CN1230220A - 半乳糖苷酶a缺乏症的治疗 - Google Patents
半乳糖苷酶a缺乏症的治疗 Download PDFInfo
- Publication number
- CN1230220A CN1230220A CN97197909A CN97197909A CN1230220A CN 1230220 A CN1230220 A CN 1230220A CN 97197909 A CN97197909 A CN 97197909A CN 97197909 A CN97197909 A CN 97197909A CN 1230220 A CN1230220 A CN 1230220A
- Authority
- CN
- China
- Prior art keywords
- cell
- galactosidase
- gal
- sequence
- human alpha
- 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.)
- Granted
Links
- 238000002560 therapeutic procedure Methods 0.000 title abstract description 7
- 230000007812 deficiency Effects 0.000 title description 3
- 101000718525 Homo sapiens Alpha-galactosidase A Proteins 0.000 claims abstract description 42
- 102000043404 human GLA Human genes 0.000 claims abstract description 42
- 208000024720 Fabry Disease Diseases 0.000 claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims description 236
- 108090000623 proteins and genes Proteins 0.000 claims description 64
- 108090000790 Enzymes Proteins 0.000 claims description 55
- 102000004190 Enzymes Human genes 0.000 claims description 55
- 108020004414 DNA Proteins 0.000 claims description 48
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 47
- 238000000034 method Methods 0.000 claims description 40
- 102000004169 proteins and genes Human genes 0.000 claims description 39
- NBSCHQHZLSJFNQ-QTVWNMPRSA-N D-Mannose-6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@@H]1O NBSCHQHZLSJFNQ-QTVWNMPRSA-N 0.000 claims description 38
- 108010000521 Human Growth Hormone Proteins 0.000 claims description 37
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 33
- 102000002265 Human Growth Hormone Human genes 0.000 claims description 32
- 239000000854 Human Growth Hormone Substances 0.000 claims description 32
- 230000014509 gene expression Effects 0.000 claims description 32
- 239000011347 resin Substances 0.000 claims description 25
- 229920005989 resin Polymers 0.000 claims description 25
- 238000011282 treatment Methods 0.000 claims description 25
- 238000001890 transfection Methods 0.000 claims description 23
- 229920001184 polypeptide Polymers 0.000 claims description 21
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 21
- 230000002018 overexpression Effects 0.000 claims description 11
- 230000028327 secretion Effects 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 230000001225 therapeutic effect Effects 0.000 claims description 8
- 230000008488 polyadenylation Effects 0.000 claims description 7
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 6
- 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 6
- 210000003725 endotheliocyte Anatomy 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 5
- 239000003981 vehicle Substances 0.000 claims description 5
- 230000002209 hydrophobic effect Effects 0.000 claims description 4
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 3
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 3
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 3
- 206010028980 Neoplasm Diseases 0.000 claims description 3
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 201000011510 cancer Diseases 0.000 claims description 3
- 238000011210 chromatographic step Methods 0.000 claims description 3
- 210000002919 epithelial cell Anatomy 0.000 claims description 3
- 125000000524 functional group Chemical group 0.000 claims description 3
- 229920000669 heparin Polymers 0.000 claims description 3
- 229960002897 heparin Drugs 0.000 claims description 3
- 210000002510 keratinocyte Anatomy 0.000 claims description 3
- 210000005229 liver cell Anatomy 0.000 claims description 3
- 239000002243 precursor Substances 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- 238000013519 translation Methods 0.000 claims description 3
- 206010006187 Breast cancer Diseases 0.000 claims description 2
- 208000026310 Breast neoplasm Diseases 0.000 claims description 2
- 206010033128 Ovarian cancer Diseases 0.000 claims description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 2
- 108020005038 Terminator Codon Proteins 0.000 claims description 2
- 208000032839 leukemia Diseases 0.000 claims description 2
- 201000001441 melanoma Diseases 0.000 claims description 2
- 239000002808 molecular sieve Substances 0.000 claims description 2
- 210000000663 muscle cell Anatomy 0.000 claims description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 2
- 102000053602 DNA Human genes 0.000 claims 1
- 239000003937 drug carrier Substances 0.000 claims 1
- 238000000338 in vitro Methods 0.000 claims 1
- 210000003125 jurkat cell Anatomy 0.000 claims 1
- 210000005260 human cell Anatomy 0.000 abstract description 8
- 101000718529 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) Alpha-galactosidase Proteins 0.000 description 156
- NNISLDGFPWIBDF-MPRBLYSKSA-N alpha-D-Gal-(1->3)-beta-D-Gal-(1->4)-D-GlcNAc Chemical compound O[C@@H]1[C@@H](NC(=O)C)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@@H](CO)O1 NNISLDGFPWIBDF-MPRBLYSKSA-N 0.000 description 156
- 108010030291 alpha-Galactosidase Proteins 0.000 description 57
- 102000005840 alpha-Galactosidase Human genes 0.000 description 56
- 239000012634 fragment Substances 0.000 description 55
- 229940088598 enzyme Drugs 0.000 description 54
- 230000000694 effects Effects 0.000 description 37
- 235000018102 proteins Nutrition 0.000 description 37
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 35
- 239000002299 complementary DNA Substances 0.000 description 33
- 239000000523 sample Substances 0.000 description 31
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 29
- 108091034117 Oligonucleotide Proteins 0.000 description 27
- 150000001413 amino acids Chemical class 0.000 description 25
- 108020004707 nucleic acids Proteins 0.000 description 25
- 102000039446 nucleic acids Human genes 0.000 description 25
- 150000007523 nucleic acids Chemical class 0.000 description 25
- 239000013612 plasmid Substances 0.000 description 25
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 23
- 235000001014 amino acid Nutrition 0.000 description 23
- 229940024606 amino acid Drugs 0.000 description 22
- 238000013016 damping Methods 0.000 description 22
- 239000012530 fluid Substances 0.000 description 22
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 21
- 238000003153 stable transfection Methods 0.000 description 20
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 19
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 17
- 201000010099 disease Diseases 0.000 description 17
- 239000007788 liquid Substances 0.000 description 17
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 16
- 210000002950 fibroblast Anatomy 0.000 description 16
- 210000001519 tissue Anatomy 0.000 description 16
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 15
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 15
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 238000012239 gene modification Methods 0.000 description 14
- 230000005017 genetic modification Effects 0.000 description 14
- 235000013617 genetically modified food Nutrition 0.000 description 14
- 239000000047 product Substances 0.000 description 14
- 210000003712 lysosome Anatomy 0.000 description 13
- 239000000758 substrate Substances 0.000 description 13
- 229920000936 Agarose Polymers 0.000 description 12
- 241000701022 Cytomegalovirus Species 0.000 description 12
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 12
- 230000029087 digestion Effects 0.000 description 12
- 108010085238 Actins Proteins 0.000 description 11
- 238000004587 chromatography analysis Methods 0.000 description 11
- 108010006232 Neuraminidase Proteins 0.000 description 10
- 102000005348 Neuraminidase Human genes 0.000 description 10
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- 102000012422 Collagen Type I Human genes 0.000 description 9
- 108010022452 Collagen Type I Proteins 0.000 description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 description 9
- 238000001415 gene therapy Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 102000005962 receptors Human genes 0.000 description 9
- 108020003175 receptors Proteins 0.000 description 9
- 108010031099 Mannose Receptor Proteins 0.000 description 8
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 230000001868 lysosomic effect Effects 0.000 description 8
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 8
- 102000007469 Actins Human genes 0.000 description 7
- 102000004594 DNA Polymerase I Human genes 0.000 description 7
- 108010017826 DNA Polymerase I Proteins 0.000 description 7
- 239000007983 Tris buffer Substances 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- 201000009431 angiokeratoma Diseases 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- 230000000875 corresponding effect Effects 0.000 description 6
- 239000007943 implant Substances 0.000 description 6
- 230000002132 lysosomal effect Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000001488 sodium phosphate Substances 0.000 description 6
- 229910000162 sodium phosphate Inorganic materials 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 6
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 5
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 5
- 241000212384 Bifora Species 0.000 description 5
- 208000027219 Deficiency disease Diseases 0.000 description 5
- -1 M6P glycan Chemical class 0.000 description 5
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 230000013595 glycosylation Effects 0.000 description 5
- 238000006206 glycosylation reaction Methods 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000001155 isoelectric focusing Methods 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 108091026890 Coding region Proteins 0.000 description 4
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000012505 Superdex™ Substances 0.000 description 4
- 239000012190 activator Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 102220369446 c.1274G>A Human genes 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 239000003636 conditioned culture medium Substances 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 210000003953 foreskin Anatomy 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 102220023258 rs387907548 Human genes 0.000 description 4
- 239000012619 Butyl Sepharose® Substances 0.000 description 3
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 206010017076 Fracture Diseases 0.000 description 3
- 102000004547 Glucosylceramidase Human genes 0.000 description 3
- 108010017544 Glucosylceramidase Proteins 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- 102100039619 Granulocyte colony-stimulating factor Human genes 0.000 description 3
- 108090001090 Lectins Proteins 0.000 description 3
- 102000004856 Lectins Human genes 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241001045988 Neogene Species 0.000 description 3
- 102000010292 Peptide Elongation Factor 1 Human genes 0.000 description 3
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 102000001708 Protein Isoforms Human genes 0.000 description 3
- 108010029485 Protein Isoforms Proteins 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 102000005936 beta-Galactosidase Human genes 0.000 description 3
- 108010005774 beta-Galactosidase Proteins 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 230000001186 cumulative effect Effects 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 230000012202 endocytosis Effects 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 230000003328 fibroblastic effect Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000013067 intermediate product Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 101150091879 neo gene Proteins 0.000 description 3
- 150000002482 oligosaccharides Chemical class 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 150000003408 sphingolipids Chemical class 0.000 description 3
- 108060007951 sulfatase Proteins 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 2
- OVSKIKFHRZPJSS-UHFFFAOYSA-N 2,4-D Chemical compound OC(=O)COC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-UHFFFAOYSA-N 0.000 description 2
- ZJBUILVYSXQNSW-YTWAJWBKSA-N Arg-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O ZJBUILVYSXQNSW-YTWAJWBKSA-N 0.000 description 2
- 102000009133 Arylsulfatases Human genes 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- 102000000844 Cell Surface Receptors Human genes 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 108010062580 Concanavalin A Proteins 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108090000394 Erythropoietin Proteins 0.000 description 2
- 102000003951 Erythropoietin Human genes 0.000 description 2
- 108010076282 Factor IX Proteins 0.000 description 2
- 108010023321 Factor VII Proteins 0.000 description 2
- 108010054218 Factor VIII Proteins 0.000 description 2
- 108010014173 Factor X Proteins 0.000 description 2
- 208000017462 Galactosialidosis Diseases 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- 102000051325 Glucagon Human genes 0.000 description 2
- 108060003199 Glucagon Proteins 0.000 description 2
- PAWIVEIWWYGBAM-YUMQZZPRSA-N Gly-Leu-Ala Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O PAWIVEIWWYGBAM-YUMQZZPRSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 2
- 102000016871 Hexosaminidase A Human genes 0.000 description 2
- 108010053317 Hexosaminidase A Proteins 0.000 description 2
- 102000003996 Interferon-beta Human genes 0.000 description 2
- 108090000467 Interferon-beta Proteins 0.000 description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 2
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 2
- 102000007330 LDL Lipoproteins Human genes 0.000 description 2
- 108010007622 LDL Lipoproteins Proteins 0.000 description 2
- ZURHXHNAEJJRNU-CIUDSAMLSA-N Leu-Asp-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O ZURHXHNAEJJRNU-CIUDSAMLSA-N 0.000 description 2
- 208000015439 Lysosomal storage disease Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010056886 Mucopolysaccharidosis I Diseases 0.000 description 2
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 2
- 229930193140 Neomycin Natural products 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000008468 bone growth Effects 0.000 description 2
- 210000000069 breast epithelial cell Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 238000002641 enzyme replacement therapy Methods 0.000 description 2
- 229940105423 erythropoietin Drugs 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 2
- 229960004666 glucagon Drugs 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229960001388 interferon-beta Drugs 0.000 description 2
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 2
- 238000001361 intraarterial administration Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 239000002523 lectin Substances 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 229950006780 n-acetylglucosamine Drugs 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 125000005629 sialic acid group Chemical group 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000012384 transportation and delivery Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- JCZPMGDSEAFWDY-SQOUGZDYSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanamide Chemical compound NC(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO JCZPMGDSEAFWDY-SQOUGZDYSA-N 0.000 description 1
- BZSALXKCVOJCJJ-IPEMHBBOSA-N (4s)-4-[[(2s)-2-acetamido-3-methylbutanoyl]amino]-5-[[(2s)-1-[[(2s)-1-[[(2s,3r)-1-[[(2s)-1-[[(2s)-1-[[2-[[(2s)-1-amino-1-oxo-3-phenylpropan-2-yl]amino]-2-oxoethyl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-hydroxy Chemical compound CC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCC)C(=O)N[C@@H](CCCC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(=O)N[C@H](C(N)=O)CC1=CC=CC=C1 BZSALXKCVOJCJJ-IPEMHBBOSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 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 1
- JUAGNSFMKLTCCT-UHFFFAOYSA-N 2-aminoacetic acid;carbonic acid Chemical compound OC(O)=O.NCC(O)=O JUAGNSFMKLTCCT-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 1
- 101150013604 ACADS gene Proteins 0.000 description 1
- 108010022579 ATP dependent 26S protease Proteins 0.000 description 1
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 0.000 description 1
- DHBKYZYFEXXUAK-ONGXEEELSA-N Ala-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 DHBKYZYFEXXUAK-ONGXEEELSA-N 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 102000013918 Apolipoproteins E Human genes 0.000 description 1
- 108010025628 Apolipoproteins E Proteins 0.000 description 1
- RATVAFHGEFAWDH-JYJNAYRXSA-N Arg-Phe-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CCCN=C(N)N)N RATVAFHGEFAWDH-JYJNAYRXSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- 102000005427 Asialoglycoprotein Receptor Human genes 0.000 description 1
- 108010002913 Asialoglycoproteins Proteins 0.000 description 1
- APYNREQHZOGYHV-ACZMJKKPSA-N Asp-Cys-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(=O)O)N APYNREQHZOGYHV-ACZMJKKPSA-N 0.000 description 1
- FIAKNCXQFFKSSI-ZLUOBGJFSA-N Asp-Ser-Cys Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(O)=O FIAKNCXQFFKSSI-ZLUOBGJFSA-N 0.000 description 1
- 206010068220 Aspartylglucosaminuria Diseases 0.000 description 1
- 102100032487 Beta-mannosidase Human genes 0.000 description 1
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 description 1
- 101100328886 Caenorhabditis elegans col-2 gene Proteins 0.000 description 1
- 101100328884 Caenorhabditis elegans sqt-3 gene Proteins 0.000 description 1
- 102000055006 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 241000238097 Callinectes sapidus Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000004652 Cardiovascular Abnormalities Diseases 0.000 description 1
- 102000004201 Ceramidases Human genes 0.000 description 1
- 108090000751 Ceramidases Proteins 0.000 description 1
- 102100022641 Coagulation factor IX Human genes 0.000 description 1
- 208000002330 Congenital Heart Defects Diseases 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 208000006069 Corneal Opacity Diseases 0.000 description 1
- UPJGYXRAPJWIHD-CIUDSAMLSA-N Cys-Asn-Leu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O UPJGYXRAPJWIHD-CIUDSAMLSA-N 0.000 description 1
- 206010011831 Cytomegalovirus infection Diseases 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241001269238 Data Species 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000991587 Enterovirus C Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108010014172 Factor V Proteins 0.000 description 1
- 102000001690 Factor VIII Human genes 0.000 description 1
- 108010088842 Fibrinolysin Proteins 0.000 description 1
- 108010029961 Filgrastim Proteins 0.000 description 1
- 208000009796 Gangliosidoses Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- KUTPGXNAAOQSPD-LPEHRKFASA-N Glu-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)N)C(=O)O KUTPGXNAAOQSPD-LPEHRKFASA-N 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- IRJWAYCXIYUHQE-WHFBIAKZSA-N Gly-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)CN IRJWAYCXIYUHQE-WHFBIAKZSA-N 0.000 description 1
- YOBGUCWZPXJHTN-BQBZGAKWSA-N Gly-Ser-Arg Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YOBGUCWZPXJHTN-BQBZGAKWSA-N 0.000 description 1
- 206010053185 Glycogen storage disease type II Diseases 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000016870 Hexosaminidase B Human genes 0.000 description 1
- 108010053345 Hexosaminidase B Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 208000015178 Hurler syndrome Diseases 0.000 description 1
- 241000521257 Hydrops Species 0.000 description 1
- 101150102264 IE gene Proteins 0.000 description 1
- 108010053927 Iduronate Sulfatase Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102100026018 Interleukin-1 receptor antagonist protein Human genes 0.000 description 1
- 101710144554 Interleukin-1 receptor antagonist protein Proteins 0.000 description 1
- 102000003815 Interleukin-11 Human genes 0.000 description 1
- 108090000177 Interleukin-11 Proteins 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 102000000646 Interleukin-3 Human genes 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 102000000853 LDL receptors Human genes 0.000 description 1
- 108010001831 LDL receptors Proteins 0.000 description 1
- 208000007177 Left Ventricular Hypertrophy Diseases 0.000 description 1
- ZTLGVASZOIKNIX-DCAQKATOSA-N Leu-Gln-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZTLGVASZOIKNIX-DCAQKATOSA-N 0.000 description 1
- JLYUZRKPDKHUTC-WDSOQIARSA-N Leu-Pro-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O JLYUZRKPDKHUTC-WDSOQIARSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 208000027933 Mannosidase Deficiency disease Diseases 0.000 description 1
- HUKLXYYPZWPXCC-KZVJFYERSA-N Met-Ala-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HUKLXYYPZWPXCC-KZVJFYERSA-N 0.000 description 1
- 201000011442 Metachromatic leukodystrophy Diseases 0.000 description 1
- 206010027727 Mitral valve incompetence Diseases 0.000 description 1
- 208000008955 Mucolipidoses Diseases 0.000 description 1
- 206010072928 Mucolipidosis type II Diseases 0.000 description 1
- 206010028095 Mucopolysaccharidosis IV Diseases 0.000 description 1
- 206010056893 Mucopolysaccharidosis VII Diseases 0.000 description 1
- 208000025915 Mucopolysaccharidosis type 6 Diseases 0.000 description 1
- 208000000149 Multiple Sulfatase Deficiency Disease Diseases 0.000 description 1
- 208000035032 Multiple sulfatase deficiency Diseases 0.000 description 1
- 241000711386 Mumps virus Species 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 description 1
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 1
- 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 1
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 1
- 108700010674 N-acetylVal-Nle(7,8)- allatotropin (5-13) Proteins 0.000 description 1
- 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 1
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 1
- 101710202061 N-acetyltransferase Proteins 0.000 description 1
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000015336 Nerve Growth Factor Human genes 0.000 description 1
- 208000014060 Niemann-Pick disease Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 241000609499 Palicourea Species 0.000 description 1
- 240000004760 Pimpinella anisum Species 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102000003923 Protein Kinase C Human genes 0.000 description 1
- 108090000315 Protein Kinase C Proteins 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 101001135767 Rattus norvegicus Parathyroid hormone Proteins 0.000 description 1
- 108010084592 Saporins Proteins 0.000 description 1
- 102000017852 Saposin Human genes 0.000 description 1
- 108050007079 Saposin Proteins 0.000 description 1
- 201000002883 Scheie syndrome Diseases 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 201000001828 Sly syndrome Diseases 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 102000011971 Sphingomyelin Phosphodiesterase Human genes 0.000 description 1
- 108010061312 Sphingomyelin Phosphodiesterase Proteins 0.000 description 1
- 102000005262 Sulfatase Human genes 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- QHUWWSQZTFLXPQ-FJXKBIBVSA-N Thr-Met-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)NCC(O)=O QHUWWSQZTFLXPQ-FJXKBIBVSA-N 0.000 description 1
- AHERARIZBPOMNU-KATARQTJSA-N Thr-Ser-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O AHERARIZBPOMNU-KATARQTJSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- IQXWAJUIAQLZNX-IHPCNDPISA-N Trp-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N IQXWAJUIAQLZNX-IHPCNDPISA-N 0.000 description 1
- 102000018594 Tumour necrosis factor Human genes 0.000 description 1
- 108050007852 Tumour necrosis factor Proteins 0.000 description 1
- 108091000117 Tyrosine 3-Monooxygenase Proteins 0.000 description 1
- 102000048218 Tyrosine 3-monooxygenases Human genes 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- LEBBDRXHHNYZIA-LDUWYPJVSA-N [(2s,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] n-[(z)-1,3-dihydroxyoctadec-4-en-2-yl]carbamate Chemical compound CCCCCCCCCCCCC\C=C/C(O)C(CO)NC(=O)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O LEBBDRXHHNYZIA-LDUWYPJVSA-N 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 102000015395 alpha 1-Antitrypsin Human genes 0.000 description 1
- 108010050122 alpha 1-Antitrypsin Proteins 0.000 description 1
- 229940024142 alpha 1-antitrypsin Drugs 0.000 description 1
- SHZGCJCMOBCMKK-PHYPRBDBSA-N alpha-D-fucose Chemical compound C[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O SHZGCJCMOBCMKK-PHYPRBDBSA-N 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 108010061314 alpha-L-Fucosidase Proteins 0.000 description 1
- 102000012086 alpha-L-Fucosidase Human genes 0.000 description 1
- 108010012864 alpha-Mannosidase Proteins 0.000 description 1
- 102000019199 alpha-Mannosidase Human genes 0.000 description 1
- 201000008333 alpha-mannosidosis Diseases 0.000 description 1
- BIGPRXCJEDHCLP-UHFFFAOYSA-N ammonium bisulfate Chemical compound [NH4+].OS([O-])(=O)=O BIGPRXCJEDHCLP-UHFFFAOYSA-N 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000002788 anti-peptide Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000004019 antithrombin Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 108010006523 asialoglycoprotein receptor Proteins 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 238000011021 bench scale process Methods 0.000 description 1
- 108010081485 beta Subunit Follicle Stimulating Hormone Proteins 0.000 description 1
- 102000035824 beta Subunit Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010068678 beta Subunit Thyrotropin Proteins 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 108010055059 beta-Mannosidase Proteins 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229940097706 buminate Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 108010089934 carbohydrase Proteins 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 229940106189 ceramide Drugs 0.000 description 1
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 description 1
- ZXFCRFYULUUSDW-OWXODZSWSA-N chembl2104970 Chemical compound C([C@H]1C2)C3=CC=CC(O)=C3C(=O)C1=C(O)[C@@]1(O)[C@@H]2CC(O)=C(C(=O)N)C1=O ZXFCRFYULUUSDW-OWXODZSWSA-N 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000030944 contact inhibition Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 231100000269 corneal opacity Toxicity 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229960004222 factor ix Drugs 0.000 description 1
- 229960000301 factor viii Drugs 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229960004177 filgrastim Drugs 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 201000006440 gangliosidosis Diseases 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 201000004502 glycogen storage disease II Diseases 0.000 description 1
- 108010082286 glycyl-seryl-alanine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 230000005831 heart abnormality Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 108010089932 heparan sulfate sulfatase Proteins 0.000 description 1
- LIIALPBMIOVAHH-UHFFFAOYSA-N herniarin Chemical compound C1=CC(=O)OC2=CC(OC)=CC=C21 LIIALPBMIOVAHH-UHFFFAOYSA-N 0.000 description 1
- JHGVLAHJJNKSAW-UHFFFAOYSA-N herniarin Natural products C1CC(=O)OC2=CC(OC)=CC=C21 JHGVLAHJJNKSAW-UHFFFAOYSA-N 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229940074383 interleukin-11 Drugs 0.000 description 1
- 229940117681 interleukin-12 Drugs 0.000 description 1
- 229940076264 interleukin-3 Drugs 0.000 description 1
- 229940028885 interleukin-4 Drugs 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011866 long-term treatment Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- ZLQJVGSVJRBUNL-UHFFFAOYSA-N methylumbelliferone Natural products C1=C(O)C=C2OC(=O)C(C)=CC2=C1 ZLQJVGSVJRBUNL-UHFFFAOYSA-N 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 208000005907 mitral valve insufficiency Diseases 0.000 description 1
- 208000020460 mucolipidosis II alpha/beta Diseases 0.000 description 1
- 201000002273 mucopolysaccharidosis II Diseases 0.000 description 1
- 208000005340 mucopolysaccharidosis III Diseases 0.000 description 1
- 208000012253 mucopolysaccharidosis IVA Diseases 0.000 description 1
- 208000000690 mucopolysaccharidosis VI Diseases 0.000 description 1
- 208000022018 mucopolysaccharidosis type 2 Diseases 0.000 description 1
- 208000011045 mucopolysaccharidosis type 3 Diseases 0.000 description 1
- 208000010978 mucopolysaccharidosis type 4 Diseases 0.000 description 1
- 208000025919 mucopolysaccharidosis type 7 Diseases 0.000 description 1
- 201000006033 mucosulfatidosis Diseases 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- 229940053128 nerve growth factor Drugs 0.000 description 1
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 230000028742 placenta development Effects 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000008476 powdered milk Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- RYVMUASDIZQXAA-UHFFFAOYSA-N pyranoside Natural products O1C2(OCC(C)C(OC3C(C(O)C(O)C(CO)O3)O)C2)C(C)C(C2(CCC3C4(C)CC5O)C)C1CC2C3CC=C4CC5OC(C(C1O)O)OC(CO)C1OC(C1OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OCC(O)C(O)C1O RYVMUASDIZQXAA-UHFFFAOYSA-N 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000033300 receptor internalization Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 102220023257 rs387907546 Human genes 0.000 description 1
- 102220023256 rs387907547 Human genes 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 210000002955 secretory cell Anatomy 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 235000017550 sodium carbonate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229940032362 superoxide dismutase Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 238000012385 systemic delivery Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 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 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 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)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/04—Centrally acting analgesics, e.g. opioids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- 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
-
- 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
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Cardiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Neurosurgery (AREA)
- Endocrinology (AREA)
- Pain & Pain Management (AREA)
- Heart & Thoracic Surgery (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Obesity (AREA)
- Hematology (AREA)
- Diabetes (AREA)
- General Engineering & Computer Science (AREA)
- Neurology (AREA)
- Immunology (AREA)
- Nutrition Science (AREA)
- Urology & Nephrology (AREA)
- Hospice & Palliative Care (AREA)
- Epidemiology (AREA)
Abstract
一种治疗怀疑患有α-半乳糖苷酶A缺乏症,如弥漫性躯体血管角质瘤病病人的治疗方法,它用(1)经基因修饰的人细胞来过度表达和分泌α-半乳糖苷酶A或(2)从培养的、基因修饰的人细胞得的纯化的人α-半乳糖苷酶A进行治疗。
Description
发明背景
本发明涉及α-半乳糖苷酶A和对α-半乳糖苷酶A缺乏症的治疗。
弥漫性躯体血管角化瘤(Fabry)病是一种X连续遗传性溶酶体贮积病,其特征在于有诸如严重肾损害、血管角化瘤和心血管异常,包括心室扩大和二尖瓣闭锁不全等体征。该病也影响外周神经***,导致极度痛苦的肢端烧灼样疼痛发作。由于α-半乳糖苷酶A(α-gal A)缺乏,导致中枢糖神经鞘脂的分解代谢受阻滞,酶底物、神经酰胺三己糖苷(trihexoside)在细胞里和血流里积聚引起此病。
由于该病是X连续遗传类型,故基本上所有的弥漫性躯体血管角化瘤病病人都是男性。虽然已观察到一些受严重影响的女性杂合体,但女性杂合体一般或没有症状,或主要向限在特征性角膜混浊的轻微体征。弥漫性躯体血管角化瘤病的一种非典型变体显示为低残留α-半乳糖苷酶A活性,以及或症状很轻微,或似乎没有与左心室肥大和心脏病病相关的弥漫性躯体血管角质瘤病的其它特征性体征(Nakano等,新英格兰医学杂志(New.Engl.J Med)333:288-293,1995)。现推测α-半乳糖苷酶A的减少可能是这类心脏异常的原因。
已分离和测序了编码人α-半乳糖苷酶A的cDNA和基因(Bishop等,Proc.Natl.Acad.Sci.USA83:4859,1986;Kornreich等,Nuc.Acids Res.17:3301,1988;Oeltjen等,哺乳动物基因组(Mammalian Genome)6:335-338.1995)。人α-半乳糖苷酶A被表达为429个氨基酸的多肽,其中N-末端的31个氨基酸构成了信号肽。人体此酶已在中国仓鼠卵巢(CHO)细胞中表达(Desnick,美国专利5,356,804;Ioannou等,细胞生物学杂志(J.Cell Biol.)119:1137,1992);在昆虫细胞(Calhoun等,美国专利5,179,023)中表达;和在COS细胞中表达(Tsuji等,Eur.J.Biochem.165:275,1987)。已报道了用衍生于人体组织的该蛋白质进行α-半乳糖苷酶A的替代疗法的小规模试验(Mapes等,Science169:987 1970;Brady等,N.Engl.J.Med.289:9,1973;Desnick等,Proc.Natl.Acad.Sci.USA76:5326,1979),但对弥漫性躯体血管角质瘤病暂时没有有效的治疗方法。
发明综述
现已发现,在培养的人细胞里表达编码人体α-半乳糖苷酶A的DNA产生了一种多肽,它恰当地被糖基化,结果它不仅有酶活性,能作用于弥漫性躯体血管角质瘤病积聚的糖神经鞘脂底物,而且能通过细胞表面受体被细胞有效地内化,此种细胞表面受体能将α-gal A精确导向此病所需的目标:受影响细胞特别是病人血管壁上的上皮细胞的溶酶体小室。该发现(将在下面作更详尽的讨论)意味着怀疑患有α-半乳糖苷酶A缺乏症,如弥漫性体躯血管角质瘤的人可用(1)已经基因修饰能过度表达和分泌人α-半乳糖苷酶A的人细胞或(2)从培养的、基因修饰的人细胞获得的纯化人α-半乳糖苷酶A来治疗。
如Selden等在WO93/09222中的所述(已纳入本文作为),通过第一途径即用修饰细胞本身的疗法,涉及体外或离体人细胞(如初级细胞,次级细胞或不死细胞)的基因操纵通过将这些细胞植入病人诱导它们表达和分泌高水平的人α-半乳糖苷酶A,
细胞进行基因修饰目的是用于基因疗法或酶替代疗法治疗Fabry病,含α-半乳糖苷酶A cDNA或基因组DNA序列的DNA分子可被包含在表达构建物中,并通过标准的转染方法,包括,但不限于,脂质体-、聚凝胺-或DEAE右旋糖苷介导的转染,电穿孔法,磷酸钙沉淀法,显微注射或速度驱动发射体(“biolistics”)引入初级或次级人体细胞(如,成纤维细胞,包括***和肠上皮细胞等上皮细胞,内皮细胞,包括淋巴细胞和骨髓细胞等血液有形成分、神经胶质细胞、肝细胞、角质化细胞、肌肉细胞、神经细胞或这些类型细胞的前体细胞)。另外,人们可使用病毒载体来递送DNA的***。已知用于基因转移的病毒包括腺病毒、腺相关病毒、疱疹病毒、腮腺炎病毒、脊髓灰质炎病毒、逆转录病毒、新培斯(Sindbis)病毒和诸如金丝崔痘病毒的痘苗病毒。虽然初级或次级细胞培养物是本发明的优选治疗方法,也可使用不死的人细胞。用于本发明的不死的人细胞系包括,但不限于,Bows黑素瘤细胞(ATCC保藏号CRL9607),Daudi细胞(ACTT保藏号:CCL213)、Hele细胞和及其衍生细胞(ACTT保藏号:CCL2,CCL2.1和CCL2.2)、HL-60细胞(ACTT保藏号CCL240)、HT1080细胞(ATCC保藏号CCL121),Jurkat细胞(ATCC保藏号TIB152)、KB癌细胞(ATCC保藏号CCL17)、K-562白血病细胞(ACTT保藏号CCL243)、MCF-7乳腺癌细胞(ATCC保藏号BTH22)、MOLT-4细胞(ACTT保藏号1582)、Namalwa细胞(ATCC保藏号CRL1432)、Raji细胞(ACTT保藏号CCL86)、RPMI8226细胞(ATCC保藏号CCL155)、U-937细胞(ATCC保藏号CRL1593)、WI-38VA13亚系2R4细胞(ATCC保藏号CLL75.1)和2780AD卵巢癌细胞(Van der Blick等,癌症研究(Cancer Res)48:5927-5932,1988)及通过人细胞和另一种动物细胞融合的异质性杂交瘤细胞。也可使用次级人成纤维细胞株,如WI-38(ATCC保藏号CCL75)和MEC-5(ATCC保藏号CCL171)。
以编码α-半乳糖苷酶A的DNA分子将人细胞基因工程(或用如下所述的另外的合适的基因修饰后)化产生了过度表达和分泌α-半乳糖苷酶A的细胞后,也许可产生基本上由许多遗传上相同培养的初级人细胞构成的克隆细胞株;或,当这些细胞不死时,可产生基本上由许多遗传上相同的不死的人细胞组成的克隆细胞系优选的是,此克隆细胞株或克隆细胞系是成纤维细胞。
然后可通过合适的方法,如Selden等(WO93/09222所述的方法)来制备基因修饰的细胞并引入病人体内。
根据本发明的基因疗法与用衍生于人体或动物组织的酶的替代疗法相比具有许多优点。例如,本发明的方法不依赖于可能不稳定的合适组织的来源,故具有治疗α-半乳糖苷酶A缺乏症的商品可行性。与用可能被已知或未知病毒和其它传染因子感染的人体组织酶的酶替代疗法相比,它相对无危险。此外,本发明的基因疗法与一般的酶替代疗法相比还有许多优点。例如,本发明方法(1)提供了长程治疗方法的好处不需要每天注射;(2)消除了通常伴随常规药物递送而出现的治疗性蛋白质的血清和组织浓度的极端波动;和(3)可能比酶替代疗法更便宜,因为不需要生产和纯化蛋白质供频繁用药。
如上所述,患有α-半乳糖苷酶A缺乏症的个体也许可用纯化的α-半乳糖苷酶A进行治疗(即酶替代疗法)。经基因修饰能过度表达人α-半乳糖苷酶A的初级、次级或不死的人细胞也可用于体外此酶蛋白的生产,以生产出可被纯化、用于酶替代疗法的该酶蛋白质。可从上述的细胞中选择次级或不死人细胞,并通过上述的转染或转导方法进行基因修饰。基因修饰后,在允许过度表达和分泌α-半乳糖苷酶A的条件下培养此细胞。通过收集细胞生长于其中的培养基和/或溶解细胞以释放其中的内容物,然后用标准的蛋白质纯化技术,从培养的细胞中分离得到此酶蛋白质。一种这类技术涉及将培养基或含人α-半乳糖苷酶A的样品通过疏水性反应树脂,如Butyl Sepharose或其它有丁基功能团部分的树脂。将样品通过这类树脂构成了第一层析步骤。若需要进一步的纯化,可将从疏水反应树脂中洗脱的含α-半乳糖苷酶A的材料通过含第二树脂柱,如HeparinSepharose固定化肝素树脂,羟基磷灰石,诸如Q Sepharose阴离子交换树脂,或诸如Superdex200的分子筛树脂。优选的是,纯化方案包括使用上述类型树脂中的每一种。另外,可在疏水性反应树脂前使用一种或多种后面的树脂,或不用疏水性树脂。
原先制备相对高纯度α-半乳糖苷酶A的方法依赖于使用亲和层析,用凝集素亲和层析(伴刀豆球蛋白(Con A)琼脂糖凝胶)和基于α-半乳糖苷酶A与琼脂糖基质交联的底物类似物N-6-氨基己酰基-α-D-半乳糖胺结合的亲和层析(Bishop等,生物化学杂志(J Biol Chem)256:1307-1316,1981)。使用蛋白质性凝集素亲和树脂和底物类似物树脂,通常伴有从固体支持物上不断沥滤的亲和剂(参见Marikar等,Anal.Biochem.201:306-310,1992),导致纯化产物污染了游离在溶液里或与洗脱蛋白质结合的亲和剂,这类污染物使该产物不宜用于药物制剂中。结合的底物类似物和凝集素对该蛋白质的酶促、功能和结构性能也有负面影响。此外,这类亲和树脂制备通常很昂贵,使之与常规的层析树脂相比更不适宜以商业规模生产。这样,开发使用在供应和质量上都很适合大规模商业用途的常规色谱层析的纯化方案是本发明的一个显著优点。
疑患有α-半乳糖苷酶A缺乏症的个体可通过标准的给药方法,包括,但不限于静注、皮下注射或肌注或作为固体植入,给予药学上可接受的纯化的人α-半乳糖苷酶A进行治疗。纯化的该蛋白质可配制成治疗组合物,此组合物由含生理上可接受的赋形剂,如人血清白蛋白载体的水溶液构成,其pH为6.5或更低。本发明因此为得到大量恰当糖基化因此可用于治疗的人α-半乳糖苷酶A提供了手段。这使得α-半乳糖苷酶A缺乏症的酶替代治疗法具有商品可行性,与用人体或动物组织酶治疗相比相对无危险性。
熟悉本发明技术领域的人员认识到,人α-半乳糖苷酶A DNA序列(cDNA或基因组DNA),或由于沉默密码子改变或由于产生保守性氨基酸取代的密码子改变而产生的与其不同的序列,可用于基因修饰培养的人细胞,使它们可过度表达和分泌此酶。也可能α-半乳糖苷酶A的DNA序列中某些突变会编码出这样的多肽,即它们保留了或显示出改进的α-半乳糖苷酶A酶活性(如本文所述,这可通过使培养细胞中突变的DNA分子表达,纯化编码的多肽和测定催化活性而显现)。例如,人们可期望保守性氨基酸替代物对其生物活性几乎没有或没有影响,特别是若它们的存在量低于蛋白质中残留物总数的10%时,更是如此。保守性替代物通常包括在下列各组中的替代:甘氨酸,丙氨酸;缬氨酸,异亮氨酸,亮氨酸;天冬氨酸,谷氨酸;天冬酰胺,谷氨酰胺;丝氨酸,苏氨酸;赖氨酸,精氨酸;和苯丙氨酸,酪氨酸。
通过特定的基因操作可使细胞产生的α-半乳糖苷酶A最大量化。例如,编码α-半乳糖苷酶A的DNA分子也可编码异源性信号肽,如人体生长激素(hGH)的信号肽,红细胞生成素,因子Ⅷ,因子Ⅸ,胰高血糖素,低密度脂蛋白(LDL)受体,或非α-半乳糖苷酶A的溶酶体酶类。优选的是,该信号肽是hGH信号肽(SED ID NO:21),位于该编码蛋白的N-端。编码信号肽的DNA序列可含诸如hGH基因的第一内含子的内含子,得到诸如SEQ ID NO:27的序列(参见图10)。此外,此DNA分子也可含3’非翻译序列(UTS),其长度至少为6个核苷酸(与人体发现的α-半乳糖苷酶A的mRNA相反,后者没有3’UTS,这要求多腺苷酸化位点在此编码序列内)。UTS处于该编码序列的末端密码子的3’旁,并包括多腺苷酸化位点。较优的长度至少为6个核苷酸,更优的是至少12个,最好至少有30个核苷酸,在所有情况下都含有序列AATAA或促进多腺苷酸化的相关序列。这里所述的DNA序列,即,编码连接于α-半乳糖苷酶A的hGH信号肽,并含有包括多腺苷酸化位点的3’UTS,和较优地包括表达控制序列,也在本发明中。在本发明的范围里还有一个DNA分子,它编码的蛋白质包括与α-半乳糖苷酶A或任何其它异源多肽(即,非hGH或非hGH类似物的多肽)连接的hGH信号肽。该异源多肽通常是哺乳动物蛋白质,例如任何医学上需要的人多肽。
本发明的其它特征和优点可从下列详尽的阐述和权利要求书中看出。
本文用于细胞的术语“基因修饰”意指包括在将编码基因产物的DNA分子和/或控制编码序列表达的调节元件引入后,表达特定基因产物的细胞。可通过基因定向引入DNA分子(即,将DNA分子引入特定的基因组位点);此外,同源重组使有缺陷的基因本身得以代替(在弥漫性躯体血管角质瘤病病人的自身细胞里有缺陷的α-半乳糖苷酶A基因或其部分可用该全基因或其部分代替)。
本文使用的术语“α-半乳糖苷酶A”指没有信号肽的α-半乳糖苷酶A,信号肽见SEQ ID NO:26(图9)。有一些迹象表示,SEQ ID NO:26(图9)的残基371到398,或373到398可在溶酶体中除去;但是,除去该推定的前肽据信不会影响酶的活性。这提示,可删去此推定前肽的任何部分而不影响活性。这样,本文的术语“α-半乳糖苷酶A”也覆盖了一个蛋白质,它具有SEQ ID NO:26,相对应的序列,除了该序列C未端28个残基外。
“α-半乳糖苷酶A缺乏症”指病人此酶的量或活性缺乏。该缺乏可能会诱发通常在患有弥漫性躯体角质瘤病男性中所见到的严重症状,或可能仅仅在杂接合的女有缺陷基因携带者中所看到的相对轻的症状。
本文使用的术语“初级细胞”包括存在于从脊椎动物组织源分离的细胞悬液里的细胞(在它们被接种前,即贴附到组织培养物如平皿或烧瓶前),在衍生于此细胞首次接种前组织的外植体中的细胞,和衍生于这些接种细胞的细胞悬液。
“次级细胞”指培养中所有后续步骤中的细胞。即第一次接种后的初级细胞从培养底物中取出后再接种(传代),它被称为次级细胞,是如同后续传代中的所有细胞。
“细胞株”由传代一次或多次的次级细胞构成;显示在培养中平均群体倍增的数量有限;显示接触-抑制性质、贴壁依赖性生长性质(在悬浮培养中繁殖的细胞除外);而且是不死的。
“不死细胞”指建立的细胞系细胞,它们在培养中显示明显无限的寿命。
术语“信号肽”指一种肽序列,它引导它所连接的新合成的多肽附着于内质网以进行下一步的翻译后加工和/或分布。
本文对α-半乳糖苷酶A使用的术语“异源信号肽”,指不是人α-半乳糖苷酶A信号肽的信号肽(即,被SEQ ID NO:18的核苷酸36-128编码)。它通常是一些非α-半乳糖苷酶A的哺乳动物蛋白的信号肽。
术语“第一层析步骤”指样品首先用的层析柱(所有与样品制备有关的步骤都除外)。
附图简述
图1是代表用于从人成纤维细胞cDNA库分离α-半乳糖苷酶A的210bp的探针(SEQ ID NO:19)。该序列来自α-半乳糖苷酶A的外显子7。该探从人体基因组DNA中通过聚合酶链反应(PCR)分离得到。图中有下划线的区域相应于扩增引物的序列。
图2代表完成α-半乳糖苷酶A cDNA克隆5’末端的DNA片断的序列(SEQID NO:20)。该片断从人体基因组DNA用PCR扩增。下划线区域相应于扩增引物的序列。也显示了NcoⅠ和SacⅡ限制内切核酸酶位点,如在实施例1A中所述用于亚克隆。
图3代表了α-半乳糖苷酶A的cDNA序列,包括编码信号肽的序列(SEQ IDNO:18)。
图4是pXAG-16的示意图,pXAG-16是α-半乳糖苷酶A的表达构建物,包括CMV(巨细胞病毒)启动子和内含子,hGH信号肽编码序列和第一内含子,α-半乳糖苷酶A的cDNA(即缺乏α-半乳糖苷酶A信号肽的序列)和hGH3’UTS。
图5是pXAG-28的示意图,pXAG-28是α-半乳糖苷酶A表达的构建物,包括胶源Iα2启动子、β-肌动蛋白内含子,hGH信号肽编码序列和第一内含子,α-半乳糖苷酶A的cDNA(即缺乏α-半乳糖苷酶A信号肽的序列)和hGH3’UTS。
图6是α-半乳糖苷酶A纯化步骤用Butyl Sepharose树脂的色谱层析图。它显示了280nm处的吸光值(普通线)和所选组分的α-半乳糖苷酶A活性(带点线)。
图7是描述Fabry成纤维细胞内化本发明制备的人α-半乳糖苷酶A的直线图。用本发明制备的增加浓度的人α-半乳糖苷酶A培养细胞后,测量细胞内α-半乳糖苷酶A活性和总蛋白质浓度。显示了潜在的内化-抑制剂甘露糖-6磷酸(M6P;空心方块)和甘露聚糖(空心圆)的作用。
图8是一个设计用来在α-半乳糖苷酶A内化后检查Fabry成纤维细胞的实验性示例的示意图。Fabry细胞在接触正常的或培养在TranswellTM***物中的α-半乳糖苷酶A过度表达的人成纤维细胞后测定该细胞的α-半乳糖苷酶A活性。“M6P”=甘露糖-6-磷酸;“UntrfHF”=未转染的人成纤维细胞;“BRS11”=转染的、α-半乳糖苷酶A过度表达的成纤维细胞株。
图9代表人体α-半乳糖苷酶A氨基酸序列(SEQ ID NO:26)。
图10代表编码hGH信号肽和含第一hGH内含子(划线部分)的DNA序列(SEQ ID NO:27)。
图11代表没有内含子的编码hGH信号肽的DNA序列(SEQ ID NO:22)。
图12代表hGH信号肽的氨基酸序列(SEQ ID NO:21)。
图13代表编码人α-半乳糖苷酶A的cDNA序列(没有信号肽)(SEQ IDNO:25)。
发明详述
诸如α-半乳糖苷酶A的溶酶体酶通过与甘露糖-6-磷酸盐(M6P)受体相互作用而导向细胞的溶酶体小室,它与(预定为)溶酶体小室酶的寡糖部分中存在的M6P残基结合(Kornfeld,S.和Mellman,I.Ann.Rev.Cell Biol.5:483-525,1989)。初级相互反应在高尔基体中发生,其中与高尔基体M6P受体结合的酶被分开隔离,以运送到溶酶体。次级相互反应据信发生在细胞外α-半乳糖苷酶A和细胞表面的M6P受体之间。被细胞内化的细胞外物质以胞吞小泡经细胞质运送,小泡与初级溶酶体融合并将其内含物排空入溶酶体。在该过程中,细胞表面的M6P受体也掺入胞吞小泡并运送到溶酶体。
在细胞外环境里的α-半乳糖苷酶A,若带有M6P残基,则结合到细胞表面M6P受体,从而与受体一起运送入溶酶体小室。一旦由于此捕获通路而进入溶酶体小室里,此酶可执行它合适的功能。这样,即使细胞有基因缺陷不能产生其自体的α-半乳糖苷酶A,也存在摄入外源产生酶的机制。条件是(a)此酶是恰当糖基化的,和(b)缺陷的细胞带有M6P受体。
在弥漫性躯体血管角质瘤病中,肾脏和心脏的血管内皮细胞已显示出严重的组织病理学异常,导致该病的临床病理学变化;不携带M6P受体的这些细胞,是本发明的特定目标。本发明生产的α-半乳糖苷酶A可通过基因疗法(即在病人体内种植表达和分泌糖化酶的基因修饰细胞)或通过常规的给药途径经局部或全身递送给受影响细胞。以N-连接的寡糖形式存在于α-半乳糖苷酶A中的M6P对于本发明的疗法极为重要。
此外,α-半乳糖苷酶A的N-连接的寡糖的被唾液酸化修饰的程度也是很重要的。缺乏合适的唾液酸化时,α-半乳糖苷酶A由于被肝无唾液酸糖蛋白受体结合而被迅速从循环中清除,然后被肝细胞内化并分解(Ashwell和Harford,Ann.Rev.Biochem.51:531-554,1982)。这减少了循环中可与细胞上M6P受体结合的α-半乳糖苷酶A的量,所述的细胞与弥漫性躯体血管角质瘤病的临床病理学变化相关,如肾脏和心脏的血管内皮细胞。令人惊奇的是,本申请人业已发现,通过稳定转染的人细胞所分泌的α-半乳糖苷酶A具有糖化性质,它适合用基因疗法或常规地给予纯化的分泌蛋白质来治疗弥漫性躯体血管角质瘤病。这与最佳研究的溶酶体酶、葡糖脑苷脂酶的情况相反,其中从人体胎盘纯化的此酶或由转染的CHO细胞分泌到体内临床相关细胞的此酶的递送,要求在纯化后对此酶进行复杂的酶促修饰(参见Beutiler,New Engl.J.Med.325:1354-1360,1991)。
本发明的疗法可以两种一般方法进行:通过给病人引入治疗有效量的纯化的从培养的基因修饰过能过度表达和分泌此酶的人细胞中得到的、人α-半乳糖苷酶A,或通过给病人引入过度表达的细胞本身。下面讨论进行必须的基因修饰的技术,纯化的方法、配制和治疗。
实施例Ⅰ.用来传递和表达α-gal A的构建物的制备及其应用
构建两个表达质粒pXAG-16和pXAG-28。这些质粒含有编码α-gal A酶398个氨基酸(没有α-gal A信号肽)的人α-gal A cDNA;人生长激素(hGH)信号肽基因组DNA序列,它被hGH基因的第一个内含子所间断;以及hGH基因的含有聚腺苷酸化信号的3′非翻译序列(UTS)。质粒pXAG-16有人巨细胞病毒立即早期(CMVIE)启动子和第一个内含子(侧接非编码的外显子序列),而pXAG-28是由胶原Iα2启动子驱动的,它还含有β-肌动蛋白基因的5′非翻译序列,该序列中含有β-肌动蛋白基因的第一个内含子。
A.全长α-gal A cDNA的克隆以及α-gal A表达质粒pXAG-16的构建
从按照下述方式构建的人成纤维细胞cDNA文库中克隆出人α-gal AcDNA。从全部RNA中分离出Poly-A+mRNA,根据生产商的指导(Stratagene Inc.,LaJolla,CA)用λZapⅡ***用的试剂合成cDNA。简言之,在含有内部XhoⅠ限制性内切酶位点的寡脱氧胸苷引物的存在下,逆转录产生cDNA的“第一链”。用RNA酶H处理后,用DNA聚合酶Ⅰ将cDNA切口平移,产生双链cDNA。用T4DNA聚合酶将该cDNA变成平头,连接到EcoRⅠ衔接头上。该连接产物用T4 DNA激酶处理并用XhoⅠ消化。cDNA用Sephacryl-400层析组分。合并大的和中等大小的组分,将cDNA连接到EcoRⅠ和XhoⅠ消化的λZapⅡ臂上。然后包装该连接产物并滴定。该初级文库的滴度为1.2×107pfu/ml,***物平均大小为925bp。
用人α-gal A基因外显子7的210bp的探针(图1,SEQ ID NO:19)来分离该cDNA。探针本身是采用下列寡核苷酸通过聚合酶链反应(PCR)从基因组DNA中分离获得的:5′-CTGGGCTGTAGCTATGATAAAC-3′(寡核苷酸1;SEQ ID NO:1)和5′-TCTAGCTGAAGCAAAACAGTG-3′(寡核苷酸2;SEQ ID NO:2)。然后用PCR产物来筛选成纤维细胞的cDNA文库,分离阳性克隆作进一步的性质鉴定。根据生产商的指导,对一个阳性克隆噬菌体3A进行λZapⅡ***切割步骤(Stratagene,Inc.,La Jolla,CA)。该步骤产生了质粒pBSAG3A,该质粒在pBluescriptSK-TM质粒骨架中含有α-gal A cDNA序列。DNA测序揭示了该质粒不含此cDNA序列完整的5′端。因此,用从人基因组DNA扩增得到的PCR片段重新构建5′端。为了实现这一过程,用下列寡核苷酸扩增268bp的基因组DNA片段(图2,SEQ ID NO:20):5′-ATTGGTCCGCCCCTGAGGT-3′(寡核苷酸3;SEQID NO:3)和5′-TGATGCAGGAATCTGGCTCT-3′(寡核苷酸4;SEQ ID NO:4)。将该片段亚克隆入“TA”克隆质粒(Invitrogen Corp.,San Diego,CA)中,产生质粒pTAAGEI。用SacⅡ和NcoⅠ各自消化含有大部分α-gal A cDNA序列的质粒pBSAG3A和含有α-gal A cDNA 5′末端的质粒pTAAGEI。扩增的DNA片段中相关的SacⅡ和NcoⅠ位点的位置显示在图2中。分离pTAAGEI中0.2kb的SacⅡ-NcoⅠ片段并连接到同样方式消化的pBSAG3A中。该质粒pAGAL含有全部的α-gal AcDNA序列,包括编码α-gal A信号肽的序列。对cDNA进行完整的测序(如图3所示,包括α-gal A信号肽;SEQ ID NO:18),发现与人α-gal A cDNA的公开序列(Genbank sequence HUMGALA)相同。
如下所示,通过几个中间产物构建质粒pXAG-16。首先,用SacⅡ和XhoⅠ消化pAGAL并使其成平头。其次,使全长α-gal A cDNA的末端与XbaⅠ接头连接,并亚克隆入XbaⅠ消化的pEF-BOS(Mizushima等,Nucl.Acads Res.18:5322,1990)中,产生pXAG-1。该构建物含有人粒细胞集落刺激因子(G-CSF)的3′非翻译序列和侧接编码α-gal A加上α-gal A信号肽的cDNA的人延伸因子-1α(EF-1α)启动子,使得α-gal A cDNA的5′末端与EF-1α启动子融合。为了产生具有CMV IE启动子和第一内含子的构建物,从pXAG-1中取出α-gal A cDNA和G-CSF3′UTS作为2kb的XbaⅠ-BamHⅠ片段。使该片段变成平头,与BamHⅠ接头连接并***BamHⅠ消化的pCMVflpNeo(按照下述方式构建)中。取向是α-gal AcDNA的的5′末端融合到CMV IE启动子区域中。
按如下所示生产pCMVflpNeo。用作为模板的CMV基因组DNA和下列寡核苷酸通过PCR扩增获得CMV IE基因启动子片段:5′-TTTTGGATCCCTCGAGGACATTGATTATTGACTAG-3′(SEQ ID NO:23)和5′-TTTTGGATCCCGTGTCAAGGACGGTGAC-3′(SEQ ID NO:24)。所得产物(1.6kb片段)用BamHⅠ消化,产生具有BmaHⅠ消化的粘性末端、含有CMV启动子的片段。从质粒pMClneopA(Stratagene Inc.,La Jolla,CA)分离获得neo表达单元,为1.1kb的XhoⅠ-BamHⅠ片段。将含有CMV启动子的片段和neo片段***BamHⅠ、XhoⅠ消化的质粒(pUC12)中。值得注意的是,pCMVflpNeo含有CMV IE启动子区域(从Genbank sequence HS5MISP的核苷酸546开始到核苷酸2205结束)以及紧靠CMV IE启动子片段5′端的受单纯疱疹病毒(HSV)胸苷激酶启动子(Thneo基因)驱动的新霉素抗性基因。neo基因的转录方向与CMV启动子片段相同。该中间构建物称为pXAG-4。
为了加入hGH3′非翻译序列,从pXAG-4中取出成为XbaⅠ-SmaⅠ片段的GCSF3′非翻译序列,并使pXAG-4的末端变成平头。从pXGH5(Selden等,Mol.Cellular Biol.6:3173-3179,1986)中取出hGH3′非翻译序列,它是0.6kb的SmaⅠ-EcoRⅠ片段。在将片段变成平头后,将其连接到pXAG-4中紧靠pXAG-4的平头XbaⅠ位点后。该中间产物称为pXAG-7。从该质粒中取出TKneo片段(HindⅢ-ClaⅠ片段),通过“填入”DNA聚合物Ⅰ的Klenow片段使该质粒末端变平。连接入来自pcDNeo(Chen等,Mol.Cellular biol.7:2745-2752,1987)消化物的受SV40早期启动子驱动的新霉素抗性基因(ClaⅠ-BsmBⅠ平头片段),将neo转录单元放在α-galA转录单元的相同取向上。该中间产物称为pXAG-13。
为了完成有26个氨基酸hGH信号肽编码序列和hGH基因的第一个内含子的pXAG-16,首先除去pXAG-13中2.0kb的EcoRⅠ-BamHⅠ片段。该片段包括α-gal AcDNA和hGH3′非翻译序列。该大片段用3个片段代替。第一个片段由pXGH5的0.3kb PCR产物组成,它含有hGH信号肽编码序列,并包括hGH的第一个内含子序列,从正好位于Kozak共有序列上游的合成的BamHⅠ位点到hGH信号肽编码序列末端。用下列寡核苷酸来扩增该片段(片段1):5′-TTTTGGATCCACCATGGCTA-3′(寡核苷酸HGH101;SEQ ID NO:5)和5′-TTTTGCCGGCACTGCCCTCTTGAA-3′(寡核苷酸HGH102;SEQ ID NO:6)。第二个片段由0.27kb的PCR产物组成,该产物含有对应于从编码398个氨基酸(即缺少α-gal A信号肽)的α-gal A酶的cDNA起始部位到NheⅠ位点的序列。用下列寡核苷酸来扩增该片段(片段2):5′-TTTTCAGCTGGACAATGGATTGGC-3′(寡核苷酸AG10;SEQ ID NO:7)和5′-TTTTGCTAGCTGGCGAATCC-3′(寡核苷酸AG11;SEQ ID NO:8)。第三个片段由含有其余α-gal A序列以及hGH3′非翻译序列的pXAG-7的NheⅠ-EcoRⅠ片段组成(片段3)。
使片段1(用BamHⅠ和NaeⅠ消化)、片段2(用PvuⅡ和NheⅠ消化)、片段3与6.5kb、含有neo基因和CMV IE启动子的pXAG-13的BamHⅠ-EcoRⅠ片段混合,并连接在一起形成质粒pXAG-16(图4)。
B.α-gal A的构建
质粒pXAG-28的表达
分离获得人胶原Iα2启动子,以用于下述的α-gal A表达构建物pXAG-28中。用下列寡核苷酸分离含有人胶原Iα2启动子部分的人基因组DNA的408bp PCR片段:5′-TTTTGGATCCGTGTCCCATAGTGTTTCCAA-3′(寡核苷酸72;SEQ IDNO:9)和5′-TTTTGGATCCGCAGTCGTGGCCAGTACC-3′(寡核苷酸73;SEQ IDNO:10)。
用该片段来筛选EMBL3中的人白细胞库(Clontech Inc.Palo Alto,CA)。分离获得含有3.8kb EcoRⅠ片段的阳性克隆(噬菌体7H),并将其克隆入pBSIISK+(Stratagene Inc.,La Jolla,CA)的EcoRⅠ位点上(产生pBS/7H.2)。通过SpeⅠ消化使pBSIISK+多接头断裂,“填入”DNA聚合酶Ⅰ的Klenow片段,***寡核苷酸5′-CTAGTCCTAGGA-3′(SEQ ID NO:11),在PBSIISK+中引入一个AvrⅡ位点。用BamHⅠ和AvrⅡ消化该pBSIISK+变体,将其连接到上述最初的408bp胶原Iα2启动子PCR片段的121bp BamHⅠ-AvrⅡ片段上,产生pBS/121COL.6。
用XbaⅠ消化质粒pBS/121COL.6,使其在pBSIISK+多接头序列中断裂,“填入”DNA聚合酶Ⅰ的Klenow片段,用ArvⅡ消化。分离获得pBS/7H.2中3.8kb的BamHⅠ-AvrⅡ片段,通过Klenow酶处理使BamHⅠ位点变成平头。然后用AvrⅡ消化该片段,并将其连接到AvrⅡ消化的载体上,从而产生胶原启动子质粒pBS/121bpCOL7H.18。
然后,将胶原启动子融合到含有人β-肌动蛋白基因第一个内含子的人β-肌动蛋白基因5′非翻译序列上。为了分离该序列,用下列寡核苷酸从人基因组DNA中分离获得2kb PCR片段:5′-TTTTGAGCACAGAGCCTCGCCT-3′(寡核苷酸BAl;SEQ ID NO:12)和5′-TTTTGGATCCGGTGAGCTGCGAGAATAGCC-3′(寡核苷酸BA2;SEQ ID NO:13)。
用BamHⅠ和BsiHKAⅠ消化该片段,释放出含有β-肌动蛋白5′非翻译序列和内含子的0.8kb片段。然后按如下所述从胶原启动子质粒pBS/121bpCOL7H.18中分离获得3.6kb的SalⅠ-SrfⅠ片段。用BamHⅠ部分消化pBS/121bpCOL7H.18(BamHⅠ位点在胶原Iα2启动子片段的5′末端),通过Klenow片段处理使其变为平头,连接到SalⅠ接头(5′-GGTCGACC-3′)上,从而将SalⅠ位点放在胶原Io2启动子的上游。然后用SalⅠ和SrfⅠ消化该质粒(SrfⅠ位点在胶原Iα2启动子CAP位点上游110bp处),分离获得3.6kb的片段。使0.8kb和3.6kb片段与SalⅠ和BamHⅠ消化的pBSIISK-(Stratagene Inc.,La Jolla,CA)、以及由下列四个寡核苷酸一起退火(形成有平头和BsiHKAL末端的片段)组成的一个片段结合,四个寡核苷酸是:5′-GGGCCCCCAGCCCCAGCCCTCCCATTGGTGGAGGCCCTTTTGGAGGCACCCTAGGGCCAGGAAACTTTTGCCGTAT-3′(寡核苷酸COL-1;SEQ ID NO:14)、5′-AAATAGGGCAGATCCGGGCTTTATTATTTTAGCACCACGGCCGCCGAGACCGCGTCCGCCCCGCGAGCA-3′(寡核苷酸COL-2;SEQ ID NO:15)、5′-TGCCCTATTTATACGGCAAAAGTTTCCTGGCCCTAGGGTGCCTCCAAAAGGGCCTCCACCAATGGGAGGGCTGGGGCTGGGGGCCC-3′(寡核苷酸COL-3;SEQID NO:16)和5′-CGCGGGGCGGACGCGGTCTCGGCGGCCGTGGTGCTAAAATAATAAAGCCCGGATC-3′(寡核苷酸COL-4;SEQ ID NO:17)。这四个寡核苷酸在退火时对应于始于胶原启动子SrfⅠ位点并延续到β-肌动蛋白启动子BsiHKAⅠ位点的区域。所得质粒命名为pCOL/β-肌动蛋白。
为了完成pXAG-28的构建,分离获得含有胶原Iα2启动子和β-肌动蛋白5′非翻译序列的pCOL/β-肌动蛋白的SalⅠ-BamHⅠ片段。将该片段连接到来自pXAG-16的两个片段(参见实施例1A和图4)上:(1)6.0kb BamHⅠ片段(含有neo基因、质粒骨架、编码398个氨基酸α-gal A酶的cDNA以及hGH3′非翻译序列);和(2)0.3kb的BamHⅠ-XhoⅠ片段(其含有pcDneo的SV40聚腺苷酸序列)。pXAG-28含有与人β-肌动蛋白5′非翻译序列融合的人胶原Iα2启动子、hGH信号肽(被hGH第一内含子隔断)、编码α-gal A酶的cDNA以及hGH3′非翻译序列。图5显示了完整的表达构建物pXAG-28的图谱。
C.用α-gal A表达质粒电穿孔转染成纤维细胞和选择
为了在成纤维细细胞中表达α-gal A,根据已在颁布的步骤(Selden等,WO93/09222)培育并转染次级成纤维细胞。
将质粒pXAG-13,pXAG-16和pXAG-28通过电穿孔转染到人***成纤维细胞中,以产生稳定转染的克隆细胞株,如实施例ID所述监测所得α-gal A表达水平。正常的***成纤维细胞分泌α-gal A的速度在2-10单位/106细胞/24小时的范围内。相反,经转染的成纤维细胞表现出的平均表达水平如表1所示:表1: α-gal A平均表达水平(±标准偏差)pXAG-13 420±344U/106细胞/天
N=26个克隆株(范围为3-1133U/106细胞/天)pXAG-16 2051±1253U/106细胞/天
N=24个克隆株(范围为422-5200U/106细胞/天)pXAG-28 141±131U/106细胞/天
N=38个克隆株(范围为20-616U/106细胞/天)
这些数据显示所有这三个表达构建物均能使α-gal A表达水平比未转染的成纤维细胞提高许多倍。用pXAG-13(它编码与α-gal A信号肽相连的α-gal A)稳定转染的成纤维细胞的表达比用pXAG-16转染的成纤维细胞的表达低得多,其不同之处只是信号肽是hGH信号肽,hGH信号肽的编码序列被hGH基因的第一个内含子隔断。
在转染的细胞每次传代时,测定分泌的α-gal A活性,计数细胞,并计算细胞的密度。根据收获的细胞数以及α-gal A分泌的时间,确定了α-gal A的表达比速度(specific expression rate),并报告在表2和3中,以每106个细胞每24小时期间分泌的α-gal A单位表示。基因治疗所需的或用于生产纯化α-gal A的材料所需的细胞株,应在几次传代后呈现稳定的生长和表达。细胞株的数据显示在表2和3中,它们用α-gal A表达构建物pXAG-16稳定转染,这说明了在几次传代后α-gal A表达保持稳定的事实。
表2含有α-gal A表达构建物pXAG-16的BRS-11细胞的生长和表达
BRS-11 | ||
传代次数 | 表达(单位/106细胞/24小时) | 细胞密度(细胞/cm2) |
13 | 2601 | 4.80×104 |
14 | 1616 | 4.40×104 |
15 | 3595 | 4.40×104 |
表3含有α-gal A表达构建物pXAG-16的HF503-242细胞的生长和表达
HF503-242 | ||
传代次数 | 表达(单位/106细胞/24小时) | 细胞密度(细胞/cm2) |
5 | 4069 | 2.80×104 |
6 | 7585 | 3.55×104 |
7 | 5034 | 2.48×104 |
D.α-gal A表达的定量分析
用Ioannou等(J.Cell Biol.119:1137-1150,1992)所述的方案的改进方法,以4-甲基伞形基(umbelliferyl)-α-D-半乳糖吡喃糖苷(4-MUF-gal;Research Products-Inc.)作为水溶性底物,测定α-gal A活性。将底物溶解在底物缓冲液(0.1M柠檬酸磷酸盐,pH4.6)中至浓度为1.69mg/ml(5mM)。通常是将10μl培养上清液加入75μl底物溶液中。将试管加盖,使其在37℃水浴中培育60分钟。在培育的最后,用2ml甘氨酸-碳酸盐缓冲液(130mM甘氨酸,83mM碳酸钠,pH10.6)来终止反应。用TK0100型荧光计(Hoefer Scientific Instruments)(此仪器有365nm的固定激发(excitation)波,并检测460nm的固定发射波)测定每个样品的相对荧光度。将样品的读数与从1μM甲基伞形酮(Sigma Chemical Co.)储存液制得的标准品作比较,计算水解的底物量。α-gal A的活性用单位表示;一单位的α-gal A活性等效于37℃下每小时水解1纳摩尔底物。细胞表达数据大致上用106细胞/24小时分泌的α-gal A活性/单位表示。该试验也用来测定细胞裂解物中以及α-gal A纯化各步骤取(如下所述)样中的α-gal A活性。
实施例Ⅱ从稳定转染的人细胞株条件培养基中纯化α-gal A
实施例ⅡA-ⅡE描述了α-gal A可以从培育的人细胞株的条件培养基中纯化至接近匀质,该人细胞株已被稳定转染以生产酶。
A.使用丁基琼脂糖(Butyl Sepharose)层析作为纯化α-gal A的第一步
将冷的条件培养基(1.34升)离心并过滤通过0.45μm乙酸纤维素滤膜(以玻璃纤维预过滤),使澄清。在搅拌时,滴加入1N HCl,将冷的过滤的培养基pH调至5.6,通过滴加入3.9M超纯硫酸铵贮备液(室温),使得加入硫酸铵最终浓度为0.66M。将培养基4℃再搅拌5分钟,如前所述且样过滤,并上样于丁基琼脂糖4快速柱(81ml柱体积,2.5×16.5cm;Pharmacia,Uppsala,Sweden)中,此柱已经用含有0.66M硫酸铵的pH5.6、10mM MES-Tris(缓冲液A)平衡过。层析于4℃在Gradi-FracTM***(Pharmacia,Uppsala,Sweeden)中进行,该***装备有串联的UV(280nm)和电导率监测器分别用于评估总蛋白和盐浓度。在以10ml/min的流速施加完样品后,用10倍柱体积的缓冲液A洗柱。用14倍柱体积的从缓冲液A(含有硫酸铵)至pH5.6,10mM MES-Tris的线性梯度液将α-gal A从丁基琼脂糖TM柱上洗脱下来。用4-MUF-gal试验测定组分的α-gal A活性,合并含有显著酶活的那些组分。如图6和纯化总结(表3)所示,该步骤除去了大约99%的杂蛋白(柱纯化前样品为8.14g总蛋白;柱纯化后样品为0.0638g总蛋白)。
表4:从稳定转染的人成纤维细胞的条件培养基纯化α-gal A
纯化步骤 | 体积(ml) | α-gal A活性(×106单位) | 总蛋白(mg) | 比活(×106单位/mg) | 纯化倍数(累积) | 回收百分数 |
培养上清 | 1340 | 14.6 | 8140 | 0.0018 | =1 | =100 |
丁基琼脂糖 | 417 | 14.1 | 63.8 | 0.221 | 123 | 96.6 |
肝素琼脂糖 | 134 | 12.1 | 14.6 | 0.829 | 436 | 82.9 |
羟基磷灰石 | 47 | 9.73 | 4.46 | 2.18 | 1220 | 66.6 |
Q琼脂糖 | 31.5 | 8.91 | 3.31 | 2.69 | 1503 | 61.0 |
Superdex200 | 10 | 8.58 | 2.93 | 2.92 | 1634 | 59.0 |
B.使用肝素琼脂糖层析作为α-gal A纯化的第二步
4℃用pH5.6,10mM MES-Tris(4升)液透析了基琼脂糖柱峰组分(换液一次)。4℃按需加入水或NaCl,将透析物电导率调至1.0mMHO。随后,将样品加入肝素琼脂糖6快速柱(Pharmacia,Uppsala,sweden;29ml柱体积,2.5×6cm)中,该柱已用含有9mM NaCl的pH5.6,10mM MES-Tris(缓冲液B)平衡过。加样在4℃下进行,流速为10ml/min。用串联的UV(280nm)和电导率监测器测定总蛋白和盐浓度。加样后,用10倍柱体积的缓冲液B,然后是3倍柱体积至8%缓冲液C/92%缓冲液B(其中缓冲液C是含有250mM NaCl的pH5.6,10mM MES-Tris)的线性梯度液洗柱,再用10倍柱体积的8%缓冲液C洗柱。然后用1.5倍柱体积至29%缓冲液C的线性梯度液,随后用10倍柱体积至35%缓冲液C的线性梯度液洗脱α-gal A。测定组分的α-gal A活性,合并含有显著活性的那些组分。
C.使用羟基磷灰石层析作为α-gal A纯化步骤
过滤肝素柱合并物,直接加入陶土羟基磷灰石HC柱(40μm;AmericanInternational Chemical,Natick,MA;12ml柱体积,1.5×6.8cm)中,该柱已用pH6.0.1mM磷酸钠(缓冲液D)平衡过。层析在室温下、杂交Gradi-FracTM/FPLC***(Pharmacia,Uppsala,Sweden)中进行,该***装备有串联的UV(280nm)和电导率监测器。加入样品(5ml/min)后,用10倍柱体积的缓冲液D洗柱。用7倍柱体积至42%缓冲液E/58%缓冲液D(其中缓冲液E是pH6.0,250mM磷酸钠)的线性梯度液,然后用10倍柱体积至52%缓冲液E的梯度液洗脱α-gal A。测定组分的α-gal A活性,合并含有显著活性的组分。
D.用琼脂糖阴离子交换层析作为α-gal A纯化的一个步骤
在室温下用水将羟基磷灰石柱合并物稀释约1.5倍至最终电导率为3.4-3.6mMHO。过滤后,将样品加入Q琼脂糖HP柱(Pharmacia,Uppsala,Sweden:5.1ml柱体积,1.5×2.9cm)中,该柱已用10%缓冲液G/90%缓冲液F平衡过,其中缓冲液F是pH6.0,25M磷酸钠,缓冲液G是含250mM NaCl的pH6.0,25mM磷酸钠。层析在室温下、Gradi-FracTM/FPLC杂交***(Pharmacia,Uppsala,Sweden)中进行,总蛋白和盐浓度由串联的监测器测定。样品以5ml/min的流速加入,然后进行下列步骤:(1)用5倍柱体积的10%缓冲液G,(2)7倍柱体积的12%缓冲液G,(3)3倍柱体积至50%缓冲液G的线性梯度液,(4)10倍柱体积至53%缓冲液G的线性梯度液,(5)3倍柱体积液至100%缓冲液G的梯度液和(6)10倍柱体积的100%缓冲液G洗柱。α-gal A主要在步骤3和4时被洗脱。合并含有显著活性的组分(“Q合并物”)。
E.用Superdex-200凝胶过滤层析作为α-gal A纯化的一个步骤
用Centriprep-10离心浓缩装置(Amicon,Beverly,MA)将Q合并物浓缩大约5倍,并上样于Superdex200柱(Pharmacia,Uppsala,Sweden;189ml柱体积,1.6×94cm)中。该柱用含150mM NaCl的pH6.0.25mM磷酸钠液平衡并洗脱。在室温下FPLC***(Pharmacia,Uppsala,Sweden)中进行层析,用串联的UV监测器(280nm)来跟踪蛋白质的洗脱。加入柱中的样品体积≤2ml,流速为0.5ml/min,组分体积为2ml。进行多个柱操作;测定组分的α-gal A活性,合并含有显著活性的组分。
用Centriprep-10装置浓缩来自Superdex200柱的合并组分,分成等分,快速冷冻,并在-80℃下保藏较短的时间。这一纯化α-gal A的实施例的小结显示在表3中。α-gal A的最终得率为原料活性的59%,纯化产物的比活为2.92×106单位/毫克蛋白。在还原条件下4-15%SDS-聚丙烯酰胺凝胶上进行电泳,然后银染,所得产物显示出高纯度。
实施例Ⅲ.纯化α-gal A的配方和保藏
当高度纯化的α-gal A以纯化蛋白稀溶液(≤1mg蛋白/ml)保存时,不能稳定地保藏较长时间。因此,设计了一种配方以提高α-gal A在长时间保藏(即持续几周到至少几个月)时的稳定性。用离心浓缩器将纯化酶浓缩到至少为1mg/ml(在含25mM磷酸钠(pH6.0)和150mM NaCl组成的酶缓冲液中)。加入人血清白蛋白(HSA;Buminate,Baxter-Hyland)至最终浓度为2.5mg/ml。然后用附有注射器的0.2μm乙酸纤维素滤膜(Schleicher和Schuell)除菌过滤此蛋白质溶液。将α-gal A溶液分装到无菌、无热原的玻璃管中,用Telfon封口带密封,快速冷冻,保藏在-20℃下。
3个月内用4-MUF-gal试验来评价α-gal A活性的稳定性。表5中所示数据表明在测试期间酶活性没有损失。配方的酸性pH对高度纯化的酶的稳定性是非常关键的。
表5:配制的α-gal A在-20℃下的稳定性
样品 | 比活(单位/毫克总蛋白) |
开始时 | 2.24×106±0.33×106 |
1周 | 2.40×106±0.25×106 |
2周 | 2.42×106±0.21×106 |
3周 | 2.37×106±0.05×106 |
1个月 | 2.39×106±0.16×106 |
2个月 | 2.31×106±0.26×106 |
3个月 | 2.29×106±0.17×106 |
实施例Ⅳ,人细胞包株产生的α-gal A适用于治疗α-gal A缺乏症研究了根据本发明制得的纯化人α-gal A在结构和功能上的性质,以证明本文所述的DNA分子以及转染的人细胞株表达产生的相应糖蛋白可分别用于基因或酶替代治疗。
A.稳定转染的人细胞在培养中产生的α-gal A的大小
通过MALDI-TOF质谱分析来估计α-gal A的分子量。这些结果表明二聚物的分子量为102353道尔顿,而单体的分子量为51002道尔顿。根据氨基酸组成,预计单体的分子量为45400道尔顿。因此,可以推断此酶的糖含量可达5600道尔顿分子量。
对纯化蛋白进行标准的氨基酸分析,结果与下述结论是一致的,即转染的人细胞产生的此蛋白与从人组织中纯化获得的此蛋白在氨基酸水平上是相同的。
B.稳定转染的人细胞产生的α-gal A的N-端加工
人α-gal A cDNA核苷酸序列编码429个氨基酸。31个N-端氨基酸组成了信号肽序列,它会在新生蛋白质转运通过内质网膜时断裂(LeDonne等,Arch.Biochem.Biophys.224:186,1983;Lemansky等,J.Biol.Chem.262:2062,1987)。为了确认当α-gal A与异源信号肽序列(例如人生长激素信号序列)相联并在转染的人成纤维细胞表达时被合适地加工,对分泌的蛋白的N-端氨基酸进行微测序。对样品进行SDS-PAGE电泳,并用10mM CAPS(pH11.0),10%甲醇缓冲液***转移到ProBlott(ABI.Foster City,CA)上。用考马斯染色使ProBlott上的蛋白显色,切下合适分子量(50k道尔顿)的条带。用自动进行Edman降解的应用生物***脉冲-液相氨基酸测序仪(Applied Biosystems pulse-liquid phase amino acid sequenator)获得N-端序列。获得的N-端序列LDNGLARTPT(SEQ ID NO:28)与信号肽的适当断裂物一致,并与分泌的蛋白预计的N-端序列一致。
C.稳定转染的人细胞产生的α-gal A的C-端氨基酸
用Hewlett PackardC-端自动测序仪来鉴定根据本发明生产的分泌的α-gal A的C-端氨基酸残基。结果表明C-端为亮氨酸残基,这与以DNA序列预计的C-端氨基酸一致。
D.稳定转染的人细胞产生的α-gal A的糖修饰
对根据本发明制得的α-gal A的糖基化方式也进行了评价。适当的糖基化对α-gal A的最优体内活性是很重要的;非糖基化***表达的α-gal A是没有活性或不稳定的(Hantzopolous等,Gene 57:159,1987)。糖基化对α-gal A内化到所需靶细胞中也是重要的,并且会影响酶在体内的循环半衰期。在α-gal A的每个亚单位上有四个能够加入天冬酰胺连接糖链的位点,其中只有三个被占据(Desnick等.InThe Metabolic and Molecular Bases fInherited Disease,pp2741-2780,McGraw Hill,New York,1995)。
用从A.urafaciens分离获得的神经氨酸酶(Boehringer-Mannheim,Indianapolis,IN)处理稳定转染的细胞产生的α-gal A样品,以除去唾液酸。这一反应是在室温下,在总体积为10μl的乙酸盐缓冲溶液(ABS,20mM乙酸钠,pH5.2,150mMNaCl)中用10mU神经氨酸酶处理5μgα-gal A过夜来进行的。
稳定转染的细胞产生并经纯化的α-gal A还用碱性磷酸酶(小牛肠碱性磷酸酶.Boehringer-Mannheim,Indianapolis,IN)来去磷酸化,方法是在室温下在ABS(pH用1M Tris升高至7.5)中用15U碱性磷酸酶处理5μgα-gal A过夜。
用Western印迹法以α-gal A特异性抗体来分析样品。所用的抗体是兔多克隆抗肽抗体,该抗体是用代表α-gal A的68-81氨基酸的肽作为免疫原而制得。在将蛋白转移到PVDF(Millipore,Bedford,MA)膜上后,用2.5%Blotto无脂肪干奶粉(20mM Tris-HCl液,pH7.5,0.05%Tween-20)液对抗血清作1∶2000稀释来探测此膜。然后用交联辣根过氧化物酶的山羊的抗兔IgG(Organon Teknika/Cappel,durham,NC;1∶5000稀释)和ECL化学发光试剂盒(Amersham,Arlington Heights.IN)的试剂来检测。
用神经氨酸酶处理α-gal A导致其分子量轻微偏移(约1500-2000道尔顿或4-6个唾液酸/单体),这提示α-gal A已由唾液酸充分修饰。例如,血浆形式的α-galA每个单体有5-6个唾液酸残基,而胎盘形式的每个单体有0.5-1.0个唾液酸残基(Bishop等,J.Biol.Chem.256:1307,1981)。
另一种用来测定α-gal A的唾液酸和甘露糖-6-磷酸修饰的方法是等电点聚焦(IEF),根据样品等电点(pI)或净电荷来分离样品。这样,预计从α-gal A中除去带电荷的残基如唾液酸或磷酸,能改变此蛋白质在IEF***中的迁移。
进行IEF实验时,用神经氨酸酶和碱性磷酸酶先处理根据本发明制得的α-galA样品,然后将其按1∶1与2X Novex样品缓冲液(8M尿素,pH3.0-7.0)混合,用Pharmalyte(Pharmacia,Uppsala,Sweden;pH3.0-6.5;Pharmalyte4-6.5和2.5-5.5.每份凝胶0.25ml)加到6M尿素IEF凝胶上。另外还加入等电点标准品(Bio-Rad)。电泳后,将凝胶转移到PVDF膜上,如上所述进行Western印迹分析。
稳定转染的人成纤维细胞产生的α-gal A包括三个主要的同工型,它们的pI大约在4.4-4.65范围内。这些值与血浆形式以及脾形式的α-gal A(Bishop等,J.Biol.Chem.256:1307,1981)的pI相似。用神经氨酸酶处理此酶使得三个同工型的pI均有所提高,这表明所有的同工型均一定程度上被唾液酸修饰。这些数据提示,稳定转染的人细胞产生的α-gal A应有理想的血浆半衰期,说明该物质非常适于药理应用。另外,用碱性磷酸酶来处理经神经氨酸酶处理的α-gal A还会进一步将部分酶蛋白的pI提高至大约5.0-5.1,这说明此酶携带了一个或多个甘露糖-6-磷酸残基。该修饰是意义显著的,因为它是靶细胞有效内化α-gal A所需的。
E.从稳定转染的成纤维细胞纯化获得的α-gal A的比活
通过测定此酶的催化活性(用4-MUF-gal试验法)和蛋白浓度来计算纯化的α-gal A的效能或比活。蛋白浓度可用任何标准方法来测定,例如用BCA***(Pierce)或通过测定280nm吸光值并用2.3(从氨基酸分析测得)的mg/ml消光系数来计算数值。用这些方法测得,从转染的人成纤维细胞的条件培养基中纯化获得的α-galA比活为2.2-2.9×106单位/毫克蛋白,这与从人组织中纯化获得的α-gal A的比活相当(Bishop等,J.Biol.Chem.256:1301,1981)。
F.甘露糖或甘露糖-6-磷酸介导的α-gal A的内化
为了使稳定转染的细胞产生的α-gal A成为α-gal A缺乏症的有效治疗剂,此酶必须被受影响的细胞内化。α-gal A在生理pH水平时没有活性,不可能在血液或组织液中有效。它只有在内化进入溶酶体的酸性环境中时才会使累积的脂类底物最适地代谢。这种内化是由α-gal A与甘露糖-6-磷酸(M6P)受体的结合来介导的,该受体表达在细胞表面并将酶通过内吞通路输送给溶酶体。M6P受体有普遍表达;大多数体细胞都有一定程度的表达。对糖蛋白上外露的甘露糖残基有特异性的甘露糖受体,则普遍性较差。后一种受体通常只发现于巨噬细胞和巨噬细胞样细胞上,它们为α-gal A进入这些种类的细胞提供了额外的方式。
为了证明M6P介导α-gal A的内化,取Fabry病患者的皮肤成纤维细胞(NIGMS Human Genetic Mutant Cell Repository),在浓度渐增的本发明的纯化α-galA存在下培育该细胞过夜。一些样品含有5mM可溶的M6P,M6P竞争性地抑制了α-gal A结合甘露糖-6-磷酸受体并被此受体内化的作用。其它样品含有30μg/ml甘露聚糖,甘露聚糖抑制了α-gal A结合甘露糖受体并被此受体内化的作用。培育后,将细胞刮到裂解缓冲液(10mM Tris,pH7.2,100mM NaCl,5mM EDTA,2mMPefablocTM(Boehringer-Mannheim,Indianapolis,IN)和1%NP-40)中洗涤并收获细胞。然后测定裂解样品的蛋白浓度和α-gal A活性。结果用α-gal A活性单位/毫克细胞蛋白表示。Fabry细胞以剂量依赖方式内化α-gal A(图7)。这种内化受甘露糖-6-磷酸抑制,但不受甘露糖抑制。因此α-gal A在Fabry成纤维细胞中的内化是由甘露糖-6-磷酸受体而不是甘露糖受体来介导的。
α-gal A也可在体外被内皮细胞(为治疗Fabry病重要的靶细胞)内化。将人脐带静脉内皮细胞(HUVEC)与7500单位的α-gal A培育过夜;一些孔中含有M6P。在培育期后,如上所述收获细胞并测定α-gal A。只有与α-gal A一起培育的细胞的此酶水平几乎是对照(不与α-gal A一起培育)细胞的10倍以上。M6P抑制了α-gal A的胞内累积,揭示HUVEC内化α-gal A是由M6P受体介导的。因此,本发明的人α-gal A可被临床相关的细胞内化。
已知只有很少的培育的人细胞系能表达甘露糖受体。然而,可以用携带甘露糖受体和极少量(如果有的话)甘露糖-6-磷酸受体的小鼠巨噬细胞样细胞系(J774.E)来确定本发明的纯化α-gal A是否是通过甘露糖受体而内化(Diment等,J.Leukocyte Biol.42:485-490,1987)。在10000单位/毫升α-gal A的存在下,将J774.E细胞培育过夜。所选样品还含有2mM M6P,其它样品含有100μg/ml甘露聚糖。如上所述洗涤并收获细胞,测定每份样品的总蛋白和α-gal A活性。结果列在表5中。M6P不抑制这些细胞摄入α-gal A,而甘露聚糖使α-gal A的累积水平降低75%。因此,本发明的α-gal A可被表达这类特定细胞表面受体(甘露糖受体)的细胞中的甘露糖受体内化。
表6.J774.E细胞内化α-gal A α-Gal活性(单位/mg总蛋白)
不加入 加α-gal A 加α-gal A、 加α-gal A、甘露M6P 聚糖 |
J774.E 409±25 6444±554 6297±674 1654±323 |
[甘露聚糖]=100μg/ml
[M6P]=2mM或660μg/ml
这些实验证明了稳定转染的人细胞产生的α-gal A可以通过甘露糖或甘露糖-6-磷酸受体被细胞内化。
G.用人成纤维细胞表达α-gal A来纠正Fabry成纤维细胞
就基因治疗而言,种植产生α-gal A的自身细胞必须产生适当修饰形式的此酶,以“纠正”靶细胞中的α-gal A缺乏。为了评价转染的人成纤维细胞在Fabry细胞上生产α-gal A的效果,将取自Fabry病患者的成纤维细胞(NIGMS HumenGenetics Mutant Cell Repository)在Transwells(Costar,Cambridge,MA)中与产生α-gal A的细胞株共同培育。实验方案说明见图8。将Fabry细胞培育在12孔组织培养碟中,其中一些含有细胞能生长在其表面上的插片(insert)(Transwells,0.4μm孔径)。该插片的生长基质是多孔的,其使大分子从上层环境通过孔进入下层环境。一套插片含有分泌少量α-gal A的正常人***(HF)成纤维细胞,而另一套含有能分泌大量α-gal A的稳定转染的人成纤维细胞株BRS-11。在与产生α-gal A的细胞共同培育的孔中,α-gal A可以进入浸有Fabry细胞的培养基并能被Fabry细胞有效地内化。
表7中的数据显示,Fabry细胞能内化分泌的α-gal A。监测胞内α-gal A水平三天。单独培育(没有插片)或在非转染的***成纤维细胞(HF插片)存在下的细胞的胞内α-gal A活性水平非常低。然而,和产生α-gal A的细胞(BRS-11插片)一起培养的Fabry细胞在2天后表现出类似于正常细胞的酶水平(正常的成纤维细胞有25-80单位α-gal A/毫克蛋白)。这种纠正是由于通过M6P受体摄入了α-gal A,可用甘露糖-6-磷酸抑制来证明(BRS-11插片+M6P)。
表7用表达α-gal A的人成纤维细胞纠正Fabry成纤维细胞
α-gal A活性(单位/毫克总蛋白)
时间 | 没有插片 | HF插片 | BRS-11插片 | BRS-11插片+M6P |
第1天 | 2±1 | 2±1 | 13±1 | 4±1 |
第2天 | 2±1 | 2±1 | 40±11 | 6±2 |
第3天 | 2±1 | 5±1 | 85±1 | 9±1 |
H.其它细胞类型的使用
本文描述的方法中可采用其它类型的细胞。这些细胞可从各种组织获得,包括能维持培养的所有类型的细胞。例如,可用本发明方法转染的初级和次级细胞包括人成纤维细胞、角质化细胞、上皮细胞(如***或肠上皮细胞)、内皮细胞、神经胶质细胞、神经细胞、血液的有形成分(如淋巴细胞和骨髓细胞)、肌细胞以及这些体细胞的前体细胞。成纤维细胞是特别受注意的。初级细胞最好是从有待给予转染的初级或次级细胞的个体中获得,这样它们就不会被患者的免疫***排斥。然而,如果能适当地注意避免或抑制免疫排斥(如下所述),也可使用除患者以外的供者人细胞。这将使标准化建株的、稳定转染的细胞系细胞能用于所有患者。
Ⅰ.表达α-gal A的细胞的给予
上述细胞可以通过各种标准化的给药途径导入患者体内,因此它们可以定居在(例如)肾包囊下、皮下间隙、中枢神经***、鞘内腔、肝脏、腹膜内空腔或肌肉中。细胞也可经静脉或动脉内注入,使其在个体的血流中循环。一旦植入体内,转染的细胞就会产生并分泌治疗产物-糖基化的人α-gal A。
有待引入个体内的基因修饰细胞的数目将有所不同,但这是本领域技术人员能确定的。每位患者的年龄、体重、性别以及总的健康状况、还有分布的体积、酶的半衰期和生物利用度、以及基因修饰细胞的体内产率,这些是确定给药剂量和途径的首要考虑因素。通常,将采用1百万至10亿个细胞,表达水平为100-100000单位/106细胞/天。如果需要,该过程可以重复或作改变,直至达到所需结果,例如解除Fabry病有关的症状。
如上所述,采用的细胞通常对患者是特异性的,即从有待给予转染的初级或次级细胞的个体获得的细胞,以使细胞不被患者的免疫***排斥。然而,如果这种情况不可能或不需要,则可以从其它个体获取细胞,如本文所述那样进行基因修饰,并再植入患α-gal A缺乏症的患者体内。
用受者以外的个体的细胞可能需要给予免疫抑制剂、改变组织相容性抗原,或采用屏蔽装置(barrier device)来防止植入细胞被排斥。屏蔽装置可用一种材料(例如膜,如Amicon,Beverly,MA的XM-50)制成,该种材料允许分泌产物进入受者循环或组织,但可防止植入细胞与受者免疫***接触,从而防止了受者对细胞的免疫应答(以及可能产生的排斥)。关于基因治疗的进一步的指导可以参见Selden等的WO93/09222。
或者,细胞可以包埋在基质或凝胶材料中,如共同拥有的美国专利08/548/002中所描述的那样,采用杂交基质植入物,或如Jain等在PCT专利申请WO95/19430中描述的那样,将分泌细胞高度胶囊化(macroencapsulation)在疏水凝胶材料中(这些内容均纳入本文作参考)。
J.常规给予α-gal A蛋白的药物配方
本文所述的由稳定转染的(或者说是遗传上修饰的)人细胞表达和分泌并经纯化的α-gal A蛋白可给予不能产生或产生很少α-gal A蛋白的个体。该蛋白可以配制在药学上可接受的pH低于6.5的载体中,例如如实施例Ⅲ所述的配方中。可加入配方中的赋形剂的例子为缓冲液,例如柠檬酸盐缓冲液、磷酸盐缓冲液、乙酸盐缓冲液和碳酸氢盐缓冲液、氨基酸、尿素、醇、抗坏血酸、磷脂、蛋白质如血清白蛋白和明胶、EDTA、氯化钠、脂质体、聚乙烯吡咯烷酮、甘露糖醇、山梨糖醇、甘油、丙二醇和聚乙二醇(如PEG-4000,PEG-6000)。给药途径例如可以是静脉内、动脉内、皮下、腹膜内、脑内、肌内、肺内或经粘膜给药。给药途径和蛋白质递送量可以通过本领域技术人员能进行评价的几个因素来决定。另外,本领域技术人员知道,对于确定的患者,给药途径和治疗蛋白的剂量可以不同,直至获得治疗剂量水平。通常,每公斤体重将给予0.01-100毫克剂量的α-galA。预计在患者的一生中需要有规律地重复给予此蛋白剂量。
K.酶缺乏引起的其它疾病的治疗
同样,由α-gal A以外的溶酶体贮积酶的缺乏引起的其它疾病也适合用类似于本文所述的那些方法来治疗。在这些情况下,编码缺乏酶的功能形式的DNA可以在本文揭示的表达构建物中代替编码α-gal A的DNA。表8中列出了已经确定且适合本文所述治疗方法的酶缺乏综合征的例子。该表中的信息来自E.Neufeld的Ann.Rev.Biochem.(60:257-280,1991),这些内容纳入本文作参考。
表8.溶酶体贮积病的概述疾病 初级缺乏[次级缺乏]鞘酯降解疾病Fabry病 α-半乳糖苷酶Farber病 神经酰胺酶(ceramidase)Gaucher病 葡糖脑苷脂酶GM1神经节苷脂沉积症 β-半乳糖苷酶GM2神经节苷脂沉积症 β-氨基己糖苷酶,α-亚单位Tay-Sachs病 [氨基己糖苷酶A]Sandhoff病 β-氨基己糖苷酶,β-亚单位
[氨基己糖苷酶A和B]激活剂缺乏症 GM2激活剂Krabbe病 半乳糖神经酰胺酶异染性脑白质营养不良酶缺乏形式 芳基硫酸酯酶A激活剂缺乏形式 硫脑苷脂激活剂/皂草素(saposin)粘脂病Ⅳ型 初级缺乏未知
[神经节苷脂唾液酸酶]多硫酸酯酶缺乏症 初级缺乏未知
[缺乏所有的硫酸酯酶]Niemann-Pick病 鞘磷脂酶Schindler病 α-N-乙酰氨基半乳糖苷酶糖蛋白降解疾病天冬氨酰氨基葡糖胺尿 天冬氨酰氨基葡糖苷酶(aspartylglycosaminuria)岩藻糖苷(贮积)病 α-L-岩藻糖苷酶半乳糖唾液酸沉积症 保护性蛋白/组织蛋白酶(galactosialidosis)
[β-半乳糖苷酶和唾液酸酶]α-甘露糖苷过多症 α-甘露糖苷酶β-甘露糖苷过多症 β-甘露糖苷酶唾液酸过多症 唾液酸酶葡萄糖胺聚糖降解疾病Hunter综合征 艾杜糖醛酸硫酸酯酶Hurler和Scheie综合征 α-L-艾杜糖醛酸酶Maroteaux-Lamy综合征 GalNAc 4-硫酸酯酶/芳基硫酸酯酶Morquio综合征A-亚型 Gal 6-硫酸酯酶B-亚型 β-半乳糖苷酶Sanfilippo综合征A-亚型 类肝素N-硫酸酯酶B-亚型 α-N-乙酰基葡糖胺糖苷酶C-亚型 乙酰基CoA:葡糖胺N-乙酰基转移酶D-亚型 GlcNAc 6-硫酸酯酶Sly综合征 β-葡糖苷酸酶其它单一酶缺乏病Pompe病(糖原贮积病Ⅱ) α-葡糖苷酶Wolman病 酸性脂肪酶溶酶体酶生物合成疾病Ⅰ-细胞病和假Hurler病 6-磷-N-乙酰基葡糖胺
转移酶多种营养不良(polystrophy) [许多溶酶体酶的错位]溶酶体膜转运疾病胱氨酸(代谢)病 胱氨酸转运
唾液酸贮积和Salla病唾液酸转运
Ⅴ.其它实施例
本文所述的发明已经部分列举了用转染后(即在引入编码基因产物的、具有控制编码序列表达的调控元件的构建物后)表达特定基因产物的细胞来治疗的方法。这些方法也可采用由其它方法遗传修饰的细胞来实现(包括基因寻靶和基因激活(参见Treco等,WO95/31560,纳入本文作参考);另见Selden等,WO93/09222)。
hGH信号肽可以与α-gal A以外的异源蛋白一起使用,以提高异源蛋白的表达和分泌水平。这些蛋白的例子包括α-1抗胰蛋白酶、抗凝血酶Ⅲ、载脂蛋白E、载脂蛋白A-1、凝血因子Ⅴ、Ⅶ、Ⅷ、Ⅸ、Ⅹ和ⅩⅢ、骨生长因子-2、骨生长因子-7、降钙素、催化性抗体、DNA酶、红细胞生成素、FSH-β、球蛋白、胰高血糖素、葡糖脑苷脂酶、G-CSF、GM-CSF、生长激素、免疫应答修饰因子、免疫球蛋白、胰岛素、促胰岛素、***、干扰素-β、干扰素-β神经生长因子、白细胞介素-1、白细胞介素-2、白细胞介素-3、白细胞介素-4、白细胞介素-6、白细胞介素-11、白细胞介素-12、IL-2受体、IL-1受体拮抗物、低密度脂蛋白受体、M-CSF、甲状旁腺激素、蛋白激酶C、可溶性CD4、超氧化物歧化酶、组织纤溶酶原活化因子、TGF-β、肿瘤坏死因子、TSHβ、酪氨酸羟化酶和尿激酶。
其它实施例在下列权利要求中。
序列表
(1)一般信息
(ⅰ)申请人:Transkaryotic Therapies,Inc.
(ⅱ)发明名称:半乳糖苷酶A缺乏症的治疗
(ⅲ)序列数目:28
(ⅳ)通信地址:
(A)收件人:Fish&Richardson P.C.
(B)街道:225 Franklin Street
(C)城市:Boston
(D)州:MA
(E)国家:USA
(F)邮编:02110-2804
(ⅴ)计算机可读形式:
(A)记录介质类型:磁盘
(B)计算机:IBM兼容型
(C)操作***:PC-DOS/MS-DOS
(D)软件:Patent In Eelease#1.0,Verasion#1.30
(ⅵ)本申请资料:
(A)申请号:
(B)申请日:
(ⅶ)在先申请资料:
(A)申请号:08/712,614
(B)申请日:13-SEP-1996
(ⅷ)律师/代理人信息:
(A)姓名:Fraser,J anis K
(B)登记号:34,819
(C)参考/案卷号:07236/003WO1
(ⅸ)通讯信息:
(A)电话:617/542-5070
(B)电传:617/542-8906
(C)电报:200154
(2)SEQ ID NO:1的信息:
(ⅰ)序列特征:
(A)长度:22碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:1:CTGGGCTGTA GCTATGATAA AC 22
(2)SEQ ID NO:2的信息:
(ⅰ)序列特征:
(A)长度:21碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:2:TCTAGCTGAA GCAAAACAGT G 21
(2)SEQ ID NO:3的信息:
(ⅰ)序列特征:
(A)长度:19碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:3:ATTGGTCCGC CCCTGAGGT 19
(2)SEQ ID NO:4的信息:
(ⅰ)序列特征:
(A)长度:20碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:4:TGATGCAGGA ATCTGGGCTCT 20
(2)SEQ ID NO:5的信息:
(ⅰ)序列特征:
(A)长度:20碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:5:TTTTGGATCC ACCATGGCTA 20
(2)SEQ ID NO:6的信息:
(ⅰ)序列特征:
(A)长度:24碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:6:TTTTGCCGGC ACTGCCCTCT TGAA 24
(2)SEQ ID NO:7的信息:
(ⅰ)序列特征:
(A)长度:24碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:7:TTTTCAGCTG GACAATGGAT TGGC 24
(2)SEQ ID NO:8的信息:
(ⅰ)序列特征:
(A)长度:20碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:8:TTTTGCTAGC TGGCGAATCC 20
(2)SEQ ID NO:9的信息:
(ⅰ)序列特征:
(A)长度:30碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:9:TTTTGGATCC GTGTCCCATA GTGTTTCCAA 30
(2)SEQ ID NO:10的信息:
(ⅰ)序列特征:
(A)长度:28碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:10:TTTTGGATCC GCAGTCGTGG CCAGTACC 28
(2)SEQ ID NO:11的信息:
(ⅰ)序列特征:
(A)长度:12碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:11:CTAGTCCTAG GA 12
(2)SEQ ID NO:12的信息:
(ⅰ)序列特征:
(A)长度:22碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:12:TTTTGAGCAC AGAGCCTCGC CT 22
(2)SEQ ID NO:13的信息:
(ⅰ)序列特征:
(A)长度:30碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:13:TTTTGGATCC GGTGAGCTGC GAGAATAGCC 30
(2)SEQ ID NO:14的信息:
(ⅰ)序列特征:
(A)长度:76碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:14:GGGCCCCCAG CCCCAGCCCT CCCATTGGTG GAGGCCCTTT TGGAGGCACC CTAGGGCCAG 60GAAACTTTTG CCGTAT 76
(2)SEQ ID NO:15的信息:
(ⅰ)序列特征:
(A)长度:69碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:15:AAATAGGGCA GATCCGGGCT TTATTATTTT AGCACCACGG CCGCCGAGAC CGCGTCCGCC 60CCGCGAGCA 69
(2)SEQ ID NO:16的信息:
(ⅰ)序列特征:
(A)长度:86碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:16:TGCCCTATTT ATACGGCAAA AGTTTCCTGG CCCTAGGGTG CCTCCAAAAG GGCCTCCACC 60AATGGGAGGG CTGGGGCTGG GGGCCC 86
(2)SEQ ID NO:17的信息:
(ⅰ)序列特征:
(A)长度:55碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO№1 7:CGCGGGGCGG ACCGCGGTCTC GGCGGCCGTG GTGCTAAAAT AATAAAGCCC GGATC 55
(2)SEQ ID NO:18的信息:
(ⅰ)序列特征:
(A)长度:1343碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:18:CCGCGGGAAA TTTATGCTGT CCGGTCACCG TGACAATGCA GCTGAGGAAC CCAGAACTAC 60ATCTGCGCTG CGCGCTTGCG CTTCGCTTCC TGGCCCTCGT TTCCTGGAGC ATCCCTGGGG 120CTAGAGCACT GGACAATGGA TTGGCAAGGA CGCCTACCAT GGGCTGGCTG CACTGGGAGC 180GCTTCATGTG CAACCTTGAC TGCCAGGAAG AGCCAGATTC CTGCATCAGT GAGAAGCTCT 240TCATGGAGAT GGCAGAGCTC ATGGTCTGAG AAGGCTGGAA GGATGCAGGT TATGAGTACC 300TCTGCATTGA TGACTGTTGG ATGGCTCCCC AAAGAGATTC AGAAGGCAGA CTTCAGGCAG 360ACCCTCAGCG CTTTCCTCAT GGGATTCGCC AGCTAGCTAA TTATGTTCAC AGCAAAGGAC 420TGAAGCTAGG GATTTATGCA GATGTTGGAA ATAAAACCTG CGCAGGCTTC CCTGGGAGTT 480TTGGATACTA CGACATTGAT GCCCAGACCT TGGCTGACTG GGGAGTAGAT CTGCTAAAAT 540TTGATGGTTG TTACTGTGAC AGTTTGGAAA ATTTGGCAGA TGGTTATAAG CACATGTCCT 600TGGCCCTGAA TAGGACTGGC AGAAGCATTG TGTACTCCTG TGAGTGGCCT CTTTATATGT 660GGCCCTTTCA AAAGCCCAAT TATACAGAAA TCCGACAGTA CTGCAATCAC TGGCGAAATT 720TTGCTGACAT TGATGATTCC TGGAAAGGTA TAAAGAGTAT CTTGGACTGG ACATCTTTTA 780ACCAGGAGAG AATTGTTGAT GTTGCTGGAC CAGGGGGTTG GAATGACCCA GATATGTTAG 840TGATTGGCAA CTTTGGCCTC AGCTGGAATC AGCAAGTAAC TCAGATGGCC CTCTGGGCTA 900TCATGGCTGC TCCTTTATTC ATGTCTAATG ACCTCCGACA CATCAGCCCT CAAGCCAAAG 960CTCTCCTTCA GGATAAGGAC GTAATTGCCA TCAATCAGGA CCCCTTGGGC AAGCAAGGGT 1020ACCAGCTTAG ACAGGGAGAC AACTTTGAAG TGTGGGAACG ACCTCTCTCA GGCTTAGCCT 1080GGGCTGTAGC TATGATAAAC CGGCAGGAGA TTGGTGGACC TCGCTCTTAT ACCATCGCAG 1140TTGCTTCCCT GGGTAAAGGA GTGGCCTGTA ATCCTGCCTG CTTCATCACA CGCTCCCTCC 1200CTGTGAAAAG GAAGCTAGGG TTCTATGAAT GGACTTCAAG GTTAAGAAGT CACATAAATC 1260CCACAGGCAC TGTTTTGCTT CAGCTAGAAA ATACAATGCA GATGTCATTA AAAGACTTAC 1320TTTAAAAAAA AAAAAAACTC GAG 1343
(2)SEQ ID NO:19的信息:
(ⅰ)序列特征:
(A)长度:210碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:19:CTGGGCTGTA GCTATGATAA ACCGGCAGGA GATTGGTGGA CCTCGCTCTT ATACCATCGC 60AGTTGCTTCC CTGGGTAAAG GAGTGGCCTG TAATCCTGCC TGCTTCATCA CACAGCTCCT 120CCCTGTGAAA AGGAAGCTAG GGTTCTATGA ATGGACTTCA AGGTTAAGAA GTCACATAAA 180TCCCACAGGC ACTGTTTTGC TTCAGCTAGA 210
(2)SEQ ID NO:20的信息:
(ⅰ)序列特征:
(A)长度:268碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:20:ATTGGTCCGC CCCTGAGGTT AATCTTAAAA GCCCAGGTTA CCCGCGGAAA TTTATGCTGT 60CCGGTCACCG TGACAATGCA GCTGAGGAAC CCAGAACTAC ATCTGGGCTG CGCGCTTGCG 120CTTCGCTTCC TGGCCCTCGT TTCCTGGGAC ATCCCTGGGG CTAGAGCACT GGACAATGGA 180TTGGCAAGGA CGCCTACCAT GGGCTGGCTG CACTGGGAGC GCTTCATGTG CAACCTTGAC 240TGCCAGGAAG AGCCAGATTC CTGCATCA 268
(2)SEQ ID NO:21的信息:
(ⅰ)序列特征:
(A)长度:26氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ⅱ)分子类型:肽
(ⅹⅰ)序列描述:SEQ ID NO:21:Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu1 5 10 15Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala
20 25
(2)SEQ ID NO:22的信息:
(ⅰ)序列特征:
(A)长度:78碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:22:ATGGCTACAG GCTCCCGGAC GTCCCTGCTC CTGGCTTTTG GCCTGCTCTG CCTGCCCTGG 60CTTCAAGAGG GCAGTGCC 78
(2)SEQ ID NO:23的信息:
(ⅰ)序列特征:
(A)长度:35碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:23:TTTTGGATCC CTCGAGGACA TTGATTATTG ACTAG 35
(2)SEQ ID NO:24的信息:
(ⅰ)序列特征:
(A)长度:28碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:24:TTTTGGATCC CGTGTCAAGG ACGGTGAC 28
(2)SEQ ID NO:25的信息:
(ⅰ)序列特征:
(A)长度:1197碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述:SEQ ID NO:25:CTG GAC AAT GGA TTG GCA AGG ACG CCT ACC ATG GGC TGG CTG CAC TGG 48GAG CGC TTC ATG TGC AAC CTT GAC TGC CAG GAA GAG CCA GAT TCC TGC 96ATC AGT GAG AAG CTC TTC ATG GAG ATG GCA GAG CTC ATG GTC TCA GAA 144GGC TGG AAG GAT GCA GGT TAT GAG TAC CTC TGC ATT GAT GAC TGT TGG 192ATG GCT CCC CAA AGA GAT TCA GAA GGC AGA CTT CAG GCA GAC CCT CAG 240CGC TTT CCT CAT GGG ATT CGC CAG CTA GCT AAT TAT GTT CAC AGC AAA 288GGA CTG AAG CTA GGG ATT TAT GCA GAT GTT GGA AAT AAA ACC TGC GCA 336GGC TTC CCT GGG AGT TTT GGA TAC TAC GAC ATT GAT GCC CAG ACC TTT 384GCT GAC TGG GGA GTA GAT CTG CTA AAA TTT GAT GGT TGT TAC TGT GAC 432AGT TTG GAA AAT TTG GCA GAT GGT TAT AAG CAC ATG TCC TTG GCC CTG 480AAT AGG ACT GGC AGA AGC ATT GTG TAC TCC TGT GAG TGG CCT CTT TAT 528ATG TGG CCC TTT CAA AAG CCC AAT TAT ACA GAA ATC CGA CAG TAC TGC 576AAT CAC TGG CGA AAT TTT GCT GAC ATT GAT GAT TCC TGG AAA AGT ATA 624AAG AGT ATC TTG GAC TGG ACA TCT TTT AAC CAG GAG AGA ATT GTT GAT 672GTT GCT GGA CCA GGG GGT TGG AAT GAC CCA GAT ATG TTA GTG ATT GGC 720AAC TTT GGC CTC AGC TGG AAT CAG CAA GTA ACT CAG ATG GCC CTC TGG 768GCT ATC ATG GCT GCT CCT TTA TTC ATG TCT AAT GAC CTC CGA CAC ATC 816AGC CCT CAA GCC AAA GCT CTC CTT CAG GAT AAG GAC GTA ATT GCC ATC 864AAT CAG GAC CCC TTG GGC AAG CAA GGG TAC CAG CTT AGA CAG GGA GAC 912AAC TTT GAA GTG TGG GAA CGA CCT CTC TCA GGC TTA GCC TGG GCT GTA 960GCT ATG ATA AAC CGG CAG GAG ATT GGT GGA CCT CGC TCT TAT ACC ATC 1008GCA GTT GCT TCC CTG GGT AAA GGA GTG GCC TGT AAT CCT GCC TGC TTC 1056ATC ACA CAG CTC CTC CCT GTG AAA AGG AAG CTA GGG TTC TAT GAA TGG 1104ACT TCA AGG TTA AGA AGT CAC ATA AAT CCC ACA GGC ACT GTT TTG CTT 1152CAG CTA GAA AAT ACA ATG CAG ATG TCA TTA AAA GAC TTA CTT TAA 1197
(2)SEQ ID NO:26的信息:
(ⅰ)序列特征:
(A)长度:398氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ⅱ)分子类型:肽
(ⅴ)片段类型:内部
(ⅹⅰ)序列描述:SEQ ID NO:26:Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp1 5 10 15Glu Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys
20 25 30Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu
35 40 45Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp
50 55 60Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln65 70 75 80Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys
85 90 95Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala
100 105 110Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe
115 120 125Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp
130 135 140Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu145 150 155 160Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr
165 170 175Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys
180 185 190Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile
195 200 205Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp
210 215 220Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly225 230 235 240Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp
245 250 255Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile
260 265 270Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile
275 280 285Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp
290 295 300Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val305 310 315 320Ala Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile
325 330 335Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe
340 345 350Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp
355 360 365Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu
370 375 380Gln Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu385 390 395
(2)SEQ ID NO:27的信息:
(ⅰ)序列特征:
(A)长度:338碱基对
(B)类型:核酸
(C)涟型:单链
(D)拓扑结构:线性
(ⅹⅰ)序列描述SEQ ID NO:27:ATGGCTACAG GTAAGCGCCC CTAAAATCCC TTTGGGCACA ATGTGTCCTG AGGGGAGAGG 60CAGCGACCTG TAGATGGGAC GGGGGCACTA ACCCTCAGGT TTGGGGCTTC TGAATGTGAG 120TATCGCCATG TAAGCCCAGT ATTTGGCCAA TCTCAGAAAG CTCCTGGTCC CTGGAGGGAT 180GGAGAGAGAA AAACAAACAG CTCCTGGAGC AGGGAGAGTG CTGGCCTCTT GCTCTCCGGC 240TCCCTCTGTT GCCCTCTGGT TTCTCCCCAG GCTCCCGGAC GTCCCTGCTC CTGGCTTTTG 300GCCTGCTCTG CCTGCCCTGG CTTCAAGAGG GCAGTGCC 338
(2)SEQ ID NO:28的信息:
(ⅰ)序列特征:
(A)长度:10氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ⅱ)分子类型:肽
(ⅹⅰ)序列描述:SEQ ID NO:28:Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr1 5 10
Claims (35)
1.一种治疗方法,包括鉴别怀疑患有α-半乳糖苷酶A缺乏症的病人,和给病人导入基因修饰的人细胞以过度表达和分泌人α-半乳糖苷酶A。
2.根据权利要求1所述的方法,其中细胞以一个DNA分子体外转染,所述DNA分子具有一个编码序列,该序列编码包含人α-半乳糖苷酶A的一个多肽(SEQID NO:26)。
3.根据权利要求2所述的方法,其中的多肽还包括一个异源信号肽。
4.根据权利要求3所述的方法,其中的异源信号肽是人生长激素(hGH)信号肽(SED ID NO:21)。
5.根据权利要求3所述的方法,其中DNA分子包含一个位于编码信号肽序列中的内含子。
6.根据权利要求3所述的方法,其中DNA分子包含至少有6个核苷酸的非翻译序列,它正好位于3到该编译码序列末端密码子处,所述的作翻译序列包括一个多腺苷酸化位点。
7.一种DNA分子,包括
编码hGH信号肽的第一序列(SEQ ID NO:21),所述的第一序列包括一个内含子;和
连接到第一序列的3’末端的第二序列,此第二序列编码含人α-半乳糖苷酶A的一个多肽。
8.根据权利要求7所述的DNA分子,它还包括含多腺苷酸化位点的3’非翻译序列。
9.一种表达构建物,它包括权利要求7所述的DNA分子,它适合在人细胞中表达。
10.一种培养的人体细胞,它含DNA分子,所述DNA分子(a)编码含有与异源信号肽连接的人α-半乳糖苷酶A的一个多肽,和(b)允许此多肽在细胞中表达。
11.根据权利要求10所述的细胞,所述的细胞是成纤维细胞。
12.根据权利要求10所述的细胞,其中所述的细胞选自上皮细胞、内皮细胞、骨髓细胞、神经胶质细胞、肝细胞、角质化细胞、肌肉细胞、神经细胞或这些类型细胞的前体。
13.根据权利要求10所述的细胞,其中所述的信号肽是hGH信号肽(SEQ IDNO:21)。
14.一种克隆细胞株,它基本上由许多培养的人细胞构成,所述的培养的人细胞被一个DNA分子转染,此DNA分子(a)编码含有与异源信号肽连接的人α-半乳糖苷酶A的一个多肽,和(b)允许此多肽在细胞里表达。
15.根据权利要求14所述的克隆细胞株,该细胞是成纤维细胞。
16.一种克隆细胞系,它基本上由许多不死的人细胞构成,所述的不死细胞被一个DNA分子转染,此DNA分子(a)编码含有与异源信号肽连接的人α-半乳糖苷酶A的一个多肽,和(b)允许此多肽在细胞里表达。
17.根据权利要求16所述的克隆细胞系,其中该细胞选自Bows黑素瘤细胞、Daudi细胞,Hela细胞、HL-60细胞,HT1080细胞,Jurkat细胞,KB癌瘤细胞,K-562白血病细胞,MCF-7乳腺癌细胞,MOLT-4细胞,Namalwa细胞,Raji细胞,RPMI8226细胞,U-937细胞,WI-38VA13亚系2R4细胞和2780AD卵巢癌细胞。
18.一种蛋白质,它包括(a)hGH信号肽(SEQ ID NO:21),此信号肽被肽键连接到(b)人α-半乳糖苷酶A上。
19.一种制备糖化的人α-半乳糖苷酶A的方法,它包括
在允许所述的DNA分子表达人α-半乳糖苷酶A的条件下培养权利要求11所述的细胞,并分泌糖化的人α-半乳糖苷酶A到细胞的培养介质中;和
从培养介质分离出糖化的人α-半乳糖苷酶A。
20.用权利要求19所述的方法制取纯化的糖化人α-半乳糖苷酶A。
21.一种从样品中纯化人α-半乳糖苷酶A的方法,它包括第一层析步骤,其中样品通过一个疏水反应树脂。
22.根据权利要求21所述的方法,其中树脂上的功能团部分包括丁基。
23.一种制备糖化的人α-半乳糖苷酶A的方法,它包括
在允许所述的DNA分子表达人α-半乳糖苷酶A的条件下培养权利要求11所述的细胞,并分泌糖化的人α-半乳糖苷酶A到细胞的培养介质中;和
从培养介质分离出糖化的人α-半乳糖苷酶A。
24.根据权利要求21所述的方法,它还包括使样品通过第二树脂的步骤,所述的第二树脂选自固定了肝素的树脂,羟基磷灰石,阴离子交换树脂,和分子筛树脂。
25.一种治疗方法,它包括
鉴别怀疑患有α-半乳糖苷酶A缺乏症的病人,和
给予病人权利要求20所述的纯化的糖化人α-半乳糖苷酶A。
26.一种治疗组合物,它包括药学上可接受的纯化的人α-半乳糖苷酶A,此酶在药学上可接受的载体里有N-连接的甘露糖-6-磷酸盐。
27.一种治疗组合物,它包括权利要求20所述的纯化的人α-半乳糖苷酶A和药学上可接受的赋形剂。
28.根据权利要求27所述的治疗组合物,它在pH6.5或更低pH下配制。
29.根据权利要求28所述的治疗组合物,其中药学上可接受的赋形剂是人血清白蛋白。
30.一种编码一种蛋白质的DNA分子,该蛋白包括连接到非hGH的一个多肽上的hGH的信号肽(SEQ ID NO:21)。
31.根据权利要求30所述的DNA分子,其中该DNA分子包括一个位于编码信号肽序列中的内含子。
32.一种培养的人细胞,它含DNA分子,所述的DNA分子(a)编码含有与异源信号肽连接的人α-半乳糖苷酶A的一个多肽,和(b)允许此多肽在细胞里过度表达。
33.一种制备糖化人α-半乳糖苷酶A的方法,它包括培养含DNA分子的人细胞,此DNA分子在允许表达人α-半乳糖苷酶A的条件下编码含人α-半乳糖苷酶A的多肽。
34.根据权利要求26所述的治疗组合物,它还包括药学上可接受的赋形剂。
35.一种从样品中纯化人α-半乳糖苷酶A的方法,它包括将所述的样品通过疏水反应树脂,所述的树脂含有丁基作为功能团部分。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US71261496A | 1996-09-13 | 1996-09-13 | |
US08/712,614 | 1996-09-13 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1230220A true CN1230220A (zh) | 1999-09-29 |
CN1268741C CN1268741C (zh) | 2006-08-09 |
Family
ID=24862863
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB97197909XA Expired - Lifetime CN1268741C (zh) | 1996-09-13 | 1997-09-12 | 半乳糖苷酶a缺乏症的治疗 |
Country Status (21)
Country | Link |
---|---|
EP (4) | EP0935651B1 (zh) |
JP (7) | JP4001925B2 (zh) |
KR (2) | KR20050084473A (zh) |
CN (1) | CN1268741C (zh) |
AT (1) | ATE286120T1 (zh) |
AU (1) | AU4424497A (zh) |
CA (1) | CA2265464C (zh) |
DE (1) | DE69732129T2 (zh) |
DK (2) | DK2374876T3 (zh) |
ES (3) | ES2458292T3 (zh) |
HK (3) | HK1022173A1 (zh) |
HU (2) | HU227189B1 (zh) |
IL (2) | IL128960A (zh) |
MX (1) | MX340738B (zh) |
NO (4) | NO328443B1 (zh) |
NZ (2) | NZ506214A (zh) |
PL (1) | PL190041B1 (zh) |
PT (1) | PT1538202E (zh) |
RU (1) | RU2179034C2 (zh) |
TW (1) | TW585919B (zh) |
WO (1) | WO1998011206A2 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107980063A (zh) * | 2015-05-11 | 2018-05-01 | Ucl商业有限公司 | 法布里病基因治疗 |
CN112852784A (zh) * | 2013-10-23 | 2021-05-28 | 建新公司 | 重组糖蛋白及其用途 |
Families Citing this family (41)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6458574B1 (en) * | 1996-09-12 | 2002-10-01 | Transkaryotic Therapies, Inc. | Treatment of a α-galactosidase a deficiency |
US6083725A (en) | 1996-09-13 | 2000-07-04 | Transkaryotic Therapies, Inc. | Tranfected human cells expressing human α-galactosidase A protein |
IL135578A0 (en) * | 1997-10-29 | 2001-05-20 | Genzyme Corp | Compositions and methods for treating lysosomal storage disease |
EP1658857A1 (en) * | 1997-10-29 | 2006-05-24 | Genzyme Corporation | Compositions and methods for treating lysosomal storage disease |
US6274597B1 (en) | 1998-06-01 | 2001-08-14 | Mount Sinai School Of Medicine Of New York University | Method of enhancing lysosomal α-Galactosidase A |
AU2016250357B2 (en) * | 1999-03-11 | 2018-11-01 | Shire Human Genetic Therapies, Inc. | Treatment of alpha-Galactosidase A deficiency |
AU2004242550B2 (en) * | 1999-03-11 | 2008-04-03 | Shire Human Genetic Therapies, Inc. | Treatment of alpha-Galactosidase A deficiency |
AU2012241170B2 (en) * | 1999-03-11 | 2016-07-28 | Shire Human Genetic Therapies, Inc. | Treatment of alpha-Galactosidase A deficiency |
EP2275559A3 (en) * | 1999-09-28 | 2011-03-23 | Shire Human Genetic Therapies, Inc. | Optimized messenger RNA |
WO2002064799A2 (en) | 1999-09-28 | 2002-08-22 | Transkaryotic Therapies, Inc. | Optimized messenger rna |
AU1647501A (en) | 1999-12-10 | 2001-06-18 | Cytos Biotechnology Ag | Replicon based activation of endogenous genes |
BR0108077A (pt) | 2000-02-04 | 2002-10-22 | Children S Hospital Res Founda | Processo para reduzir placas ateroscleróticas em um mamìfero, método para tratamento da aterosclerose em um mamìfero, composição, métodos para prover proteìna ou polipeptìdeo hidrolisante de lipìdeo biologicamente ativo ou mistura deles, para células de um mamìfero tendo deficiência na proteìna ou polipeptìdeo hidrolisante de lipìdeo biologicamente ativo , para prover lipase de ácido lisossomal biologicamente ativa para células de um mamìfero tendo deficiência de lipase de ácido lisossomal biologicamente ativa, para prover lipase de ácido lisossoal biologicamente ativa para células de um mamìfero com aterosclerose, para tratamento da doença de wolman em um mamìfero, para tratamento de doença de armazenagem de colesteril éster em um mamìfero, e, para tratamento da aterosclerose em um mamìfero |
WO2001097829A2 (en) | 2000-06-19 | 2001-12-27 | Genzyme Corporation | Combination enzyme replacement, gene therapy and small molecule therapy for lysosomal storage diseases |
US7138262B1 (en) * | 2000-08-18 | 2006-11-21 | Shire Human Genetic Therapies, Inc. | High mannose proteins and methods of making high mannose proteins |
IL140110A0 (en) * | 2000-12-05 | 2002-02-10 | Applied Research Systems | Improvement of homogeneity and secretion of recombinant proteins in mammalian systems |
NZ536361A (en) | 2002-04-25 | 2008-05-30 | Shire Human Genetic Therapies | Treatment of alpha-galactosidase a deficiency |
ATE521701T1 (de) | 2003-01-22 | 2011-09-15 | Univ Duke | Verbesserte konstrukte zur expression lysosomaler polypeptide |
WO2007058381A1 (ja) * | 2005-11-18 | 2007-05-24 | Tokyo Metropolitan Organization For Medical Research | 基質特異性を変換した新規高機能酵素 |
AR059089A1 (es) | 2006-01-20 | 2008-03-12 | Genzyme Corp | Administracion intraventricular de una enzima para enfermedades de almacenamiento lisosomal |
ES2391657T3 (es) | 2006-02-07 | 2012-11-28 | Shire Human Genetic Therapies, Inc. | Composiciones estabilizadas de proteínas que tienen un resto tiol libre |
JP2009526066A (ja) | 2006-02-09 | 2009-07-16 | ジェンザイム・コーポレーション | 遅速性脳室内送達 |
US8569032B2 (en) | 2007-05-18 | 2013-10-29 | Tokyo Metropolitan Institute Of Medical Science | Proteins having acquired A-galactosidase activity |
SI2889043T1 (sl) | 2008-12-16 | 2019-08-30 | Genzyme Corporation | Sintetične vmesne spojine za pripravo konjugatov oligosaharid-protein |
RU2733466C2 (ru) | 2009-07-28 | 2020-10-01 | Шайр Хьюман Дженетик Терапиз | Композиции и способы для лечения болезни гоше |
MX338426B (es) | 2010-04-23 | 2016-04-15 | Synageva Biopharma Corp | Enzima de enfermedad del almacenamiento lisosomico. |
US9453847B2 (en) | 2010-07-19 | 2016-09-27 | Shire Human Genetic Therapies, Inc. | Mannose receptor C type 1 (MRC1) codon optimized cell line and uses thereof |
EP2613798B2 (en) | 2010-09-09 | 2018-01-24 | Synageva BioPharma Corp. | Use of lysosomal acid lipase for treating lysosomal acid lipase deficiency in patients |
WO2012112681A1 (en) | 2011-02-15 | 2012-08-23 | Shire Human Genetic Therapies, Inc. | Methods for treating lysosomal acid lipase deficiency |
KR20140091721A (ko) * | 2011-11-02 | 2014-07-22 | 제넨테크, 인크. | 오버로드 및 용리 크로마토그래피 |
KR20140138850A (ko) | 2012-03-02 | 2014-12-04 | 샤이어 휴먼 지네틱 테라피즈 인크. | Iii형 고셔병을 치료하기 위한 조성물 및 방법 |
CN104507503A (zh) * | 2012-06-29 | 2015-04-08 | 康斯乔最高科学研究公司(Csic) | 用于递送生物活性化合物的功能化脂质体 |
WO2014016873A1 (en) * | 2012-07-26 | 2014-01-30 | Jcr Pharmaceuticals Co., Ltd. | Method for production of recombinant human alpha-galactosidase a |
JP6226435B2 (ja) * | 2012-07-26 | 2017-11-08 | Jcrファーマ株式会社 | 組換えヒトα−ガラクトシダーゼAの製造方法 |
CN111172135A (zh) | 2012-10-24 | 2020-05-19 | 建新公司 | 使用mes和mops作为流动相改性剂从多峰树脂洗脱生物分子 |
BR112017013300A2 (pt) * | 2014-12-22 | 2018-02-27 | Codexis Inc | alfa galactosidase a recombinante e/ou fragmento de alfa galactosidase a recombinante biologicamente ativo, composição, sequência de polinucleotídeos recombinantes, vetor de expressão, célula hospedeira, métodos para produzir uma variante de alfa galactosidase a e para tratar e/ou prevenir os sintomas da doença de fabry, composição farmacêutica, e, uso das composições. |
WO2016116966A1 (en) | 2015-01-22 | 2016-07-28 | Jcr Pharmaceuticals Co., Ltd. | Method for purification of recombinant human alpha-galactosidase a from material containing contaminant host cell proteins |
EP3646899A4 (en) | 2017-06-30 | 2020-07-08 | FUJIFILM Corporation | TREATMENT AGENT FOR LYSOSOMAL DISEASE |
ES2716305B2 (es) | 2017-12-11 | 2019-11-27 | Fund Biomedica Galicia Sur | Uso de chaperonas farmacologicas para el tratamiento de enfermedades de deposito lisosomal |
BR112021011750A2 (pt) | 2018-12-20 | 2021-08-31 | Codexis, Inc. | Alfa-galactosidase a recombinante e/ou fragmento de alfa-galactosidase a recombinante, composição, sequência de polinucleotídeo recombinante, vetor de expressão, célula hospedeira, métodos para produzir uma variante de alfa-galactosidase a e para tratar e/ou prevenir os sintomas da doença de fabry, composição farmacêutica, e, uso das composições |
CN111308095A (zh) * | 2020-03-04 | 2020-06-19 | 北京师范大学 | 用于诊断***癌的尿液蛋白标记物 |
WO2024044697A2 (en) * | 2022-08-24 | 2024-02-29 | Walking Fish Therapeutics, Inc. | Compositions and methods for treatment of fabry disease |
Family Cites Families (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2619719B1 (fr) * | 1987-09-01 | 1991-05-10 | Sanofi Sa | Procede d'obtention d'interleukine-2 a partir de cellules eucaryotes, vecteurs necessaires a sa mise en oeuvre et lignees cellulaires hautement productrices |
FI96121C (fi) * | 1987-10-02 | 1996-05-10 | Zymogenetics Inc | DNA-rakenne ja menetelmä heterologisen proteiinin tuottamiseksi |
WO1990011353A1 (en) * | 1989-03-24 | 1990-10-04 | Research Corporation Technologies, Inc. | Recombinant alpha-galactosidase, a therapy for fabry disease |
US5179023A (en) | 1989-03-24 | 1993-01-12 | Research Corporation Technologies, Inc. | Recombinant α-galactosidase a therapy for Fabry disease |
NZ234453A (en) * | 1989-07-13 | 1993-01-27 | Sanofi Sa | Recombinant dna encoding urate oxidase, and vector, host, protein and pharmaceutical compositions associated therewith |
IL95330A0 (en) * | 1989-08-11 | 1991-06-30 | Lilly Co Eli | Expression of a functional insect specific toxin gene in mammalian cells |
EP0550675A1 (en) * | 1990-09-28 | 1993-07-14 | Cephalon, Inc. | Transgenic animals with alzheimer's amyloid precursor gene |
US5401650A (en) * | 1990-10-24 | 1995-03-28 | The Mount Sinai School Of Medicine Of The City University Of New York | Cloning and expression of biologically active α-galactosidase A |
US5356804A (en) | 1990-10-24 | 1994-10-18 | Mount Sinai School Of Medicine Of The City Of New York | Cloning and expression of biologically active human α-galactosidase A |
WO1993008292A1 (en) * | 1991-10-16 | 1993-04-29 | Agracetus, Inc. | Particle-mediated transformation of animal somatic cells |
PT101031B (pt) * | 1991-11-05 | 2002-07-31 | Transkaryotic Therapies Inc | Processo para o fornecimento de proteinas por terapia genetica |
US5641670A (en) | 1991-11-05 | 1997-06-24 | Transkaryotic Therapies, Inc. | Protein production and protein delivery |
SE9300105D0 (sv) * | 1993-01-15 | 1993-01-15 | Kabi Pharmacia Ab | Stable protein solution |
ATE229980T1 (de) * | 1993-07-02 | 2003-01-15 | Incyte Pharma Inc | Glycosilierte und nichtglycolisierte bakterizide/die permeabilität erhoehende proteine und methoden zu ihrer herstellung |
ATE275194T1 (de) | 1994-01-13 | 2004-09-15 | Rogosin Inst | Makroeingekapselte sekretorische zellen |
JP2984552B2 (ja) * | 1994-09-02 | 1999-11-29 | 大和化成株式会社 | ラッカーゼおよびその生産方法 |
US5541087A (en) * | 1994-09-14 | 1996-07-30 | Fuji Immunopharmaceuticals Corporation | Expression and export technology of proteins as immunofusins |
JPH08196293A (ja) * | 1995-01-25 | 1996-08-06 | Mitsui Toatsu Chem Inc | タンパク質の製造方法 |
CN1161468C (zh) * | 1995-02-15 | 2004-08-11 | 安姆根有限公司 | Mpl配体类似物 |
-
1997
- 1997-09-12 AU AU44244/97A patent/AU4424497A/en not_active Abandoned
- 1997-09-12 NZ NZ506214A patent/NZ506214A/xx not_active IP Right Cessation
- 1997-09-12 PT PT40301079T patent/PT1538202E/pt unknown
- 1997-09-12 EP EP97942567A patent/EP0935651B1/en not_active Expired - Lifetime
- 1997-09-12 CN CNB97197909XA patent/CN1268741C/zh not_active Expired - Lifetime
- 1997-09-12 EP EP10181859.9A patent/EP2374876B1/en not_active Expired - Lifetime
- 1997-09-12 ES ES04030107.9T patent/ES2458292T3/es not_active Expired - Lifetime
- 1997-09-12 WO PCT/US1997/016603 patent/WO1998011206A2/en active Application Filing
- 1997-09-12 ES ES10181859.9T patent/ES2581828T3/es not_active Expired - Lifetime
- 1997-09-12 DK DK10181859.9T patent/DK2374876T3/en active
- 1997-09-12 EP EP04030107.9A patent/EP1538202B1/en not_active Expired - Lifetime
- 1997-09-12 ES ES97942567T patent/ES2234032T3/es not_active Expired - Lifetime
- 1997-09-12 HU HU9904666A patent/HU227189B1/hu unknown
- 1997-09-12 HU HU1000445A patent/HU230275B1/hu unknown
- 1997-09-12 EP EP10181991A patent/EP2327775A3/en not_active Withdrawn
- 1997-09-12 IL IL128960A patent/IL128960A/en not_active IP Right Cessation
- 1997-09-12 DE DE69732129T patent/DE69732129T2/de not_active Expired - Lifetime
- 1997-09-12 DK DK04030107.9T patent/DK1538202T3/en active
- 1997-09-12 JP JP51400498A patent/JP4001925B2/ja not_active Expired - Lifetime
- 1997-09-12 NZ NZ334721A patent/NZ334721A/xx not_active IP Right Cessation
- 1997-09-12 KR KR1020057011793A patent/KR20050084473A/ko not_active Application Discontinuation
- 1997-09-12 KR KR1019997002088A patent/KR100547402B1/ko not_active IP Right Cessation
- 1997-09-12 PL PL97332188A patent/PL190041B1/pl unknown
- 1997-09-12 CA CA002265464A patent/CA2265464C/en not_active Expired - Lifetime
- 1997-09-12 MX MX2014010892A patent/MX340738B/es unknown
- 1997-09-12 RU RU99107287/14A patent/RU2179034C2/ru active
- 1997-09-12 AT AT97942567T patent/ATE286120T1/de active
- 1997-09-13 TW TW086113342A patent/TW585919B/zh not_active IP Right Cessation
-
1999
- 1999-03-12 NO NO19991225A patent/NO328443B1/no not_active IP Right Cessation
-
2000
- 2000-02-23 HK HK00101063A patent/HK1022173A1/xx not_active IP Right Cessation
-
2005
- 2005-09-05 HK HK05107760.8A patent/HK1074058A1/zh not_active IP Right Cessation
- 2005-09-05 HK HK12102808.4A patent/HK1162580A1/zh not_active IP Right Cessation
-
2006
- 2006-07-18 JP JP2006195854A patent/JP4313381B2/ja not_active Expired - Lifetime
-
2007
- 2007-06-13 JP JP2007155943A patent/JP4313405B2/ja not_active Expired - Lifetime
- 2007-07-16 IL IL184637A patent/IL184637A/en not_active IP Right Cessation
-
2008
- 2008-11-26 JP JP2008300335A patent/JP5590788B2/ja not_active Expired - Lifetime
- 2008-11-26 JP JP2008300426A patent/JP2009060918A/ja not_active Withdrawn
-
2009
- 2009-02-06 NO NO20090583A patent/NO330687B1/no not_active IP Right Cessation
-
2011
- 2011-03-31 NO NO20110496A patent/NO340408B1/no not_active IP Right Cessation
- 2011-09-06 JP JP2011193635A patent/JP2012019793A/ja not_active Withdrawn
-
2014
- 2014-09-29 NO NO20141168A patent/NO20141168L/no not_active Application Discontinuation
- 2014-10-15 JP JP2014210566A patent/JP2015044830A/ja active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112852784A (zh) * | 2013-10-23 | 2021-05-28 | 建新公司 | 重组糖蛋白及其用途 |
CN107980063A (zh) * | 2015-05-11 | 2018-05-01 | Ucl商业有限公司 | 法布里病基因治疗 |
US11103596B2 (en) | 2015-05-11 | 2021-08-31 | Ucl Business Plc | Fabry disease gene therapy |
CN107980063B (zh) * | 2015-05-11 | 2021-12-10 | Ucl商业有限责任公司 | 法布里病基因治疗 |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1268741C (zh) | 半乳糖苷酶a缺乏症的治疗 | |
CN1267550C (zh) | 血管内皮生长因子2 | |
CN1033760C (zh) | 产生具有第八因子活性的第八因子缺失突变体的方法 | |
CN1354796A (zh) | α-半乳糖苷酶缺陷的治疗 | |
CN1062908C (zh) | 新多肽和编码它们的脱氧核糖核酸 | |
CN1151258C (zh) | 通过内源基因活化制备*** | |
CN1278183A (zh) | 可溶性mhc复合体及其使用方法 | |
CN1125401A (zh) | ***类似物组合物和方法 | |
CN1326467A (zh) | 表达以及分泌制管张素和endostatin的免疫融合物 | |
CN1086262A (zh) | 新的dna序列 | |
CN1170415A (zh) | 组织蛋白酶o2蛋白酶 | |
CN1258315A (zh) | 哺乳动物细胞因子样因子7 | |
CN1541275A (zh) | 糖蛋白及其制备方法 | |
CN1161468C (zh) | Mpl配体类似物 | |
CN1236393A (zh) | 几丁质酶材料和方法 | |
CN1057096C (zh) | 蛋白质pl40多肽及编码该多肽的DNA | |
CN1179976C (zh) | 产生凝血因子ⅷ的生产方法和宿主细胞 | |
CN1240836C (zh) | 转化生长因子αHI | |
CN1402783A (zh) | 新型多肽及其编码基因 | |
CN1100957A (zh) | 重组产品 | |
CN1234728C (zh) | 新的人淋巴因子、其编码序列及用途 | |
CN1230993A (zh) | 制备血小板生成素多肽的表达载体、细胞和方法 | |
CN1371421A (zh) | 坦科聚合酶2材料和方法 | |
CN1213069C (zh) | 发育促进因子、其制法及用途 | |
CN1166776C (zh) | 人血管ibp样生长因子在制备用于诊断血管疾病或与肿瘤形成相关的新血管化的诊断剂中的应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
REG | Reference to a national code |
Ref country code: HK Ref legal event code: GR Ref document number: 1061215 Country of ref document: HK |
|
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CX01 | Expiry of patent term |
Granted publication date: 20060809 |
|
CX01 | Expiry of patent term |