 0.1 M NaCl by using Centricon centrifugal filter device with Millipore ultrafiltration YM-10 filter. The dialyzed fractions were then concentrated and stored at ⁇ 80° C. until use.
- Hydroxyapatite binding assay-Hydroxyapatite beads (Sigma-Aldrich) were suspended in 25 mM Tris-HCl buffered saline (TBS), pH 7.4, at concentration of 100 ⁇ g/100 ⁇ l.
- the purified enzyme was mixed with the hydroxyapatite suspension at a final concentration of 1.0, 2.5, 5.0, and 10.0 ⁇ g/ml.
- the mixture was mixed at 37° C. for 1 h, and centrifuged at 14,000 ⁇ rpm for 10 min to separate unbound enzyme and bound enzyme. To determine unbound enzyme, enzyme activity in supernatant was measured, and bound enzyme was determined from the amount of total enzyme and unbound enzyme. Binding constant (K b ) and maximal binding rate (B max ) were determined from double-reciprocal plots.
- TBS used here was 10 mM Tris-HCl, pH 8.0, supplemented with 0.5 M sodium chloride, 1 mM calcium chloride, 1 mM magnesium chloride, 1 mM manganese chloride and 0.01 mM zinc chloride.
- the column of the concanavalin A ( Canavalia ensiformis , ConA)-sepharose 4B (Sigma-Aldrich) and the wheat germ agglutinin ( Triticum vulgaris , WGA)-agarose CL-4B (Fluka) were equilibrated with TBS at a flow rate of 0.2 ml/min.
- Lectin affinity chromatography was performed as described previously [43]. Briefly, the purified enzyme in 0.6 ml of TBS was applied to the ConA and WGA columns, and left to stand for 3 h at room temperature. Three fractions were obtained by using two different concentrations, 0.01 M and 0.5 M of ⁇ -methyl-D-mannopyranoside ( ⁇ MM) (Sigma-Aldrich) from ConA column, and 0.1 M and 0.5 M of N-acetyl-D-glucosamine (GlcNAc) (Sigma-Aldrich) from the WGA column: unbound fraction, weakly-bound fraction, and strongly-bound fraction.
- ⁇ MM ⁇ -methyl-D-mannopyranoside
- GlcNAc N-acetyl-D-glucosamine
- Tagged and non-tagged rhTNSALPs were digested with ⁇ (2 3, 6, 8, 9) neuraminidase ( Arthrobacter ureafaciens ) (Sigma-Aldrich) to clarify the content of sialic acids at the carbohydrate chain. Twenty units of each purified TNSALP enzyme were exposed to 0.01 unit of neuraminidase in 250 mM sodium phosphate, pH 6.0, overnight at room temperature. The digested enzyme was then analyzed for polyacrylamide gel electrophoresis and lectin affinity chromatography, as described above.
- One mg/ml of purified enzymes were labeled with Alexa Fluor 546 Protein Labeling Kit following manufacture's instruction (Molecular Probes).
- the Alexa-labeled enzyme was injected to B6 mice (6-7 weeks old) from tail vein at a dose of 1 mg/kg of body weight. Mice were sacrificed at 6, 24, 72, and 168 h after a single infusion, and multiple tissues including brain, lung, heart, liver, spleen, kidney, and leg were dissected. The tissues were immersion-fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned. Tissues were studied by fluorescence microscopy for evaluation of enzyme distribution, and the areas of fluorescence from three fields of fluorescent images around growth plate were quantitated by using AlphaEaseFC (Alpha Innotech Corp.).
- in vitro mineralization experiments were performed using bone marrow cells derived from a hypophosphatasia patient with an infantile form (10 month old).
- the bone marrow cells were seeded into 150 ⁇ 25 mm tissue culture dishes. These cells were allowed to attach without disturbance for seven days in growth medium consisting of minimum essential medium alpha (MEM ⁇ ) supplemented with 10% FBS, 50 units/ml penicillin, and 50 ⁇ g/ml streptomycin sulfate. The medium was then replaced to fresh growth medium at 3-day intervals. When the cells reached confluency, they were subcultured in the 12-well plates at a density of 10,000 cells/cm 2 .
- MEM ⁇ minimum essential medium alpha
- the growth medium was replaced with the differentiation medium: with MEM ⁇ supplemented with 10% FBS, 50 units/ml penicillin, 50 ⁇ g/ml streptomycin sulfate, 0.3 mM ascorbic acid, and 100 nM dexamethasone.
- the differentiation medium also included 2.5 mM P i or ⁇ -glycerophosphate as a phosphate source as well as either anchorless rhTNSALP at 2.5 or 5.0 units/ml.
- 50 ⁇ M PP i was added always with each enzyme to the bone marrow cell culture throughout the differentiation period.
- the differentiation medium was replaced at 3-day intervals. At 12 days after the initiation of the differentiation of the cells, the cells were fixed with 4% paraformaldehyde, followed by staining with Alizarin Red S to detect calcium phosphate deposits [46].
- ERT Long term ERT was performed using the anchorless rhTNSALP enzyme described above.
- the cephalic vein is the preferred injection rout at birth but is not visible after about 1 week. Intraperitoneal injections were administered from 1 to 4 weeks until the tail vain became visible. Three litermates remained untreated. Two mice (Specimens 1 and 2) received treatments of 5 mg/kg of body weight. Specimen received enzyme by cephalic vein injection on the day following birth, followed by weekly intraperitoneal injections at 0, 1, 2, 3, and 4 weeks and tail vein injections from 5 through 10 weeks. Similarly a Specimen 2 received enzyme on the day following birth by cephalic vein injection, followed by weekly intraperitoneal injections at 1, 2, and 3, weeks after which injection was administered though the tail vein From 4 through 10 weeks.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physical Education & Sports Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Diabetes (AREA)
- Rheumatology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
- This application claims benefit of priority to U.S. Provisional Patent Application No. 60/725,563, filed Oct. 11, 2005.
- A paper copy of the sequence listing and a computer readable form of the same sequence listing are appended below and herein incorporated by reference. The information recorded in computer readable form is identical to the written sequence listing, according to 37 C.F.R. 1.821(f).
- 1. Field of the Invention
- This invention relates generally to compositions and methods of enzyme replacement therapy (ERT). More specifically, the invention is directed to compositions and methods for treatment of enzyme deficient disease such as hypophosphatasia using a genetically modified polynucleotide to produce in an active secretory form of alkaline phosphatase.
- 2. Description of the Related Art
- Alkaline phosphatase (ALP) is a ubiquitous plasma membrane-bound enzyme.
- Hypophosphatasia is an inherited metabolic disorder of defective bone mineralization caused by deficiency of a form of ALP know as tissue-nonspecific alkaline phosphatase (TNSALP). Clinical severity is remarkably variable, ranging from death in utero to merely premature loss of dentition in adult life [1, 2]. Despite the presence of TNSALP in bone, kidney, liver, and adrenal tissue in healthy individuals, clinical manifestations in patients with hypophosphatasia are limited to defective skeletal mineralization that manifests as rickets in infants and children and osteomalacia in adults [2]. In the most pernicious form of hypophosphatasia, the perinatal lethal variant, profound skeletal hypomineralization results in caput membranaceum with shortened and deformed limbs noted. Some affected neonates survive for several days or weeks. They often succumb to respiratory failure brought on by pulmonary hypoplasia and structural failure of the weakened skeleton from demineralization [3].
- Osteoblasts modulate the composition of the bone matrix, where they deposit mineral in the form of hydroxyapatite. Specialized buds from the osteoblasts' plasma membrane are called matrix vesicles (MVs). The initiation of matrix calcification by osteoblasts and chondrocytes appears to be mediated by release of MVs, which serve as a sheltered environment for hydroxyapatite crystal formation [4-7]. MVs are alkaline phosphatase enriched, extracellular, membrane-invested bodies. Inside MVs the first crystals of hydroxyapatite bone mineral are generated. TNSALP hydrolyzes inorganic pyrophosphate (PPi) to monophosphate (inorganic phosphate; Pi), which is important for growth of the hydroxyapatite crystal [4, 5, 8-10]. Thus ALP functions as an inorganic pyrophosphatase (PPi-ase) [14, 15]. PPi itself impairs the growth of hydroxyapatite crystals as an inhibitor of mineralization [8, 11-13]. Insufficient TNSALP activity fails to hydrolyze PPi and the resulting build-up of unhydrolyzed PPi in the perivesicular matrix inhibits the proliferation of pre-formed hydroxyapatite crystals beyond the protective confines of MV membranes.
- The level of plasma PPi increases in hypophosphatasia [16-18]. Even in the absence of TNSALP, the other phosphatases (AMPase and inorganic pyrophosphatase) can hydrolyze PPi, supplying Pi for incorporation into initial mineral within MVs [19] but still be insufficient to remove excess PPi at the perimeter of MVs. Thus, despite TNSALP deficiency, initial mineral could form within MVs, while its propagation into perivesicular matrix would be inhibited by a local build-up of PPi [20, 21]. These findings suggest PPi as a plausible candidate as an inhibitor of mineralization and as a primary factor that causes clinical manifestations of hypophosphatasia.
- Enzyme replacement therapy (ERT) has proven effective in preventing or reversing lysosomal storage in patients and animal models with lysosomal storage diseases (LSDs) [22-28]. Tremendous progress in the development of ERT has been made in the last three decades. Cellular uptake of enzyme from the blood following intravenous administration requires specific oligosaccharides on the enzyme itself corresponding to oligosaccharide receptors on the target cells. Examples include the binding of high-mannose oligosaccharides of the enzyme to the mannose receptor (MR) and binding of phosphorylated high-mannose oligosaccharides of the enzyme to the cation-independent mannose 6-phosphate receptor (M6PR). Thus, LSDs have been considered potentially amenable to therapy with exogenously supplied enzymes.
- The cell-specific delivery system was also designed to enhance the clinical effectiveness of ERT. In the case of Gaucher disease, delivery of the enzyme to the affected cells was achieved by modifying the N-linked carbohydrate on the enzyme.
- This exposed core mannose residues [29, 30], enabling the enzyme to bind to the MR, which is highly abundant on cells of the reticuloendothelial system [31, 32]. These findings led to clinical management of Gaucher disease by ERT [22]. Over 3,500 patients have been treated with dramatic clinical results [33].
- However, hypophosphatasia caused by a deficiency of TNSALP seems to be a difficult disorder treated by ERT because TNSALP is a membrane-bound enzyme and is believed to require attachment at the cell surface to be functional. In fact, the results of multiple intravenous infusions of plasma ALP or purified liver ALP in patients with hypophosphatasia have been disappointing [34-38]. Administration of exogenous pyridoxal HCl delayed the onset of epileptic attacks and increased the life span of TNSALP−/−mice. Although the oldest survivor was 22 days old, all the homozygotes, however, died near weaning time, irrespective of their treatment regime [39].
- The inventors have genetically engineered a Chinese Hamster Ovarian (CHO) cell line to produce a C-terminus-anchorless TNSALP enzyme, in secreted form, [40] and showed clinical effectiveness of ERT on hypophosphatasia mice. These results indicate that the C-terminus-anchorless membrane enzyme possesses the characteristics necessary for use in ERT where the membrane-binding form is ineffective. Deletion of the C-terminus membrane anchor will be applicable to other membrane-binding proteins whose deficiency leads to other human disorders including but not limited to paroxysmal nocturnal haemoglobinuria (PNH).
- Targeted therapies have the advantage of reducing adverse effects on non-target organs as well as reducing the minimum effective systemic dose. Recently, Kasugai et al [41] has demonstrated that a small peptide consisting of a stretch of acidic amino acids (L-Aspartic acid or L-Glutamic acid) was selectively delivered to and retained in bone after a systemic administration. Furthermore, a small molecule, an estrogen, conjugated with an acidic-oligopeptide, has been selectively targeted to bone, leading to dramatic improvement of the bone mineral density in ovariectomized mice with no or few adverse effects to liver and uterus [42]. However, whether such a bone-targeting system with an acidic oligopeptide could be applied to a large molecule such as an enzyme in a manner such that the enzyme is functional and efficiently produced remains unsolved.
- The inventors have sought to address the issue of enzyme replacement therapy using membrane bound enzymes genetically modified to be synthesized in an active secretory form. In particular the inventors have applied this method to TNSALP as a treatment for hypophosphatasia. This method of releasing membrane bound enzymes in a functional form will offer new avenues for therapeutic strategies to combat disease of enzyme deficiency.
- The inventors have made the surprising discovery that removal of the nucleotide sequence encoding the C-terminus glycosylphosphatidylinositol (GPI) anchoring signal peptide of a membrane bound enzyme and expressing that nucleotide sequence in a host cell, will result in the synthesis and extracellular release of an active enzyme in a soluble form. Furthermore, a membrane bound enzyme such as tissue-nonspecific alkaline phosphatase (TNSALP) in an anchorless form is useful in enzyme replacement therapy for treatment of hypophosphatasia.
- Hypophosphatasia, caused by deficient activity of TNSALP results in defective bone mineralization. Plasma infusions of TNSALP have not achieved clinical improvement. No definitive treatment is presently available. Enzyme replacement therapy for hypophosphatasia was not thought to be feasible since TNSALP exists as a membrane-bound enzyme and functions physiologically when the enzyme is present at the cell membrane. A tissue TNSALP knock-out mouse provides a model of infantile hypophosphatasia displaying impaired bone mineralization, epileptic seizures, apnoea, and abnormal apoptosis in the thymus, abnormal lumbar nerve roots, and postnatal death before the weaning.
- To investigate the clinical effectiveness of ERT for hypophosphatasia, the inventors deleted the C-terminus of TNSALP cDNA encoding the GPI anchoring signal peptide sequence and transfected the modified nucleotide into the Chinese hamster ovary (CHO) cell line. The result was a secreted form of anchorless recombinant human TNSALP (anchorless rhTNSALP) produced by CHO cells, which was subsequently purified and characterized in vitro.
- An in vivo study was carried out, which utilized weekly infusions of anchorless rhTNSALP into TNSALP knockout mice. In vitro mineralization assays with anchorless rhTNSALP in the presence of high concentrations of pyrophosphate provided evidence of bone mineralization with bone marrow from a hypophosphatasia patient.
- Administration of the purified anchorless rhTNSALP enzyme into TNSALP knockout mice increased life span and increased body weight, showing that the treated mice lived approximately 4 and 7 times longer compared to the untreated mice. Treated mice had no epileptic seizures until at least 3 months old.
- These results show the C-terminus anchorless rhTNSALP functions bioactively in vivo and that is a good candidate for ERT for hypophosphatasia. This invention can be applied to other diseases deficient in membrane-bound proteins.
- Targeted therapies are often advantageous because they can reduce overall total effective dose and in turn adverse consequences to patients. To this purpose the inventors tagged anchorless rhTNSALP enzymes with an acidic oligopeptide, of six or eight residues of L-Aspartic acid, to provide high affinity binding to hydroxyapatite which is abundant in bone. The inventors characterized the biochemical properties of the purified tagged enzymes in comparison with the untagged enzyme to evaluate the feasibility of bone-directional delivery. CHO cell lines were established producing the tagged anchorless rhTNSALP enzymes as a secreted form. It was found that specific activities of the purified enzymes tagged with the acidic oligopeptide were almost the same as the untagged enzyme. In vitro affinity measurements indicated that the poly-aspartic acid tagged enzymes had an approximately 10-fold higher affinity to hydroxyapatite than the untagged TNSALP enzyme. Lectin affinity chromatography showed little difference among the tagged and untagged enzymes in carbohydrate structure except the tagged enzymes had fewer sialic acid residues. Biodistribution pattern analysis by infusion of the fluorescence-labeled enzymes into mice showed that the amount of the tagged enzymes retained in bone was 4-fold higher than that of the untagged enzyme at 6 hours post-infusion. The tagged enzymes were retained at higher levels continuously up to one week.
- These results indicate that the enzymes tagged with an acidic oligopeptide are delivered more specifically to bone and possess a high affinity for hydroxyapatite, suggesting the potential use of the tagged enzymes in targeted ERT for hypophosphatasia.
- Therefore, an object of this invention is a method of modifying a membrane bound protein by eliminating the GPI anchor such the protein is not bound to the cell membrane and may exist extracellularly in a soluble active form.
- In another embodiment, the object of this invention is a TNSALP, modified so that it does not comprise a GPI anchor, and that this anchorless TNSALP is not bound to the cell membrane and may exist extracellularly in a soluble active form such that it may be used therapeutically in enzyme replacement therapy for ALP deficient diseases such as hypophosphatasia.
- In another embodiment, the object of this invention is a TNSALP, modified such that the TNSALP does not comprise a GPI anchor and this anchorless TNSALP is not bound to the cell membrane and may exist extracellularly in a soluble active form, and further comprises an acidic oligopeptide sequence, such as poly-aspartic acid, providing a high affinity for bone tissue so that it may be used therapeutically in ERT for ALP deficient diseases such as hypophosphatasia.
- In another embodiment, the invention is drawn to a method of manufacturing an ALP ERT factor, comprising the steps of a) deleting the GPI anchor signal peptide encoding sequence form a nucleotide, b) transfecting a cell with said modified nucleotide, c) culturing the cell, and d) purifying the ALP ERT factor form the culture media.
- In yet another embodiment the invention is drawn to a method of treating a patient with hypophosphatasia using ALP ERT factors.
- It is envisioned that the instant ALP ERT factors (supra) may be administered to patients in vivo, in a pharmaceutically acceptable formulation as a therapy for the treatment of hypophosphatasia, or encoded a nucleotide sequence to be expressed in cells within a patient to supply the aforementioned factors.
-
FIG. 1 Construct of anchorless TNSALP. The glycosylphosphatidylinositol (GPI) anchoring signal peptide sequence of TNSALP was deleted from (A) the full-length of TNSALP cDNA to produce (B) the secreted form of the enzyme. -
FIG. 2 . SDS-PAGE of ALP ERT factors from condition medium. The purified enzymes (0.2 μg) were subjected to SDS-PAGE under reducing condition and stained with silver. A Single band appeared in all the three enzymes. The molecular mass of the untagged anchorless rhTNSALP (lane 1) was approximately 80 kDa, while those of CD6- and CD8-TNSALP were larger (lanes -
FIG. 3 . Concentration-dependent binding curves of anchorless TNSALP and tagged anchorless TNSALP to hydroxyapatite. Purified enzymes were mixed with a hydroxyapatite suspension at a final concentration of 1.0, 2.5, 5.0, and 10.0 μg/ml. The mixture was mixed at 37° C. for 1 h, and centrifuged at 14,000×rpm for 10 min to separate bound and unbound enzymes. To determine the amount of the unbound enzyme, the enzyme activity in supernatant was measured. The amount of bound enzyme was determined by measuring both total and unbound enzymes. Affinity for hydroxyapatite for oligo Aspartic acid tagged enzymes was 10-fold higher than that for the untagged enzyme. Also binding to hydroxyapatite was seen at lower concentrations of Aspartic acid tagged enzyme. -
FIG. 4 . ConA affinity chromatography of three ALP ERT factors. Anchorless rhTNSALP (A), CD6-TNSALP (B), and CD8-TNSALP (C) were applied to a ConA affinity column. After washing the column, two fractions were eluted by two different concentrations, 0.01 M (arrow; a) and 0.5 M (arrow; b) of αMM. There was no difference in the elution profile among the three enzymes. -
FIG. 5 . WGA affinity chromatography of ALP ERT factors. ALP ERT factors before (A-C) and after (D-F) the neuraminidase digestion were applied to the WGA affinity chromatography. The anchorless rhTNSALP (A and D), CD6-TNSALP (B and E), and CD8-TNSALP (C and F) enzymes were applied to the WGA column. After washing the column, two fractions were eluted by the two different concentrations, 0.1 M (arrow; a) and 0.5 M (arrow; b) of GlcNAc. -
FIG. 6 . SDS-PAGE of ALP ERT factors before and after neuraminidase digestion. The enzymes (0.3 μg) were subjected to SDS-PAGE under reducing condition and stained with silver. A single band was observed at all the lanes. After the treatment with neuraminidase, the molecular mass of the three enzymes decreased in a similar proportion. -
FIG. 7 . Biodistribution of fluorescence-conjugated ALP ERT factors to bone. Fluorescence-labeled ALP ERT factors, (A) anchorless rhTNSALP, (B) CD6-TNSALP, and (C) CD8-TNSALP, were infused to mice from tail vein at the dose of 1 mg/kg of body weight. At theindicated time points -
FIG. 8 . Relative area of fluorescence around growth plate after a single infusion of fluorescence-ALP ERT factors. The average of the relative areas of fluorescence from three fields of the fluorescent images at epiphyseal region was quantitated. -
FIG. 9 . In vitro mineralization experiment with anchorless rhTNSALP enzyme. The bone marrow cells derived from a hypophosphatasia patient were seeded in 12-well plate at a density of 10,000 cells/cm2, and differentiated under existing 2.5 mM Pi or 2.5 mM β-glycerophosphate as a phosphate source. The effect on mineralization of anchorless rhTNSALP enzyme was evaluated in the presence of PPi. The calcium deposits were visualized 12 days after the initiation of differentiation of bone marrow cells. -
FIG. 10 . Clinical phenotype of TNSALP (−/−) mouse treated by anchorless rhTNSALP. The upper mouse is a wild-type from the same littermate while the lower mouse is treated with anchorless rhTNSALP for 6 weeks. The stature and appearance of treated mouse is nearly the same as the wild-type control mouse. -
FIG. 11 Growth curve of mice injected with anchorless rhTNSALP of 5 mg/kg. A)Specimen 1,B) Specimen 2. TNSALP (−/−) mouse which received enzyme on the day after birth, followed by further weekly injection up to 10 weeks. At 0, 1, 2, 3, 4 weeks, the enzyme was injected by intraperitoneal. After 5 weeks through 10 weeks, enzyme was injected through tail vein weekly (black diamond line). The wild-type littermates of the treated TNSALP (−/−)(open circles). The untreated TNSALP (−/−). The untreated mice died before the weaning (x−x). - In vivo, TNSALP is bound to plasma membranes by a GPI anchor, which is added after removal of a C-terminus peptide during post-translational processing. TNSALP functions as an ectoenzyme. In this study, the inventors have removed the nucleotide sequence encoding the GPI anchor signal from human TNSALP cDNA in order to express and secrete an anchorless form of TNSALP into the culture medium of overexpressing CHO—K1 cells. This study demonstrates that removal of the GPI anchoring signal peptide sequence from the C-terminus of TNSALP cDNA allows the overexpressing CHO—K1 cells to produce sufficient amounts of recombinant human enzyme in a secreted form, and that this anchorless recombinant human TNSALP enzyme is bioactive and able to initialize bone mineralization in bone marrow from hypophosphatasia patients. In addition, when anchorless rhTNSALP was infused into the sublethal form of TNSALP (−/−) mouse, it improved clinical features and increased both life span and growth, further indicating the feasibility of enzyme replacement therapy for hypophosphatasia.
- Hypophosphatasia is a metabolic bone disease that establishes an important role for alkaline phosphatase (ALP) in skeletal mineralization. Subnormal serum ALP activity (hypophosphatasemia) constitutes the biochemical hallmark and reflects a generalized deficiency of activity of the tissue-nonspecific (liver/bone/kidney) ALP isoenzyme (TNSALP). Activities of the three tissue-specific ALP isoenzymes in humans—intestinal, placental, and germ-cell (placental-like) ALP—are not diminished. TNSALP is a zinc metalloglycoprotein that is catalytically active as a multimer of identical subunits. It is bound to plasma membranes by GPI linkage.
- Hypophosphatasia is characterized clinically by defective skeletal mineralization that manifests as rickets in infants and children and osteomalacia in adults. Clinical expressivity is, however, extremely variable. Stillbirth can occur from in utero onset in the perinatal (“lethal”) form, which is apparent in newborns and associated with the most severe skeletal hypomineralization and deformity. The infantile form presents as a developmental disorder by
age 6 months. It may cause craniosynostosis and nephrocalcinosis from hypercalcemia and hypercalciuria and is often fatal. Premature loss of deciduous teeth and rickets are the cardinal clinical features of childhood hypophosphatasia. Adult hypophosphatasia typically results in recurrent metatarsal stress fractures and pseudofractures in long bones and occasionally produces arthritis from calcium pyrophosphate dihydrate (CPPD) and perhaps calcium phosphate crystal deposition. Odontohypophosphatasia refers to especially mildly affected individuals who have dental, but no skeletal, manifestations. - Three phosphocompounds [phosphoethanolamine (PEA), PPi, and
pyridoxal 5′-phosphate (PLP)] accumulate endogenously in hypophosphatasia and are inferred to be natural substrates for TNSALP. A variety of evidence shows that PLP, a cofactor form of vitamin B6, collects extracellularly; intracellular levels of PLP are normal. This observation explains the absence of symptoms of deficiency or toxicity of vitamin B6 and indicates that TNSALP functions as an ectoenzyme. Extracellular accumulation of PPi, which at low concentrations promotes calcium phosphate deposition but at high concentrations acts as an inhibitor of hydroxyapatite crystal growth, appears to account for the associated CPPD deposition and perhaps calcific periarthritis, as well as the defective mineralization of bones and teeth. There is no established medical treatment. Enzyme replacement by IV infusion of ALP from various tissue sources has generally not been of significant clinical benefit [34-38]. Therefore, it has long been thought that since TNSALP is a membrane-bound protein, via GPI linkage, TNSALP needs to be attached to the membrane to provide a physiological function. - In this study the inventors have established a newly designed ERT for hypophosphatasia with C-terminus anchorless recombinant human TNSALP and have shown clinical effectiveness with the TNSALP (−/−) mouse model. This strategy is applicable to other GPI-anchored proteins whose dysfunction leads to the human disorders such as paroxysmal nocturnal haemoglobinuria (PNH) and prion diseases.
- Bone Targeted Anchorless rhTNSALP
- The development of selective drug delivery to bone will enhance the clinical effectiveness of bioactive enzymes used in ERT. To this purpose, the inventors have invented an acidic-oligopeptide-tagged bone-directional anchorless rhTNSALPs for use in ERT, and have characterized these enzymes for their bone-targeting properties. The inventors tagged the anchorless rhTNSALP enzymes with an acidic oligopeptide (a six or eight stretch of L-Aspartic acid), to provide a high affinity for hydroxyapatite, which is abundant in bone. The inventors characterized the biochemical properties of the purified tagged enzymes in comparison with the untagged enzyme to evaluate the feasibility of the bone-directional delivery.
- CHO cell lines producing tagged (six or eight residues of L-Aspartic acid) and untagged anchorless rhTNSALP enzymes were established. The specific activity of purified enzymes tagged with the acidic oligopeptides was almost identical with the untagged enzyme. In vitro affinity assays showed that the tagged anchorless rhTNSALPs had a 10-fold higher affinity for hydroxyapatite than the untagged anchorless rhTNSALP. Lectin affinity chromatography showed little difference in carbohydrate structure among the tagged and untagged enzymes except for fewer sialic acid residues on the tagged enzymes. The examination of biodistribution patterns after a single infusion of fluorescence-labeled ALP ERT factors into mice showed that the amount of tagged enzymes retained in bone were 4-fold higher than that of the untagged enzyme at 6 hours post-infusion. The tagged enzymes were retained continuously at a higher level up to one week.
- These results show that ALP ERT factors tagged with an acidic oligopeptide are characterized with a more specific affinity binding to the hydroxyapatite, suggesting the potential use of the tagged enzymes for ERT on hypophosphatasia.
- Therefore, the invention is drawn to (1) a method of producing an anchorless membrane bound protein in a soluble active form, by deleting the GPI anchoring signal peptide, (2) composition and manufacture of an anchorless human recombinant TNSALP (anchorless rhTNSALP) for treatment of hypophosphatasia by deleting the GPI anchoring signal peptide nucleic acid sequence from cDNA and transfecting a host cell for high yield expression and release of the enzyme, (3) a method and composition for an acidic oligopeptide tagged variant of anchorless rhTNSALP for targeted delivery to bone, and (4) methods of using anchorless rhTNSALP and oligopeptide tagged variants of anchorless rhTNSALP to treat hypophosphatasia in a patient.
- The term “anchorless recombinant human TNSALP” or “anchorless rhTNSALP” refers to a TNSALP which has been modified by deletion of the GPI anchor. The term “TNSALP” generally referees to tissue non-specific alkaline phosphatase. As used in
FIGS. 1, 3 , 6, and 8 as well as the provisional application to which this application claims priority, TNSALP or rhTNSALP, where it is applicability described, is equivalent to, anchorless human recombinant TNSALP or anchorless rhTNSALP. - The terms “CD6-TNSALP” and “CD8-TNSALP” or “CD6” and “CD8” refer to “anchorless recombinant human TNSALP or anchorless rhTNSALP which have been tagged with 6 or 8 L-aspartic acid residues respectively. The term “tagged” or “oligopeptide tagged” means the act of adding to, in this case, referring to the adding of six or eight aspartic acids residues to anchorless rhTNSALP through genetic engineering or other chemical means.
- The term “ALP” refers to the family of alkaline phosphatase enzymes generally.
- The term “ERT” refers to enzyme replacement therapy for treatment of disease. A disease caused by enzyme deficiency treated through replacement of the deficient enzyme. As used here it refers to replacement of the deficient enzyme, by way of explanation but not of limitation, inter venous infusion or administration of a corrective gene or cell containing a corrective gene to produce the deficient enzyme in a patient.
- The term “ALP ERT factors” refers generally to alkaline phosphatase enzymes useful in enzyme replacement therapy. More specifically this term is meant to include all compositions of anchorless rhTNSALP, CD6-TNSALP and CD8-TNSALP disclosed herein.
- The term “GPI anchor” is meant to refer to glycosylphosphatidylinositol attached at or near the C-terminus of a membrane bound protein, thereby binding the membrane bound protein to the membrane via its lipidphilic affinity with the membrane.
- The term “GPI anchor signal peptide” is meant to refer to the C-terminus amino acid sequence recognized during post-translational processing as a single for adding GPI and thereby anchoring the protein.
- The term “GPI anchor single peptide sequence” refers to a nucleotide sequence encoding the GPI anchor signal peptide.
- The term “active” means a functional state of a molecule where it performs as it would in vivo, including reactions the enzymes is know to facilitate or binding or blocking functions receptors may be know to possess. Active also includes any pro-active state, pro-enzymes which normally exist in a precursor from; that is not capable of carrying out their known function until activated by another factor or co-factor.
- Sequence identity or percent identity is intended to mean the percentage of same residues between two sequences. The two reference sequences used are the entire peptide sequence of human tissue non-specific alkaline phosphatase precursor (residues 1-524), or the GPI anchor single peptide of human tissue non-specific alkaline phosphatase precursor (residues 506-524). In all sequence comparisons, the two sequences being compared are aligned using the Clustal method (Higgins et al, Cabios 8:189-191, 1992) of multiple sequence alignment in the Lasergene biocomputing software (DNASTAR, INC, Madison, Wis.). In this method, multiple alignments are carried out in a progressive manner, in which larger and larger alignment groups are assembled using similarity scores calculated from a series of pairwise alignments. Optimal sequence alignments are obtained by finding the maximum alignment score, which is the average of all scores between the separate residues in the alignment, determined from a residue weight table representing the probability of a given amino acid change occurring in two related proteins over a given evolutionary interval. Penalties for opening and lengthening gaps in the alignment contribute to the score. The default parameters used with this program are as follows: gap penalty for multiple alignment=10; gap length penalty for multiple alignment=10; k-tuple value in pairwise alignments; gap penalty in pairwise alignment=3; window value in pairwise alignment5; diagonals saved in pairwise alignment=5. The residue weight table used for the alignment program is PAM250 (Dayhoff et al., in Atlas of Protein Sequence and Structure, Dayhoff, Ed., NBRF, Washington, Vol. 5, suppl. 3, p. 345, 1978).
TABLE 1 Percent Identity of ALPs. Shown are calculations of percent identity for comparison of alkaline phosphatase from various mammalian species relative to human tissue non-specific alkaline phosphatase precursor. Species Accession number Percent Identity Human tissue non- NP_000469 100 specific alkaline phosphatase precursor Rhesus tissue non- XP_001109717 97 specific alkaline phosphatase Rat tissue-nonspecific NP_037191 90 alkaline phosphatase Dog tissue non-specific AAF64516 89 alkaline phosphatase Pig alkaline phosphatase AAN64273 88 -
TABLE 2 Percent Identity of GPI anchor single peptide. Shows are calculations of identity for comparisons of GPI anchor single peptide sequences from various mammalian species relative to the GPI binding signal peptide of human tissue non-specific alkaline phosphatase precursor. Species Accession number Percent Identity Human tissue non- NP_000469 100 specific alkaline phosphatase precursor (residues 506-524) Rhesus tissue non- XP_001109717 84 specific alkaline phosphatase residues (634 652) Pig alkaline phosphatase AAN64273 75 (residues 237-253) Dog tissue non-specific AAF64516 68 alkaline phosphatase (residues 487-502) Rat tissue-nonspecific NP_037191 NP_599169 68 alkaline phosphatase (residues 509-524) - Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.
- The GPI anchoring signal peptide 19 amino acid (SEQ:1 residues 506-524) sequence was removed from the C-terminal of the human TNSALP cDNA (SEQ:1) to release the enzyme in the media of CHO—K1 cells. (
FIG. 1 ). The resultant anchorless rhTNSALP enzyme (>95%) was mainly secreted to culture medium in a transient expression study (data is not shown). Acidic oligopeptide-tagged enzymes (CD6-TNSALP and CD8-TNSALP), which also lack the GPI anchoring signal peptide, were secreted in to the culture medium as well. Constructs for the CD6- to CD8-TNSALP cDNA were made and transfected into CHO—K1 cells for transient expression. Cells stably expressed and secreted active TNSALP enzymes into the medium in linear fashion for 12 h. However expression of enzyme plateaued after 12 hours. The inventor's previous work with oligopeptide-tagged enzymes showed that increasing the number of Aspartic acid residues beyond eight caused a substantial reduction of enzyme activity secreted into culture media in the transient expression (data not shown). The inventors chose the 6 and 8 aspartic acid tagged enzymes (CD6-TNSALP and CD8-TNSALP) for further evaluation as their experience had shown that these molecules will exhibit superior expression characteristics. - The purification of these enzymes was performed by a two-step column chromatography method, using DEAE-Sepharose and Sephacryl S-400, as summarized in Table 3. The overall purification yields of anchorless rhTNSALP, CD6-TNSALP, and CD8-TNSALP were 32%, 62%, and 56% of the total enzymes in the culture media, respectively, and the specific activities of each enzyme were 2744, 2411, and 2374 units/mg, respectively. The lower purification yield of anchorless rhTNSALP than those of the tagged enzymes was apparently due to a broader peak eluted from the DEAE column.
TABLE 3 Purification of rhTNSALP and acidic oligopeptide-tagged TNSALP from condition medium Protein concen- Total Total Specific tration protein activity activity Purifi- (mg/l) (mg) (units/mg) (units/mg) cation Yield rhTNSALP Crude 5.26 115 3003 26.1 1 100 media DEAE 18.3 0.66 1555 2354 90 52 column Sephacryl 15.4 0.35 973 2744 105 32 S-400-HR Column CD6-TNSALP Crude 6.27 127 3022 23.9 1 100 media DEAE 32.3 1.01 2073 2043 86 69 column Sephacryl 22.1 0.77 1862 2711 101 62 S-400-HR Column CD8-TNSALP Crude 3.85 184 3065 16.6 1 100 media DEAE 29.1 1.00 2028 2035 123 66 column Sephacryl 22.4 0.72 1702 2374 143 56 S-400-HR Column - When the purified anchorless rhTNSALP was subjected to SDS-PAGE under reducing conditions, a single band with approximately 80 kDa of molecular mass was detected (
FIG. 2 ). An increase of molecular mass associated with an additional acidic oligopeptide was observed in CD6- and CD8-TNSALP. - There was little difference among anchorless rhTNSALP, CD6-TNSALP, and CD8-TNSALP in Michaelis constant (KM), as defined by the pNPP substrate with double-reciprocal plots (0.37, 0.39, and 0.37 mM, respectively), or in chemical inhibition by L-phenylalanine (10 mM; 83%, 86%, and 86% of remaining enzyme activity, respectively) and L-homoarginine (10 mM; 12%, 13%, and 12% of remaining enzyme activity, respectively).
- Affinity for Hydroxyapatite
- A remarkable difference between the tagged and untagged enzymes was observed in their affinity to hydroxyapatite. Affinity to hydroxyapatite for the tagged enzymes was 10-fold higher than that for the untagged enzyme and the binding to hydroxyapatite was seen even at low concentration of the tagged enzyme (
FIG. 3 ). The binding parameters, Kb and Bmax, are shown in Table 4. The values of Kb and Bmax of the tagged enzymes were 10- and 3-fold, respectively, higher than those of the untagged enzyme. Although no significant difference was observed between CD6- and CD8-TNSALP.TABLE 4 Binding parameters of three enzymes to hydroxyapatite. Each value represents the mean ± S.D. of 3 experiments. Kb binding constant and Bmax maximum binding rates were determined form double- reciprocal plots. Kb Bmax (ug/100 ug (ug−1 ml) hydroxyapatite) rhTNSALP 1.7 ± 1.0 0.5 ± 0.2 CD6-TNSALP 36.7 ± 7.9 1.6 ± 0.3 CD8-TNSALP 44.6 ± 4.6 1.9 ± 0.7
Elution Profiles of Enzymes by Lectin Affinity Chromatography - Three enzymes, rhTNSALP, CD6-, and CD8-TNSALP, were subjected to ConA affinity chromatography. (
FIG. 4 ). ConA affinity chromatography indicated there was little unbound enzyme, whereas weakly-bound and strongly-bound enzymes were detected. Overall the elution profiles of these enzymes did not differ when two different concentrations of competitive sugars were added. Since ConA has a high reactivity to the mannosyl residues, the inventors concluded that these enzymes did not differ with respect to mannosyl residue composition. In contrast, the WGA elution profiles between the tagged and untagged enzymes were remarkably different in the ratio of strongly-bound enzyme and weakly-bound enzyme (FIGS. 5A-C). Table 5 shows the percentages of the relative enzyme activity of three fractions on the WGA column. Approximately 30% of the tagged enzymes were weakly bound and 70% was strongly bound to the WGA column, while 66% of the untagged enzyme was weakly bound and 34% was strongly bound to the WGA column. The content of the weakly-bound enzyme was larger in the order of rhTNSALP>CD6-TNSALP>CD8-TNSALP.TABLE 5 Percentage of Unbound, Weakly bound, and Strongly bound fractions obtained by each ConA and WGA column Percent of relative activities ConA WGA WGA + Neuraminidase rhTNSALP CD6 CD8 rhTNSALP CD6 CD8 rhTNSALP CD6 CD8 Unbound 3 4 2 0 3 1 0 2 4 Weakly 59 60 59 66 32 23 9 11 4 Bound Strongly 38 36 39 33 65 76 91 86 92 Bound - To estimate the content of the sialic acid residues of the enzyme, we treated three enzymes with neuraminidase thereby removing the sialic acid residues from the enzymes. After the treatment with neuraminidase, the molecular masses of three enzymes decreased in a similar proportion (
FIG. 6 ). The elution profile of the untagged enzyme on the WGA column changed after the neuraminidase digestion. The earlier fraction accounting for the weakly-bound enzyme shifted to the later fraction for the highly-bound enzyme (FIG. 5D ). On the other hand, the elution profiles of the tagged enzymes on the WGA column slightly changed with neuraminidase digestion (FIGS. 5E and 5F ), since the tagged enzymes originally included a less amount of weakly-bound enzyme. - Biodistribution Offluorescence-Labeled Enzymes
- To evaluate the pharmacokinetic tissue distribution pattern of these enzymes, the fluorescence-labeled enzymes were prepared by the Alexa dye. The efficiencies of labeling in each of three enzymes were approximately 10 mol/mol of protein as dye content.
FIG. 7 shows the histological pictures of biodistribution of three enzymes at the epiphyseal region at 6, 24, 72, and 168 h after a single intravenous infusion.FIG. 8 shows the average of the relative area of fluorescence. Three enzymes were distributed to the mineralized region, but not to the growth plate. At 6 h, the relative areas of fluorescence at the tagged enzymes were four-fold larger than the area at the untagged enzyme. Moreover, the fluorescence-labeled tagged enzymes retained until 168 h with two- to three-fold larger amount than the untagged enzyme. These results were consistent with the result of the in vitro hydroxyapatite affinity experiment. In liver, relatively high amount of enzyme distribution was observed compared to other tissues (data not shown). The distribution was widespread throughout the liver including hepatocytes and sinus-lining cells. The distribution patterns in liver were comparable among three enzymes. In other tissues including brain, lung, heart, spleen, and kidney, no significant difference was observed among three enzymes as well (data not shown). - Overall, the above results showed no biochemical and pharmacokinetic difference between two tagged enzymes.
- In human bone marrow cells derived from a hypophosphatasia patient, mineralization never occurred in the absence of TNSALP even when β-glycerophosphate was added. The addition of one of the enzyme resulted in marked recovery of mineralization (
FIG. 9 ). In contrast, mineralization was observed when Pi was used in the medium instead of β-glycerophosphate even in the absence of any enzyme. The presence of any of the enzymes did not provide any additive effect for the mineralization. These findings indicate that the anchorless rhTNSALP enzyme played a biological role in the mineralization process by providing free Pi released during the hydrolysis of β-glycerophosphate. We added PPi, an inhibitor of mineralization, to see whether the anchorless rhTNSALP enzyme hydrolyze PPi to restore the mineralization. PPi itself completely inhibited the mineralization even in the presence of Pi. The addition of the enzyme restored the mineralization level to PPi-free control culture. - The TNSALP gene knock-out mouse strains as models for hypophosphatasia had <1% of wild-type plasma TNSALP activity. These TNSALP−/− mice were growth impaired, develop epileptic seizures and apnea, and died before weaning as described previously [39,47,48]. Postnatal growth of TNSALP−/− mice treated with anchorless rhTNSALP at 5 mg/kg of body weight and their littermate controls are shown in
FIG. 10 . The average life span of untreated TNSALP−/− mice without anchorless rhTNSALP enzyme administration was 10 days [39,47, 48]. In treated mice, injected with anchorless rhTNSALP, no epileptic seizures appeared until at least 2 months old, in addition the mice lived approximately 4 and 7 times as long. Growth curves of TNSALP−/− mice and littermate controls without treatment are shown inFIG. 11 for comparison. One mouse treated with IP infusion for 4 weeks did not grow well (FIG. 11A ). However after IV infusion began, the mouse increased its body weight substantially. A second mouse treated with IV infusion at birth grew well at subnormal levels (FIG. 11B ). Both of these mice exhibited no abnormal activity and seizures. - Overall, ERT with the C-terminus anchorless rhTNSALP enzyme showed clinical effectiveness on TNSALP−/− mice.
- Production of human recombinant acidic oligopeptide-tagged and untagged TNSALPs (GenBank: NM—000478.2)—The GPI anchoring signal peptide sequence of TNSALP (5′-CTTGCTGCAGGCCCCCTGCTGCTCGCTCTGGCCCTCTACCCCCTGAGCGTCCTGT TC-3′: c.1516C to c.1572C: Leu506 to Phe524) was deleted from the full-length of TNSALP cDNA to produce the enzymes as a secreted form. To produce acidic oligopeptide-tagged TNSALP, a stretch of six or eight of L-Asp (six L-Asp, 5′-GACGATGACGACGATGAT-3′: eight L-Asp, 5′-GATGATGATGATGATGATGACGAC-3′) was introduced additionally at the C-terminus after c.1515C of Ser505 (CD6- or CD8-TNSALP, respectively) mediating a linker (5′-ACCGGTGAAGCAGAGGCC-3′), followed by a termination codon. The three enzymes used for the further experiments were named as anchorless rhTNSALP (human TNSALP anchorless at the C-terminal), CD6-TNSALP (human TNSALP anchorless at the C-terminal tagged with a stretch of six L-Asp), and CD8-TNSALP (human TNSALP anchorless at the C-terminal tagged with a stretch of eight L-Asp), respectively.
- For the preparation of the first strand cDNA, reverse transcriptase reaction was performed by using total RNA isolated from healthy human peripheral blood. To amplify rhTNSALP, CD6-TNSALP, and CD8-TNSALP cDNA, PCR reactions were carried out with the following primers: TNSALP, forward 5′-GAATTCACCCACGTCGATTGCATCTCTGGGCTCCAG-3′ and reverse 5′-ctcgagTCAGCTGCCTGCCGAGCTGGCAGGAGCAC-3′: CD6-TNSALP, forward 5′-GAATTCACCCACGTCGATTGCATCTCTGGGCTCCAG-3′ and reverse 5′-tcaatcatcgtcgtcatcgtcggcctctgcttcaccggtGCTGCCTGCCGAGCTGGCAGGAGCACAGTG-3′: CD8-TNSALP, forward 5′-GAATTCACCCACGTCGATTGCATCTCTGGGCTCCAG-3′ and reverse 5′-tcagtcgtcatcatcatcatcatcatcggcctctgcttcaccggtGCTGCCTGCCGAGCTGGCAGGAGCAC AGTG-3′. The nucleotide sequences compatible with six or eight of L-Asp were added to the reverse primers used here. The amplified cDNA were cloned and sequenced. The cDNA were then transferred into EcoRI cloning sites of mammalian expression vector pCXN, kindly provided by Miyazaki J., Osaka University, Suita, Japan (40).
- The anchorless rhTNSALP, CD6-TNSALP, and CD8-TNSALP cDNAs subcloned in pCXN were then transfected into Chinese hamster ovary (CHO—K1) cells with lipofectamine according to manufacture's instruction (Invitrogen). Selection of colonies was carried out in growth medium with Dulbecco's Modified Eagle Medium supplemented with 15% fetal bovine serum (FBS), plus 600 μg/ml G418 (Sigma-Aldrich) for 10-12 days. Individual clones were picked, grown to confluency, and analyzed for enzyme expression by measuring secreted enzyme activity in the medium as described below. The highest-producing clone was grown in collection medium with Ex-Cell tm 325 PF CHO Protein-free medium JRH Biosciences) and 15% FBS. When the cells reached confluency, the cells were rinsed with PBS and fed with collection media without FBS to collect enzyme for purification.
- Measurement of Alkaline Phosphatase Activity
- A 50 μl of volume of sample was combined with 250 μl of 10 mM -nitrophenyl phosphate (pNPP) (Sigma-Aldrich, Mo.) as a substrate in 1 M diethanolamine, pH 9.8, containing 1 mM magnesium chloride and 0.02 mM zinc chloride, and incubated at 37° C. The time-dependent increase in absorbance at 405 nm (reflecting p-nitrophenolate production) was measured on a plate spectrophotometer (EL800, Bio-Tek Instrument, Inc., VT). One unit of activity was defined as the quantity of enzyme that catalyzed the hydrolysis of 1 μmol substrate in 1 min.
- Enzyme Purification
- The anchorless rhTNSALP enzyme was purified by a two-step column procedure.
- Tris buffer was 25 mM Tris-HCl, pH 8.0, containing 0.1 mM magnesium chloride and 0.01 mM zinc chloride. Unless stated otherwise, all steps were performed at 4° C.
-
Step 1. The medium containing enzyme was filtered through a 0.2 μm filter, and then dialyzed against Tris buffer using Amicon stirred-cell ultrafiltration unit with Millipore ultrafiltration membrane YM-30. -
Step 2. The dialyzed medium was applied to a column of DEAE Sepharose (Sigma-Aldrich, MO) equilibrated with Tris buffer. The column was first washed with Tris buffer, and then the enzyme was eluted with 0-0.4 M NaCl in a linear gradient. -
Step 3. The active eluted fractions were pooled and dialyzed against Tris buffer containing 0.1 M NaCl by using Centricon centrifugal filter device with Millipore ultrafiltration YM-10 filter. The dialyzed fractions were then concentrated forstep 4. -
Step 4. The concentrated enzyme was applied to a column of Sephacryl S-400-HR (Sigma-Aldrich, MO) equilibrated with Tris buffer containing 0.1 M NaCl. The enzyme was eluted with Tris buffer containing 0.1 M NaCl. -
Step 5. The active eluted fractions were pooled and dialyzed against Tris buffer containing 0.1 M NaCl by using Centricon centrifugal filter device with Millipore ultrafiltration YM-10 filter. The dialyzed fractions were then concentrated and stored at −80° C. until use. - Polyacrylamide Gel Electrophoresis
- Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed, followed by silver staining [44, 45].
- Hydroxyapatite binding assay-Hydroxyapatite beads (Sigma-Aldrich) were suspended in 25 mM Tris-HCl buffered saline (TBS), pH 7.4, at concentration of 100 μg/100 μl. The purified enzyme was mixed with the hydroxyapatite suspension at a final concentration of 1.0, 2.5, 5.0, and 10.0 μg/ml. The mixture was mixed at 37° C. for 1 h, and centrifuged at 14,000×rpm for 10 min to separate unbound enzyme and bound enzyme. To determine unbound enzyme, enzyme activity in supernatant was measured, and bound enzyme was determined from the amount of total enzyme and unbound enzyme. Binding constant (Kb) and maximal binding rate (Bmax) were determined from double-reciprocal plots.
- Lectin Affinity Chromatography
- To evaluate the carbohydrate chain structure of the enzymes, we applied the enzymes to lectin affinity chromatography. TBS used here was 10 mM Tris-HCl, pH 8.0, supplemented with 0.5 M sodium chloride, 1 mM calcium chloride, 1 mM magnesium chloride, 1 mM manganese chloride and 0.01 mM zinc chloride. The column of the concanavalin A (Canavalia ensiformis, ConA)-sepharose 4B (Sigma-Aldrich) and the wheat germ agglutinin (Triticum vulgaris, WGA)-agarose CL-4B (Fluka) were equilibrated with TBS at a flow rate of 0.2 ml/min. Lectin affinity chromatography was performed as described previously [43]. Briefly, the purified enzyme in 0.6 ml of TBS was applied to the ConA and WGA columns, and left to stand for 3 h at room temperature. Three fractions were obtained by using two different concentrations, 0.01 M and 0.5 M of α-methyl-D-mannopyranoside (αMM) (Sigma-Aldrich) from ConA column, and 0.1 M and 0.5 M of N-acetyl-D-glucosamine (GlcNAc) (Sigma-Aldrich) from the WGA column: unbound fraction, weakly-bound fraction, and strongly-bound fraction.
- Neuraminidase Digestion
- Tagged and non-tagged rhTNSALPs were digested with α(23, 6, 8, 9) neuraminidase (Arthrobacter ureafaciens) (Sigma-Aldrich) to clarify the content of sialic acids at the carbohydrate chain. Twenty units of each purified TNSALP enzyme were exposed to 0.01 unit of neuraminidase in 250 mM sodium phosphate, pH 6.0, overnight at room temperature. The digested enzyme was then analyzed for polyacrylamide gel electrophoresis and lectin affinity chromatography, as described above.
- Biodistribution of Alexa-Labeled Enzymes
- One mg/ml of purified enzymes were labeled with Alexa Fluor 546 Protein Labeling Kit following manufacture's instruction (Molecular Probes). The Alexa-labeled enzyme was injected to B6 mice (6-7 weeks old) from tail vein at a dose of 1 mg/kg of body weight. Mice were sacrificed at 6, 24, 72, and 168 h after a single infusion, and multiple tissues including brain, lung, heart, liver, spleen, kidney, and leg were dissected. The tissues were immersion-fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned. Tissues were studied by fluorescence microscopy for evaluation of enzyme distribution, and the areas of fluorescence from three fields of fluorescent images around growth plate were quantitated by using AlphaEaseFC (Alpha Innotech Corp.).
- In Vitro Mineralization Assay
- To evaluate the level of bioactivity of the anchorless rhTNSALP enzyme, in vitro mineralization experiments were performed using bone marrow cells derived from a hypophosphatasia patient with an infantile form (10 month old). The bone marrow cells were seeded into 150×25 mm tissue culture dishes. These cells were allowed to attach without disturbance for seven days in growth medium consisting of minimum essential medium alpha (MEMα) supplemented with 10% FBS, 50 units/ml penicillin, and 50 μg/ml streptomycin sulfate. The medium was then replaced to fresh growth medium at 3-day intervals. When the cells reached confluency, they were subcultured in the 12-well plates at a density of 10,000 cells/cm2. On the following day, the growth medium was replaced with the differentiation medium: with MEMα supplemented with 10% FBS, 50 units/ml penicillin, 50 μg/ml streptomycin sulfate, 0.3 mM ascorbic acid, and 100 nM dexamethasone. The differentiation medium also included 2.5 mM Pi or β-glycerophosphate as a phosphate source as well as either anchorless rhTNSALP at 2.5 or 5.0 units/ml. To further investigate the effect of the three enzymes on mineralization in the presence of PPi, 50 μM PPi was added always with each enzyme to the bone marrow cell culture throughout the differentiation period. The differentiation medium was replaced at 3-day intervals. At 12 days after the initiation of the differentiation of the cells, the cells were fixed with 4% paraformaldehyde, followed by staining with Alizarin Red S to detect calcium phosphate deposits [46].
- Long Term ERT With Anchorless rhTNSALP to Evaluate Clinical Effectiveness.
- Long term ERT was performed using the anchorless rhTNSALP enzyme described above. The cephalic vein is the preferred injection rout at birth but is not visible after about 1 week. Intraperitoneal injections were administered from 1 to 4 weeks until the tail vain became visible. Three litermates remained untreated. Two mice (
Specimens 1 and 2) received treatments of 5 mg/kg of body weight. Specimen received enzyme by cephalic vein injection on the day following birth, followed by weekly intraperitoneal injections at 0, 1, 2, 3, and 4 weeks and tail vein injections from 5 through 10 weeks. Similarly aSpecimen 2 received enzyme on the day following birth by cephalic vein injection, followed by weekly intraperitoneal injections at 1, 2, and 3, weeks after which injection was administered though the tail vein From 4 through 10 weeks. - The following numbered references are cited throughout this disclosure. These references are herein incorporated by reference. Applicants reserve the right to challenge the veracity of any statement made in these references.
- [1] D. Fraser, Hypophosphatasia. Am. J. Med. 22 (1957) 730-746.
- [2] M. P. Whyte, Hypophosphatasia, in: C. R. Scriver, A. L. Beaudet, W. S. Sly, D. Valle (Eds), The Metabolic and Molecular Bases of Inherited Disease, eighth ed., McGraw-Hill, New York, 2001, pp. 5313-5329.
- [3] M. M. Silver, G. A. Vilos, K. J. Milne, Pulmonary hypophosphatasia in neonatal hypophosphatasia. Pediatr. Pathol. 8 (1988) 483-493.
- [4] S. Y. Ali, Matrix formation and mineralization in bone, in: C. C. Whitehead (Ed), Bone biology and skeletal disorders, Carfax Publishing Co., Abingdon, U. K., 1992, pp.19-38.
- [5] H. C. Anderson, Molecular biology of matrix vesicles. Clin. Orthop. Relat. Res. 314 (1995) 266-280.
- [6] A. L. Boskey, B. D. Boyan, Z. Schwartz, Matrix vesicles promote mineralization in a gelatin gel. Calcif. Tissue Int. 60 (1997) 309-315.
- [7] A. L. Boskey, Amorphous calcium phosphate: the contention of bone. J. Dent. Res. 76 (1997) 1433-1436.
- [8] H. C. Anderson, Mechanisms of pathologic calcification. Rheum. Dis. Clin. North Am. 14 (1988) 303-319.
- [9] L. F. Bonewald, Z. Schwartz, L. D. Swain, B. D. Boyan, Stimulation of matrix vesicle enzyme activity in osteoblast-like cells by 1,25(OH)2D3 and transforming growth factor beta (TGF beta). Bone Miner. 17 (1992) 139-144.
- [10] K. N. Fedde, Human osteosarcoma cells spontaneously release matrix-vesicle-like structures with the capacity to mineralize. Bone Miner. 17 (1992) 145-151.
- [11] H. Fleisch, R. G. Russell, F. Straumann, Effect of pyrophosphate on hydroxyapatite and its implications in calcium homeostasis. Nature 212 (1966) 901-903.
- [12] A. S. de Jong, T. J. Hak, P. van Duijn, The dynamics of calcium phosphate precipitation studied with a new polyacrylamide steady state matrix-model: influence of pyrophosphate collagen and chondroitin sulfate. Connect. Tissue Res. 7 (1980) 73-79.
- [13] J. L. Meyer, Can biological calcification occur in the presence of pyrophosphate? Arch. Biochem. Biophys. 15 (1984) 1-8.
- [14] D. W. Moss, R. H. Eaton, J. K. Smith, L. G. Whitby, Association of inorganic-pyrophosphatase activity with human alkaline-phosphatase preparations. Biochem. J. 102 (1967) 53-57.
- [15] F. A. Leon, L. A. Rezende, P. Ciancaglini, J. M. Pizauro, Allosteric modulation of pyrophosphatase activity of rat osseous plate alkaline phosphatase by magnesium ions. Int. J. Biochem. Cell Biol. 30 (1998) 89-97.
- [16] R. G. Russell, S. Bisaz, A. Donath, D. B. Morgan, H. Fleisch, Inorganic pyrophosphate in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta, and other disorders of bone. J. Clin. Invest. 50 (1971) 961-965.
- [17] E. Sorensen, H. Flodgaard, Adult hypophosphatasia. Acta. Med. Scand. 197 (1975) 357-360.
- [18] S. A. Sorensen, H. Flodgaard, E. Sorensen, Serum alkaline phosphatase, serum pyrophosphatase, phosphorylethanolamine and inorganic pyrophosphate in plasma and urine. A genetic and clinical study of hypophosphatasia. Monogr. Hum. Genet. 10 (1978) 66-69.
- [19] H. C. Anderson, Pyrophosphate stimulation of calcium uptake into cultureed embryonic bones. Fine structure of matrix vesicles and their role in calcification. Dev. Biol. 34 (1973) 211-227.
- [20] H. C. Anderson, H. H. Hsu, D. C. Morris, K. N. Fedde, M. P. Whyte, Matrix vesicles in osteomalacic hypophosphatasia bone contain apatite-like mineral crystals. Am. J. Pathol. 151 (1997) 1555-1561.
- [21] H. C. Anderson, J. B. Sipe, L. Hessle, R. Dhanyamraju, E. Atti, N. P. Camacho, J. L. Millan, Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice. Am. J. Pathol. 164 (2004) 841-847.
- [22] N. W. Barton, R. O. Brady, J. M. Dambrosia, A. M. Di Bisceglie, S. H. Doppelt, S. C. Hill, H. J. Mankin, G. J. Murray, R. I. Parker, C. E. Argoff, et al Replacement therapy for inherited enzyme deficiency-macrophage-targeted glucocerebrosidase for Gaucher's disease. N. Engl. J. Med. 324 (1991) 1464-1470.
- [23] M. S. Sands, C. Vogler, J. W. Kyle, J. H. Grubb, B. Levy, N. Galvin, W. S. Sly, E. H. Birkenmeier, Enzyme replacement therapy for murine mucopolysaccharidosis type VII. J. Clin. Invest. 93 (1994) 2324-2331.
- [24] R. M. Shull, E. D. Kakkis, M. F. McEntee, S. A. Kania, A. J. Jonas, E. F. Neufeld, Enzyme replacement in a canine model of Hurler syndrome. Proc. Natl. Acad. Sci. 91 (1994) 12937-12941.
- [25] A. C. Crawley, D. A. Brooks, V. J. Muller, B. A. Petersen, E. L. Isaac, J. Bielicki, B. M. King, C. D. Boulter, A. J. Moore, N. L. Fazzalari, D. S. Anson, S. Byers, J. J. Hopwood, Enzyme replacement therapy in a feline model of Maroteaux-Lamy syndrome. J. Clin. Invest. 97 (1996) 1864-1873.
- [26] E. D. Kakkis, J. Muenzer, G. E. Tiller, L. Waber, J. Belmont, M. Passage, B. Izykowski, J. Phillips, R. Doroshow, I. Walot, R. Hoft, E. F. Neufeld, Enzyme-replacement therapy in mucopolysaccharidosis I. N. Engl. J. Med. 344 (2001) 182-188.
- [27] G. Altarescu, S. Hill, E. Wiggs, N. Jeffries, C. Kreps, C. C. Parker, R. O. Brady, N. W. Barton, R. Schiffmann, The efficacy of enzyme replacement therapy in patients with chronic neuronopathic Gaucher's disease. J. Pediatr. 138 (2001) 539-547.
- [28] C. M. Eng, N. Guffon, W. R. Wilcox, D. P. Germain, P. Lee, S. Waldek, L. Caplan, G. E. Linthorst, R. J. Desnick, International Collaborative Fabry Disease Study Group, Safety and efficacy of recombinant human alpha-galactosidase A-replacement therapy in Fabry's disease. N. Engl. J. Med. 345 (2001) 9-16.
- [29] F. S. Furbish, C. J. Steer, N. L. Krett, J. A. Barranger, Uptake and distribution of placental glucocerebrosidase in rat hepatic cells and effects of sequential deglycosylation. Biochim. Biophys. Acta. 673 (1981) 425-434.
- [30] G. J. Murray, Lectin-specific targeting of lysosomal enzymes to reticuloendothelial cells. Methods Enzymol. 149 (1987) 25-42.
- [31] P. D. Stahl, J. S. Rodman, M. J. Miller, P. H. Schlesinger, Evidence for receptor-mediated binding of glycoproteins, glycoconjugates, and glycosidases by alveolar macrophages. Proc. Natl. Acad. Sci. 75 (1978) 1399-1403.
- [32] D. T. Achord, F. E. Brot, C. E. Bell, W. S. Sly, Human beta-glucuronidase: in vivo clearance and in vitro uptake by a glycoprotein recognition system on reticuloendothelial cells. Cell 15 (1978) 269-278.
- [33] J. A. Barranger, E. O'Rourke, Lessons learned from the development of enzyme therapy for Gaucher disease. J. Inherit. Metab. Dis. 24 (2001) 89-96.
- [34] M. P. Whyte, R. Valdes, L. M. Ryan, W. H. McAlister, Infantile hypophosphatasia: enzyme replacement therapy by intravenous infusion of alkaline phosphatase-rich plasma from patients with Paget bone disease. J. Pediatr. 101 (1982) 379-386.
- [35] M. P. Whyte, W. H. McAlister, L. S. Patton, H. L. Magill, M. D. Fallon, W. B. Lorentz, H. G. Herrod, Enzyme replacement therapy for infantile hypophosphatasia attempted by intravenous infusions of alkaline phosphatase-rich Paget plasma: results in three additional patients. J. Pediatr. 105 (1984) 926-933.
- [36] M. P. Whyte, H. L. Magill, M. D. Fallon, H. G. Herrod, Infantile hypophosphatasia: normalization of circulating bone alkaline phosphatase activity followed by skeletal remineralization. Evidence for an intact structural gene for tissue nonspecific alkaline phosphatase. J. Pediatr. 108 (1986) 82-88.
- 37] M. Weninger, R. A. Stinson, H. Plenk, P. Bbck, A. Pollak, Biochemical and morphological effects of human hepatic alkaline phosphatase in a neonate with hypophosphatasia. Acta Paediatr. Scand. Suppl. 360 (1989) 154-160.
- [38] M. P. Whyte, M. Landt, L. M. Ryan, R. A. Mulivor, P. S. Henthorn, K. N. Fedde, J. D. Mahuren, S. P. Coburn, Alkaline phosphatase: placental and tissue-nonspecific isoenzymes hydrolyze phosphoethanolamine, inorganic pyrophosphatem and
pyridoxal 5=40 -phosphate. Substrate accumulation in carriers of hypophosphatasia corrects during pregnancy. J. Clin. Invest. 95 (1995) 1440-1445. - [39] S. Narisawa, C. Wennberg, J. L. Millan. Abnormal vitamin B6 metabolism in alkaline phosphatase knock-out mice causes multiple abnormalities, but not the impaired bone mineralization. J Pathol. 191 (2001) 125-133.
- [40] T. Nishioka, S. Tomatsu, M. A. Gutierrez, K. I. Miyamoto, G. G. Trandafirescu, PL Lopez, G. H. Grubb, R. Kanai, H. Kobayashi, S. Yamaguchi, G. S. Gottesman, R. Cahill, A. Noguchi, K. Miyamoto, W. S. Sly. Enhancement of drug delivery to bone: Characterization of human tissue-nonspecific alkaline phosphatase tagged with an acidic oligopeptide. Mol Genet Metab. 2006 Jul; 88(3):244-255. Epub Apr. 17, 2006.
- [41] Kasugai, S., Fujisawa, R., Waki, Y., Miyamoto, K., and Ohya, K. (2000) J. Bone. Miner. Res. 15, 936-943
- [42]Yokogawa, K., Miya, K., Sekido, T., Higashi, Y., Nomura, M., Fujisawa, R., Morito, K., Masamune, Y., Waki, Y., Kasugai, S., and Miyamoto, K. (2001) Endocrinology 142, 1228-1223
- [43] Koyama, I., Sakagishi, Y., and Komoda, T. (1986) J. Chromatogr. 374 51-59
- [44] U. K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685.
- [45] C. R. Merril, D. Goldman, M. L. Van Keuren, Silver staining methods for polyacrylamide gel electrophoresis. Methods Enzymol. 96 (1983) 230-239.
- [46] S. M. McGEE-RUSSELL, Histochemical methods for calcium. J. Histochem. Cytochem. 6 (1958) 22-42.
- [47] K. G. Waymire, J. D. Mahuren, J. M. Jaje, T. R. Guilarte, S. P. Coburn, G. R. MacGregor, Mice lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B-6. Nat. Genet. 11 (1995) 45-51.
- [48] S. Narisawa, N. Frohlander, J. L. Millan, Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia. Dev. Dyn. 208 (1997) 432-446.
Claims (9)
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/484,870 US20070081984A1 (en) | 2005-10-11 | 2006-07-11 | Compositions and methods for treating hypophosphatasia |
US12/405,920 US7943126B2 (en) | 2005-10-11 | 2009-03-17 | Compositions and methods for treating hypophosphatasia |
US12/497,612 US7972593B2 (en) | 2004-06-10 | 2009-07-03 | Delivery of therapeutic agents to the bone |
US13/071,445 US8691208B2 (en) | 2005-10-11 | 2011-03-24 | Compositions and methods for treating hypophosphatasia |
US13/112,924 US20110311487A1 (en) | 2004-06-10 | 2011-05-20 | Delivery of therapeutic agents to the bone |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US72556305P | 2005-10-11 | 2005-10-11 | |
US11/484,870 US20070081984A1 (en) | 2005-10-11 | 2006-07-11 | Compositions and methods for treating hypophosphatasia |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/864,758 Continuation-In-Part US7863238B2 (en) | 2004-06-10 | 2004-06-10 | Proteins with an attached short peptide of acidic amino acids |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/245,424 Continuation-In-Part US20070081986A1 (en) | 2004-06-10 | 2005-10-07 | Beta-glucuronidase with an attached short peptide of acidic amino acids |
US12/405,920 Division US7943126B2 (en) | 2005-10-11 | 2009-03-17 | Compositions and methods for treating hypophosphatasia |
Publications (1)
Publication Number | Publication Date |
---|---|
US20070081984A1 true US20070081984A1 (en) | 2007-04-12 |
Family
ID=37911243
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/484,870 Abandoned US20070081984A1 (en) | 2004-06-10 | 2006-07-11 | Compositions and methods for treating hypophosphatasia |
US12/405,920 Active US7943126B2 (en) | 2005-10-11 | 2009-03-17 | Compositions and methods for treating hypophosphatasia |
US13/071,445 Active 2027-01-31 US8691208B2 (en) | 2005-10-11 | 2011-03-24 | Compositions and methods for treating hypophosphatasia |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/405,920 Active US7943126B2 (en) | 2005-10-11 | 2009-03-17 | Compositions and methods for treating hypophosphatasia |
US13/071,445 Active 2027-01-31 US8691208B2 (en) | 2005-10-11 | 2011-03-24 | Compositions and methods for treating hypophosphatasia |
Country Status (1)
Country | Link |
---|---|
US (3) | US20070081984A1 (en) |
Cited By (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060014687A1 (en) * | 2004-04-21 | 2006-01-19 | Philippe Crine | Bone delivery conjugates and method of using same to target proteins to bone |
WO2008138131A1 (en) * | 2007-05-11 | 2008-11-20 | Enobia Pharma Inc. | Bone targeted alkaline phosphatase, kits and methods of use thereof |
US20110250187A1 (en) * | 2005-10-11 | 2011-10-13 | Saint Louis University | Compositions and methods for treating hypophosphatasia |
WO2011134084A1 (en) * | 2010-04-30 | 2011-11-03 | Enobia Pharma Inc. | Methods, compositions, and kits for the treatment of matrix mineralization disorders |
US20110300143A1 (en) * | 2006-06-27 | 2011-12-08 | Saint Louis University | Prenatal enzyme replacement therapy |
US9266939B2 (en) | 2010-12-27 | 2016-02-23 | Alexion Pharmaceuticals, Inc. | Compositions comprising natriuretic peptides and methods of use thereof |
WO2016090251A1 (en) | 2014-12-05 | 2016-06-09 | Alexion Pharmaceuticals, Inc. | Treating seizure with recombinant alkaline phosphatase |
US10052366B2 (en) | 2012-05-21 | 2018-08-21 | Alexion Pharmaceuticsl, Inc. | Compositions comprising alkaline phosphatase and/or natriuretic peptide and methods of use thereof |
CN109152820A (en) * | 2016-04-01 | 2019-01-04 | 阿雷克森制药公司 | It is powerless with alkaline phosphatase enzyme treatment muscle |
EP3488861A1 (en) | 2011-10-19 | 2019-05-29 | Alexion Pharmaceuticals, Inc. | Compositions comprising alkaline phosphatase and/or natriuretic peptide and methods of use thereof |
CN110499285A (en) * | 2018-05-17 | 2019-11-26 | 西安组织工程与再生医学研究所 | Application of the ALPL gene in the product of preparation prevention and/or treatment low alkalinity phosphatase disease |
US10603361B2 (en) | 2015-01-28 | 2020-03-31 | Alexion Pharmaceuticals, Inc. | Methods of treating a subject with an alkaline phosphatase deficiency |
US10822596B2 (en) | 2014-07-11 | 2020-11-03 | Alexion Pharmaceuticals, Inc. | Compositions and methods for treating craniosynostosis |
US10898549B2 (en) | 2016-04-01 | 2021-01-26 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia in adolescents and adults |
US10988744B2 (en) | 2016-06-06 | 2021-04-27 | Alexion Pharmaceuticals, Inc. | Method of producing alkaline phosphatase |
US11065306B2 (en) | 2016-03-08 | 2021-07-20 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia in children |
US11116821B2 (en) | 2016-08-18 | 2021-09-14 | Alexion Pharmaceuticals, Inc. | Methods for treating tracheobronchomalacia |
US11224637B2 (en) | 2017-03-31 | 2022-01-18 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia (HPP) in adults and adolescents |
US11229686B2 (en) | 2015-09-28 | 2022-01-25 | Alexion Pharmaceuticals, Inc. | Reduced frequency dosage regimens for tissue non-specific alkaline phosphatase (TNSALP)-enzyme replacement therapy of hypophosphatasia |
US11352612B2 (en) | 2015-08-17 | 2022-06-07 | Alexion Pharmaceuticals, Inc. | Manufacturing of alkaline phosphatases |
US11400140B2 (en) | 2015-10-30 | 2022-08-02 | Alexion Pharmaceuticals, Inc. | Methods for treating craniosynostosis in a patient |
US11913039B2 (en) | 2018-03-30 | 2024-02-27 | Alexion Pharmaceuticals, Inc. | Method for producing recombinant alkaline phosphatase |
US12083169B2 (en) | 2021-02-12 | 2024-09-10 | Alexion Pharmaceuticals, Inc. | Alkaline phosphatase polypeptides and methods of use thereof |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019139891A1 (en) | 2018-01-09 | 2019-07-18 | Synthetic Biologics, Inc. | Alkaline phosphatase agents for treatment of neurodevelopmental disorders |
EP3768302A4 (en) | 2018-03-20 | 2021-12-15 | Synthetic Biologics, Inc. | Intestinal alkaline phosphatase formulations |
EP3773686B1 (en) | 2018-03-20 | 2023-06-07 | Theriva Biologics, Inc. | Alkaline phosphatase agents for treatment of radiation disorders |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6455495B1 (en) * | 1997-02-14 | 2002-09-24 | The Salk Institute For Biological Studies | Methods and compositions for delivery of therapeutic agents to bone tissue employing conjugates of negatively charged peptide oligomers with therapeutic agents |
US20050276796A1 (en) * | 2004-06-10 | 2005-12-15 | Shunji Tomatsu | Proteins with an attached short peptide of acidic amino acids |
US20060014687A1 (en) * | 2004-04-21 | 2006-01-19 | Philippe Crine | Bone delivery conjugates and method of using same to target proteins to bone |
Family Cites Families (33)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2102808A1 (en) | 1991-05-10 | 1992-11-11 | Hanne Bentz | Targeted delivery of bone growth factors |
AU4835693A (en) | 1993-08-13 | 1995-03-14 | Rijksuniversiteit Te Groningen | Pharmaceutical composition comprising phosphatase or a derivative thereof |
JPH0870875A (en) | 1994-09-05 | 1996-03-19 | Tosoh Corp | Recombined alkali phosphatase-fused protein |
US5863782A (en) | 1995-04-19 | 1999-01-26 | Women's And Children's Hospital | Synthetic mammalian sulphamidase and genetic sequences encoding same |
CA2245903A1 (en) | 1998-09-28 | 2000-03-28 | Mcgill University | Use of pex in the treatment of metabolic bone diseases |
CA2262056A1 (en) | 1999-02-24 | 2000-08-24 | Guy Boileau | Composition, methods and reagents for the synthesis of a soluble form of human pex |
EP1176985A2 (en) | 1999-04-28 | 2002-02-06 | Vectramed, Inc. | Enzymatically activated polymeric drug conjugates |
JP2000327583A (en) | 1999-05-17 | 2000-11-28 | Medei Sci Puraningu:Kk | Bone-directional hormone derivative |
EP1232276B8 (en) | 1999-11-16 | 2007-06-27 | Genzyme Corporation | Vectors and transgenes with regulatory elements for gene delivery to the liver |
US6420384B2 (en) | 1999-12-17 | 2002-07-16 | Ariad Pharmaceuticals, Inc. | Proton pump inhibitors |
AU2001287429B2 (en) | 2000-08-23 | 2005-08-18 | Alexion Pharmaceuticals, Inc. | Method and compositions for promoting osteogenesis |
US6436386B1 (en) | 2000-11-14 | 2002-08-20 | Shearwater Corporation | Hydroxyapatite-targeting poly (ethylene glycol) and related polymers |
AU2002255478A1 (en) | 2001-01-10 | 2002-09-12 | Pe Corporation (Ny) | Kits, such as nucleic acid arrays, comprising a majority of human exons or transcripts, for detecting expression and other uses thereof |
US7888372B2 (en) | 2001-03-23 | 2011-02-15 | National Institutes Of Health (Nih) | Compositions and methods for modulating bone mineral deposition |
WO2002092020A2 (en) | 2001-03-23 | 2002-11-21 | The Burnham Institute | Compositions and methods for modulating bone mineral deposition |
DE60233047D1 (en) | 2001-05-14 | 2009-09-03 | Gbp Ip Llc | LENTIVIRAL VECTORS ENCODING FLAMMABLE FACTORS FOR GENETHERAPY |
US20030158132A1 (en) | 2002-01-22 | 2003-08-21 | Genvec, Inc. | Method for enhancing bone density or formation |
CA2433479A1 (en) | 2002-07-22 | 2004-01-22 | F. Hoffmann-La Roche Ag | Conjugate of a tissue non-specific alkaline phosphatase and dextran, process for its production and use thereof |
CA2527878A1 (en) | 2003-05-30 | 2005-01-27 | Alexion Pharmaceuticals, Inc. | Antibodies and fusion proteins that include engineered constant regions |
WO2005047334A1 (en) | 2003-11-13 | 2005-05-26 | Hanmi Pharmaceutical. Co., Ltd. | Igg fc fragment for a drug carrier and method for the preparation thereof |
US20070081984A1 (en) | 2005-10-11 | 2007-04-12 | Shunji Tomatsu | Compositions and methods for treating hypophosphatasia |
US7972593B2 (en) | 2004-06-10 | 2011-07-05 | Saint Louis University | Delivery of therapeutic agents to the bone |
US20070081986A1 (en) | 2005-10-07 | 2007-04-12 | Shunji Tomatsu | Beta-glucuronidase with an attached short peptide of acidic amino acids |
US20090142347A1 (en) | 2004-09-29 | 2009-06-04 | The Burnham Institute For Medical Research | Tissue-Nonspecific Alkaline Phosphatase (TNAP): a Therapeutic Target for Arterial Calcification |
MX2007006524A (en) | 2004-12-01 | 2007-06-22 | Genzyme Corp | Methods for targeted delivery of genetic material to the liver. |
US20070042957A1 (en) | 2005-08-19 | 2007-02-22 | Mayo Foundation For Medical Education And Research | Type v phosphodiesterase inhibitors and natriuretic polypeptides |
WO2007035600A2 (en) | 2005-09-16 | 2007-03-29 | Mayo Foundation For Education And Research | Natriuretic activities |
US7625564B2 (en) | 2006-01-27 | 2009-12-01 | Novagen Holding Corporation | Recombinant human EPO-Fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo |
US7820623B2 (en) | 2006-10-25 | 2010-10-26 | Amgen Inc. | Conjugated toxin peptide therapeutic agents |
US20080181903A1 (en) | 2006-12-21 | 2008-07-31 | Pdl Biopharma, Inc. | Conjugate of natriuretic peptide and antibody constant region |
PL2662448T3 (en) | 2007-05-11 | 2017-07-31 | Alexion Pharmaceuticals, Inc. | Bone targeted alkaline phosphatase, kits and methods of use thereof |
US20100184680A1 (en) | 2007-09-11 | 2010-07-22 | Dorian Bevec | Therapeutic uses of b-type natriuretic peptide and human growth hormone 1-43 |
CA2797865A1 (en) | 2010-04-30 | 2011-11-03 | Alexion Pharma International Sarl | Methods, compositions, and kits for the treatment of matrix mineralization disorders |
-
2006
- 2006-07-11 US US11/484,870 patent/US20070081984A1/en not_active Abandoned
-
2009
- 2009-03-17 US US12/405,920 patent/US7943126B2/en active Active
-
2011
- 2011-03-24 US US13/071,445 patent/US8691208B2/en active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6455495B1 (en) * | 1997-02-14 | 2002-09-24 | The Salk Institute For Biological Studies | Methods and compositions for delivery of therapeutic agents to bone tissue employing conjugates of negatively charged peptide oligomers with therapeutic agents |
US20060014687A1 (en) * | 2004-04-21 | 2006-01-19 | Philippe Crine | Bone delivery conjugates and method of using same to target proteins to bone |
US20050276796A1 (en) * | 2004-06-10 | 2005-12-15 | Shunji Tomatsu | Proteins with an attached short peptide of acidic amino acids |
Cited By (44)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060014687A1 (en) * | 2004-04-21 | 2006-01-19 | Philippe Crine | Bone delivery conjugates and method of using same to target proteins to bone |
US7763712B2 (en) | 2004-04-21 | 2010-07-27 | Enobia Pharma Inc. | Bone delivery conjugates and method of using same to target proteins to bone |
US20100221234A1 (en) * | 2004-04-21 | 2010-09-02 | Enobia Pharma Inc. | Bone delivery conjugates and method of using same to target proteins to bone |
US20100240125A1 (en) * | 2004-04-21 | 2010-09-23 | Enobia Pharma Inc. | Bone delivery conjugates and method of using same to target proteins to bone |
US10000532B2 (en) | 2004-04-21 | 2018-06-19 | Alexion Pharmaceuticals, Inc. | Bone delivery conjugates and method of using same to target proteins to bone |
US7960529B2 (en) | 2004-04-21 | 2011-06-14 | Enobia Pharma Inc. | Bone delivery conjugates and method of using same to target proteins to bone |
US11248021B2 (en) | 2004-04-21 | 2022-02-15 | Alexion Pharmaceuticals, Inc. | Bone delivery conjugates and method of using same to target proteins to bone |
US8691208B2 (en) * | 2005-10-11 | 2014-04-08 | Saint Louis University | Compositions and methods for treating hypophosphatasia |
US20110250187A1 (en) * | 2005-10-11 | 2011-10-13 | Saint Louis University | Compositions and methods for treating hypophosphatasia |
US20110300143A1 (en) * | 2006-06-27 | 2011-12-08 | Saint Louis University | Prenatal enzyme replacement therapy |
US8784833B2 (en) * | 2006-06-27 | 2014-07-22 | Saint Louis University | Prenatal enzyme replacement therapy for hypophosphatasia |
DE202008018131U1 (en) | 2007-05-11 | 2011-12-30 | Enobia Pharma Inc. | Bone-targeted alkaline phosphatase and kits thereof |
WO2008138131A1 (en) * | 2007-05-11 | 2008-11-20 | Enobia Pharma Inc. | Bone targeted alkaline phosphatase, kits and methods of use thereof |
US20100297119A1 (en) * | 2007-05-11 | 2010-11-25 | Enobia Pharma Inc. | Bone targeted alkaline phosphatase, kits and methods of use thereof |
EP2662448A1 (en) | 2007-05-11 | 2013-11-13 | Alexion Pharma International SARL | Bone targeted alkaline phosphatase, kits and methods of use thereof |
EP2368999A1 (en) | 2007-05-11 | 2011-09-28 | Enobia Pharma Inc. | Bone targeted alkaline phosphatase, kits and methods of use thereof |
JP2013525379A (en) * | 2010-04-30 | 2013-06-20 | アレクシオン ファーマ インターナショナル エスアーアールエル | Methods, compositions and kits for treating substrate calcification disorders |
WO2011134084A1 (en) * | 2010-04-30 | 2011-11-03 | Enobia Pharma Inc. | Methods, compositions, and kits for the treatment of matrix mineralization disorders |
US9988620B2 (en) | 2010-04-30 | 2018-06-05 | Alexion Pharmaceuticals, Inc. | Methods, compositions, and kits for the treatment of matrix mineralization disorders |
US9266939B2 (en) | 2010-12-27 | 2016-02-23 | Alexion Pharmaceuticals, Inc. | Compositions comprising natriuretic peptides and methods of use thereof |
WO2012099851A3 (en) * | 2011-01-17 | 2014-04-10 | Saint Louis University | Prenatal enzyme replacement therapy |
WO2012099851A2 (en) * | 2011-01-17 | 2012-07-26 | Saint Louis University | Prenatal enzyme replacement therapy |
EP3488861A1 (en) | 2011-10-19 | 2019-05-29 | Alexion Pharmaceuticals, Inc. | Compositions comprising alkaline phosphatase and/or natriuretic peptide and methods of use thereof |
US10052366B2 (en) | 2012-05-21 | 2018-08-21 | Alexion Pharmaceuticsl, Inc. | Compositions comprising alkaline phosphatase and/or natriuretic peptide and methods of use thereof |
US10822596B2 (en) | 2014-07-11 | 2020-11-03 | Alexion Pharmaceuticals, Inc. | Compositions and methods for treating craniosynostosis |
US11224638B2 (en) | 2014-12-05 | 2022-01-18 | Alexion Pharmaceuticals, Inc. | Treating seizure with recombinant alkaline phosphatase |
US10449236B2 (en) | 2014-12-05 | 2019-10-22 | Alexion Pharmaceuticals, Inc. | Treating seizure with recombinant alkaline phosphatase |
WO2016090251A1 (en) | 2014-12-05 | 2016-06-09 | Alexion Pharmaceuticals, Inc. | Treating seizure with recombinant alkaline phosphatase |
US11564978B2 (en) | 2015-01-28 | 2023-01-31 | Alexion Pharmaceuticals, Inc. | Methods of treating a subject with an alkaline phosphatase deficiency |
US10603361B2 (en) | 2015-01-28 | 2020-03-31 | Alexion Pharmaceuticals, Inc. | Methods of treating a subject with an alkaline phosphatase deficiency |
US11352612B2 (en) | 2015-08-17 | 2022-06-07 | Alexion Pharmaceuticals, Inc. | Manufacturing of alkaline phosphatases |
US11229686B2 (en) | 2015-09-28 | 2022-01-25 | Alexion Pharmaceuticals, Inc. | Reduced frequency dosage regimens for tissue non-specific alkaline phosphatase (TNSALP)-enzyme replacement therapy of hypophosphatasia |
US11400140B2 (en) | 2015-10-30 | 2022-08-02 | Alexion Pharmaceuticals, Inc. | Methods for treating craniosynostosis in a patient |
US11065306B2 (en) | 2016-03-08 | 2021-07-20 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia in children |
US11186832B2 (en) | 2016-04-01 | 2021-11-30 | Alexion Pharmaceuticals, Inc. | Treating muscle weakness with alkaline phosphatases |
US10898549B2 (en) | 2016-04-01 | 2021-01-26 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia in adolescents and adults |
CN109152820A (en) * | 2016-04-01 | 2019-01-04 | 阿雷克森制药公司 | It is powerless with alkaline phosphatase enzyme treatment muscle |
EP3436052A4 (en) * | 2016-04-01 | 2019-10-09 | Alexion Pharmaceuticals, Inc. | Treating muscle weakness with alkaline phosphatases |
US10988744B2 (en) | 2016-06-06 | 2021-04-27 | Alexion Pharmaceuticals, Inc. | Method of producing alkaline phosphatase |
US11116821B2 (en) | 2016-08-18 | 2021-09-14 | Alexion Pharmaceuticals, Inc. | Methods for treating tracheobronchomalacia |
US11224637B2 (en) | 2017-03-31 | 2022-01-18 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia (HPP) in adults and adolescents |
US11913039B2 (en) | 2018-03-30 | 2024-02-27 | Alexion Pharmaceuticals, Inc. | Method for producing recombinant alkaline phosphatase |
CN110499285A (en) * | 2018-05-17 | 2019-11-26 | 西安组织工程与再生医学研究所 | Application of the ALPL gene in the product of preparation prevention and/or treatment low alkalinity phosphatase disease |
US12083169B2 (en) | 2021-02-12 | 2024-09-10 | Alexion Pharmaceuticals, Inc. | Alkaline phosphatase polypeptides and methods of use thereof |
Also Published As
Publication number | Publication date |
---|---|
US7943126B2 (en) | 2011-05-17 |
US20090238814A1 (en) | 2009-09-24 |
US20110250187A1 (en) | 2011-10-13 |
US8691208B2 (en) | 2014-04-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8691208B2 (en) | Compositions and methods for treating hypophosphatasia | |
Nishioka et al. | Enhancement of drug delivery to bone: characterization of human tissue-nonspecific alkaline phosphatase tagged with an acidic oligopeptide | |
Tomatsu et al. | Enzyme replacement therapy in a murine model of Morquio A syndrome | |
US10716862B2 (en) | Use of P97 as an enzyme delivery system for the delivery of therapeutic lysosomal enzymes | |
US10301369B2 (en) | Nucleic acids encoding targeted therapeutic lysosomal enzyme fusion proteins | |
Tomatsu et al. | Enhancement of drug delivery: enzyme-replacement therapy for murine Morquio A syndrome | |
Sands et al. | Enzyme replacement therapy for murine mucopolysaccharidosis type VII. | |
EP2662448B1 (en) | Bone targeted alkaline phosphatase, kits and methods of use thereof | |
JP4874954B2 (en) | Bone delivery complex and methods of use for targeting bone to proteins | |
EP1981546B1 (en) | Enzyme replacement therapy for treating lysosomal storage diseases | |
JP6623213B2 (en) | Mannose-6-phosphate containing peptide fused to lysosomal enzyme | |
US20030215432A1 (en) | Methods and compositions for delivering enzymes and nucleic acid molecules to brain, bone, and other tissues | |
WO2016077356A9 (en) | Therapeutic compositions of alpha-l-iduronidase, iduronate-2-sulfatase, and alpha-galactosidase a and methods of use thereof | |
Montaño et al. | Acidic amino acid tag enhances response to enzyme replacement in mucopolysaccharidosis type VII mice | |
WO2005094874A1 (en) | Medical use of alpha-mannosidase | |
WO2003068255A1 (en) | Compositions and methods for improving enzyme replacement therapy of lysosomal storage diseases | |
RU2811100C1 (en) | Compound containing therapeutic enzyme and transport element connected to each other directly or by using linker | |
US20240318156A1 (en) | Compositions of beta-hexosaminidase variants and uses thereof | |
EP4389772A1 (en) | Targeted delivery of therapeutic enzymes | |
US20240350653A1 (en) | Targeted delivery of therapeutic enzymes | |
EA044641B1 (en) | COMPOUND CONTAINING A THERAPEUTIC ENZYME AND A TRANSPORT ELEMENT CONNECTED TO EACH OTHER DIRECTLY OR BY USING A LINKER | |
Dunder | The application of enzyme replacement therapy in vitro and in a mouse model in aspartylglycosaminuria |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: SAINT LOUIS UNIVERSITY, MISSOURI Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:TOMATSU, SHUNJI;NISHIOKA, TATSUO;GRUBB, JEFFERY H.;AND OTHERS;REEL/FRAME:019494/0785;SIGNING DATES FROM 20070615 TO 20070628 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: KANAZAWA UNIVERSITY, JAPAN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SAINT LOUIS UNIVERSITY;REEL/FRAME:032214/0107 Effective date: 20140211 Owner name: SHIMANE UNIVERSITY, JAPAN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SAINT LOUIS UNIVERSITY;REEL/FRAME:032214/0107 Effective date: 20140211 Owner name: SAINT LOUIS UNIVERSITY, MISSOURI Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SAINT LOUIS UNIVERSITY;REEL/FRAME:032214/0107 Effective date: 20140211 |