WO1996031242A1 - RECOMBINANT β-CELL AND USES THEREOF - Google Patents
RECOMBINANT β-CELL AND USES THEREOF Download PDFInfo
- Publication number
- WO1996031242A1 WO1996031242A1 PCT/US1996/004792 US9604792W WO9631242A1 WO 1996031242 A1 WO1996031242 A1 WO 1996031242A1 US 9604792 W US9604792 W US 9604792W WO 9631242 A1 WO9631242 A1 WO 9631242A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- tetracycline
- cells
- cell
- derivative
- beta
- Prior art date
Links
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 title claims abstract description 75
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 60
- 229930101283 tetracycline Natural products 0.000 claims abstract description 59
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 47
- 239000008103 glucose Substances 0.000 claims abstract description 47
- 239000004098 Tetracycline Substances 0.000 claims abstract description 43
- 229960002180 tetracycline Drugs 0.000 claims abstract description 43
- 235000019364 tetracycline Nutrition 0.000 claims abstract description 42
- 150000003522 tetracyclines Chemical class 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 40
- 230000035755 proliferation Effects 0.000 claims abstract description 35
- 102000004877 Insulin Human genes 0.000 claims abstract description 30
- 108090001061 Insulin Proteins 0.000 claims abstract description 30
- 229940125396 insulin Drugs 0.000 claims abstract description 30
- 206010012601 diabetes mellitus Diseases 0.000 claims abstract description 20
- 230000001105 regulatory effect Effects 0.000 claims abstract description 11
- 210000004027 cell Anatomy 0.000 claims description 102
- 241000282414 Homo sapiens Species 0.000 claims description 37
- 108090000623 proteins and genes Proteins 0.000 claims description 29
- 210000004369 blood Anatomy 0.000 claims description 28
- 239000008280 blood Substances 0.000 claims description 28
- 230000014509 gene expression Effects 0.000 claims description 25
- 206010028980 Neoplasm Diseases 0.000 claims description 20
- 108020001507 fusion proteins Proteins 0.000 claims description 20
- 102000037865 fusion proteins Human genes 0.000 claims description 20
- OFVLGDICTFRJMM-WESIUVDSSA-N tetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O OFVLGDICTFRJMM-WESIUVDSSA-N 0.000 claims description 17
- 238000004519 manufacturing process Methods 0.000 claims description 13
- 239000013612 plasmid Substances 0.000 claims description 13
- 108091007433 antigens Proteins 0.000 claims description 11
- 239000000427 antigen Substances 0.000 claims description 10
- 102000036639 antigens Human genes 0.000 claims description 10
- 239000003094 microcapsule Substances 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 8
- 206010022498 insulinoma Diseases 0.000 claims description 4
- 230000010354 integration Effects 0.000 claims description 4
- 230000009261 transgenic effect Effects 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 3
- 210000000496 pancreas Anatomy 0.000 claims description 3
- 239000013603 viral vector Substances 0.000 claims description 3
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 claims description 2
- 239000004100 Oxytetracycline Substances 0.000 claims description 2
- CYDMQBQPVICBEU-UHFFFAOYSA-N chlorotetracycline Natural products C1=CC(Cl)=C2C(O)(C)C3CC4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-UHFFFAOYSA-N 0.000 claims description 2
- CYDMQBQPVICBEU-XRNKAMNCSA-N chlortetracycline Chemical compound C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-XRNKAMNCSA-N 0.000 claims description 2
- 229960003722 doxycycline Drugs 0.000 claims description 2
- 229960000625 oxytetracycline Drugs 0.000 claims description 2
- IWVCMVBTMGNXQD-PXOLEDIWSA-N oxytetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3[C@H](O)[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-PXOLEDIWSA-N 0.000 claims description 2
- 235000019366 oxytetracycline Nutrition 0.000 claims description 2
- 210000003200 peritoneal cavity Anatomy 0.000 claims description 2
- IWVCMVBTMGNXQD-UHFFFAOYSA-N terramycin dehydrate Natural products C1=CC=C2C(O)(C)C3C(O)C4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-UHFFFAOYSA-N 0.000 claims description 2
- 238000001890 transfection Methods 0.000 claims 2
- 241000699670 Mus sp. Species 0.000 description 31
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 13
- 210000003719 b-lymphocyte Anatomy 0.000 description 13
- 230000000694 effects Effects 0.000 description 13
- 238000011534 incubation Methods 0.000 description 9
- 101150061166 tetR gene Proteins 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- 241000699660 Mus musculus Species 0.000 description 8
- 108091023040 Transcription factor Proteins 0.000 description 8
- 238000002513 implantation Methods 0.000 description 8
- 238000011830 transgenic mouse model Methods 0.000 description 8
- 208000013016 Hypoglycemia Diseases 0.000 description 7
- 102000040945 Transcription factor Human genes 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 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 6
- 230000002218 hypoglycaemic effect Effects 0.000 description 6
- 101150003509 tag gene Proteins 0.000 description 6
- 108010068250 Herpes Simplex Virus Protein Vmw65 Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 230000004663 cell proliferation Effects 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 230000003914 insulin secretion Effects 0.000 description 5
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 239000003651 drinking water Substances 0.000 description 4
- 235000020188 drinking water Nutrition 0.000 description 4
- 201000001421 hyperglycemia Diseases 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000003442 weekly effect Effects 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 3
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 108091008819 oncoproteins Proteins 0.000 description 3
- 102000027450 oncoproteins Human genes 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 238000010600 3H thymidine incorporation assay Methods 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 238000011735 C3H mouse Methods 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- ZSJLQEPLLKMAKR-UHFFFAOYSA-N Streptozotocin Natural products O=NN(C)C(=O)NC1C(O)OC(CO)C(O)C1O ZSJLQEPLLKMAKR-UHFFFAOYSA-N 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000002991 immunohistochemical analysis Methods 0.000 description 2
- 208000017169 kidney disease Diseases 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 2
- 229960001052 streptozocin Drugs 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 230000005748 tumor development Effects 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000010599 BrdU assay Methods 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 208000002177 Cataract Diseases 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 208000019736 Cranial nerve disease Diseases 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 206010012689 Diabetic retinopathy Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001033280 Homo sapiens Cytokine receptor common subunit beta Proteins 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 206010056677 Nerve degeneration Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 206010037596 Pyelonephritis Diseases 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 206010047513 Vision blurred Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 210000003403 autonomic nervous system Anatomy 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 229960002170 azathioprine Drugs 0.000 description 1
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 235000021256 carbohydrate metabolism Nutrition 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 235000019365 chlortetracycline Nutrition 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- 230000014101 glucose homeostasis Effects 0.000 description 1
- 230000006377 glucose transport Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 102000055647 human CSF2RB Human genes 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002390 hyperplastic effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 210000002660 insulin-secreting cell Anatomy 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 201000001119 neuropathy Diseases 0.000 description 1
- 230000007823 neuropathy Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000000578 peripheral nerve Anatomy 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- JTJMJGYZQZDUJJ-UHFFFAOYSA-N phencyclidine Chemical compound C1CCCCN1C1(C=2C=CC=CC=2)CCCCC1 JTJMJGYZQZDUJJ-UHFFFAOYSA-N 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect 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
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
Classifications
-
- 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
- C07K14/62—Insulins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- 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
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/20—Animal model comprising regulated expression system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
- A01K2267/025—Animal producing cells or organs for transplantation
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0325—Animal model for autoimmune diseases
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
Definitions
- Diabetes mellitus is a chronic disorder of carbohydrate metabolism characterized by insufficient production of insulin by the pancreatic beta cells. Diabetes effects approximately 10 million people in the United States, with more than 250,000 new cases diagnosed each year. There are two common types of diabetes mellitus: insulin-dependent (Type-I diabetes) and non- insulin-dependent (Type-II diabetes) . Insulin-dependent diabetes is generally characterized by an absolute deficiency of insulin production, whereas non-insulin- dependent diabetes is characterized by a relatively insufficient production of insulin.
- the rate of insulin secretion by beta cells is regulated by the level of glucose in the blood.
- the islet cells are stimulated to release increased amounts of insulin into the blood, accelerating glucose transport into the cells and glucose conversion into glycogen.
- insulin release from the islets is decreased.
- insulin production is abnormally low or insuf icient, resulting in abnormally high blood glucose levels, a condition known as hyperglycemia.
- beta cell lines have been generated from insulinomas and hyperplastic islets arising in mice expressing a transgene encoding the SV40 T antigen (Tag) oncogene under the control of the insulin promoter (RIP-Tag) (1-6) .
- Several of these cell lines displayed insulin secretion characteristics similar to those observed in intact adult islets, in particular the response to glucose concentrations in the physiological range (5-15 ⁇ nol/1) .
- beta cell lines derived by other methods (10-12).
- the present invention overcomes the problems associated with the previous beta cell lines by providing a beta cell line which not only maintains blood glucose levels in the normal range, but also may be controlled to prevent unregulated proliferation.
- the present invention provides a recombinant, glucose-regulated insulin producing beta cell whose proliferation is controlled by tetracycline or a derivative thereof.
- the present invention also provides a microcapsule comprising an amount of the recombinant beta cell above sufficient to maintain physiologically acceptable levels of glucose in a subject implanted with the microcapsule.
- the present invention also provides a method for treating a subject with diabetes which comprises (a) implanting in the subject recombinant, beta cells whose proliferation is controlled by tetracycline or a derivative thereof, in an amount effective to establish and maintain physiologically acceptable levels of glucose in the blood of the subject; and (b) inhibiting proliferation of the implanted recombinant beta cells by administering to the subject an amount of tetracycline or a derivative thereof, effective to inhibit proliferation of the implanted recombinant beta cells.
- the present invention also provides a method for producing a recombinant, glucose-regulated insulin producing beta cell whose proliferation is controlled by tetracycline or a derivative thereof, comprising the steps of: (a) introducing to a first, non-human animal a first plasmid comprising a DNA encoding a tetR-VPl6 fusion protein, and an insulin promoter which controls expression of the fusion protein, such that a first, genetically- controlled, non-human animal is obtained; (b) introducing to a second, non-human animal a second plasmid comprising a DNA encoding the SV40 T antigen, and a tetracycline (Tc) operator minimal promoter, such that a second genetically- modified, non-human animal is obtained; (c) crossing the first genetically-modified, non-human animal, or offspring thereof, with the second, genetically-modified, non-human animal, or offspring thereof, to obtain progeny; (d) screening the progeny for
- the present invention also provides a method for producing recombinant, glucose-regulated insulin producing beta cells whose proliferation is controlled by tetracycline or a derivative thereof, comprising the steps of: (a) introducing into a beta cell, a first gene comprising a DNA encoding a TetR-VPl6 fusion protein, and an insulin promoter which controls expression of the fusion protein, and a second gene comprising a DNA encoding SV40 T antigen, and a tetracycline operator minimal promoter, such that stable integration of both genes is acheived; and (b) screening for cells whose proliferation is controlled by tetracycline or a derivative thereof.
- the present invention further provides a method for producing recombinant cells whose proliferation is controlled by tetracycline or a derivative thereof, comprising the steps of: (a) introducing to a first, non- human animal a first plasmid comprising a DNA encoding a tetR-VPl6 fusion protein, and a promoter specific to said cell which controls expression of said fusion protein, such that a first, genetically-modified, non-human animal is obtained; (b) introducing to a second, non-human animal a second plasmid comprising a DNA encoding SV40 T antigen, and a tetracycline operator minimal promoter, such that a second, genetically-modified, non-human animal is obtained; (c) crossing said first, genetically-modified, non-human animal, or offspring thereof, with said second, genetically-modified, non-human animal, or offspring thereof, to obtain progeny thereof; (d) screening said progeny for double-transgenic,
- the present invention provides a method for producing recombinant cells whose proliferation is controlled by tetracycline or a derivative thereof, comprising the steps of: (a) introducing into a cell, a first gene comprising a DNA encoding a TetR-VP16 fusion protein, and a promoter specific to said cell which controls expression of said fusion protein, and a second gene comprising a DNA encoding SV40 T antigen, and a tetracycline operator minimal promoter, such that stable integration of both genes is achieved; and (b) screening for cells whose proliferation is controlled by tetra ⁇ cycline or a derivative thereof.
- Figure 1 is comprised of Figures 1A and IB, and represents gene constructs used in the conditional transformation strategy.
- Figure 1A depicts the tet-Tag construct.
- the SV40 Tag gene was placed under the control of a tandem array of Tc operator sequences and a minimal promoter (hatched box) .
- Figure IB depicts the RIP-tTA construct.
- a fusion gene encoding the tetR and the activating domain of the HSV VP16 protein was placed under the control of the RIP promoter, downstream of an intron element (int) and upstream of a polyadenylation signal
- Figure 2 is comprised of Figures 2A, 2B, 2C, 2D,
- FIG. 2B of 1 ⁇ g/ml Tc and photographed in a phase contrast microscope. Similar cells were incubated in 16- well slides for 7 days in the absence ( Figures 2C and 2E) or presence ( Figures 2D and 2F) of 1 ⁇ g/ l Tc. Cells were pulsed for 1 hour with BrdU ( Figures 2C and 2D) , and stained with an anti-BrdU monoclonal antibody. Cells in separate wells were stained with a Tag antiserum ( Figures 2E and 2F) . The bound antibodies were visualized with horseradish peroxidase-conjugated second antibodies. The cells shown are representative of 3 independent experiments. Magnification is X200.
- Figure 3 represents the growth arrest of BTC-tet cells following incubation with Tc and anhydrotetracycline (ATc) .
- 2xl0 4 cells in quadruplicate wells were incubated for 7 days in the presence of the indicated concentration of Tc (circles) or ATc (squares) . They were then pulsed with [ 3 H]thymidine for 6 h, followed by quantitation of the radioactivity incorporated into DNA. Values represent percent of counts in the absence of drugs, averaging 4xl0 4 cp per well.
- Figure 4 is comprised of Figures 4A, 4B, 4C, and
- mice with BTC-tet tumors received regular drinking water ( Figures 4A and 4C) or water containing Tc ( Figures 4B and 4D) for 7 days. They were then pulsed with BrdU ( Figures 4A and 4B) and Tag ( Figures 4C and 4D) antisera. The bound antibodies were visualized with horseradish peroxidase-conjugated second antibodies. Magnification is X230.
- FIG. 5 shows that BTC-tet cells maintain normal blood glucose levels in diabetic recipients.
- Mice made diabetic by treatment with streptozotocin were implanted intraperitoneally with 2xl0 6 cells (circles, squares) or received no cell implant (triangles) .
- the time of cell implantation is shown as day 0.
- Blood glucose levels were measured weekly. When blood glucose was corrected, mice in one group (circles) were implanted with slow-release Tc pellets (arrow) . Blood glucose levels in this group remained stable, while in the gro * up that was not treated with Tc (squares) blood glucose continued to decrease as a result of uncontrolled proliferation of the insulin-secreting cells.
- the present invention provides a recombinant, human or animal, glucose-regulated insulin producing beta cell whose proliferation can be controlled by incubating the cell with tetracycline or one of its derivatives.
- the recombinant beta cell is contained within the cell line designated BTC-tet, which was deposited under the terms of the Budapest Treaty on March 31, 1995 with the American Type Culture Collection (ATCC) , Rockville, Maryland, and assigned ATCC Accession Number CRL-11869. Vitality of the cell line was confirmed on April 6, 1995.
- the present invention also provides the beta cell line deposited with the ATCC under Accession Number CRL-11869.
- the recombinant beta cell of the present invention may be produced by crossing two lineages of non- human transgenic animals such as cows, pigs, mice, and are preferably mice.
- the beta cells of one lineage contain a fusion protein consisting of the tetR and the activating domain of the HSV VP16 protein under the control of an insulin promoter.
- the combination of tetR and the HSV VP16 sequence converts the tetR into a transcription activator.
- the other lineage of transgenic mice contains the Tag gene under the control of a tandem array of Tc operator sequences, and a minimal promoter. The minimal promoter alone is incapable of directing expression of the Tag gene.
- Tag gene expression is activated.
- tTA tetracycline- controlled transactivator
- Tag is expressed and this expression results in beta cell tumors. Beta cells tumors whose proliferation are inhibited by tetracycline or one of its derivatives, are then selected to obtain the beta cell of the present invention.
- the recombinant beta cell of the present invention may also be produced by the introduction into beta cells in tissue culture of a first gene encoding a fusion protein consisting of the tetR and the activating domain of the HSV VP16 protein under the control of an insulin promoter together with a second gene encoding the SV40 T antigen under the control of a tandem array of Tc operator sequences, and a minimal promoter.
- the beta cells can be, but are not limited to, beta cells of cow, pig, mouse or human origin, and are preferably of human origin.
- the genes can be introduced by stable transfection of the beta cells by methods well known to those skilled in the art such as calcium phosphate precip ⁇ itation, cationic liposome fusion or electroporation.
- the genes can be introduced into the beta cells using viral vectors such as herpes virus-, adenovirus- or retrovirus-based vectors by techniques well known to those skilled in the art.
- viral vectors such as herpes virus-, adenovirus- or retrovirus-based vectors by techniques well known to those skilled in the art.
- Beta cells whose proliferation is inhibited by tetracycline or one of its derivatives are then selected to obtain the beta cell of the present invention.
- the recombinant beta cell by virtue of its retention of normal beta cell characteristics with regard to insulin secretion and blood glucose regulation, offers an alternative to insulin administration in the treatment of diabetes in both animals and humans.
- the present invention also provides a method for treating a diabetic subject.
- the method comprises implanting recombinant beta cells in the body of the diabetic subject in an amount effective to establish and maintain physiologically acceptable levels of blood glucose; and then inhibiting the proliferation of the recombinant beta cells by administering tetracycline or one of its derivatives in an amount sufficient to inhibit the proliferation of the recombinant beta cells.
- the beta cells may be implanted in any feasible location within the body where they come in contact with the blood stream of the recipient.
- Suitable locations include but are not limited to the peritoneal cavity and the pancreas. Other locations would be apparent to one skilled in the art.
- the beta cells may be implanted by methods known to those skilled in the art such as by surgical means, injection and the like.
- the effective amount of beta cells is preferably about 100 to about 300 million cells.
- the effective amount of beta cells will depend upon the method of implantation, the pharmacokinetic characteristics of the subject treated, and/or the presence of other diseases or conditions. Such amounts are readily determined by one skilled in the art.
- Rejection of the implanted beta cells may be controlled by administration of immunosuppressant drugs such as cyclosporine or azathioprine and the like.
- the beta cells may be microencapsulated prior to implantation.
- microencapsulation means any method which may be used to protect foreign cells introduced into the body of a recipient from destruction by the recipient's immune system.
- Micro ⁇ encapsulation methods include but are not limited to the methods described in Patent Nos. 5,389,535, 5,334,640, and tissue inplant systems described in U.S. Patent Nos. 5,314,471, and 5,344,454, which are hereby incorporated by reference.
- Other means for icroencapsulating the beta cells or alternative tissue inplant systems would be apparent to one skilled in the art.
- the present invention also provides a microcapsule comprising an amount of the above beta cell which is sufficient to maintain physiologically acceptable levels of glucose in a subject implanted with the microcapsule.
- beta cells are obtained from the cell line deposited with the ATCC under Accession Number CRL-11869.
- the amount of beta cells may be about 100 to about 300 million cells. Again, the actual amount will depend upon the method of implantation, the pharmacokinetic characteristics of the subject treated, and/or the presence of other diseases or conditions. Such amounts are readily determined by one skilled in the art.
- microcapsule as used herein means any vehicle, polymer composition or like means used in a microencapsulation process for implantation into the body of a subject.
- Tetracycline is commercially available from Sigma Chemical Co., St. Louis, Missouri. Tetracycline derivatives include but are not limited to anhydrotetracycline, 7-chloro-tetracycline, 4-Epi-7- chloro-tetracycline, oxy-tetracycline, doxycycline, 6- deoxy-6-demethyl-tetracycline, and 7-azido-6-deoxy-6- demethyl-tetracycline. These derivatives and others are commercially available. Anhydrotetracycline is preferred because it binds tetR more efficiently than tetracycline and has a lower antibiotic activity, thereby enabling longterm administration.
- Tetracycline and its derivatives can be administered orally, by intravenous or intramuscular injection. Other modes of administration would be apparent to one skilled in the art.
- the amount of tetracycline is an amount effective to inhibit the proliferation of beta cells.
- the actual dosage will depend upon the route of administration and the individual pharmacokinetic parameters of the subject treated. The actual dosage is readily determined by one skilled in the art.
- the present invention also provides a general method for producing recombinant cell lines from a variety of cell types in addition to beta cells. Such cell lines may be produced by either of the methods previously described for generating recombinant beta cells. In producing such cells, however, the insulin promoter used to control the expression of the TetR-VP16 fusion protein in the beta cell, is replaced by a promoter specific to the cell of type of interest. Such cell-specific promoters are well known to those skilled in the art. For example, in producing recombinant liver cells of the present invention, a liver-specific promoter such as the albumin promoter could be utilized. The present invention is described in the following Experimental Details Section which is set forth to aid in the understanding of the invention, and should not be construed to limit in any way the invention as defined in the claims which follow thereafter.
- Plasmid constructs Plasmid constructs. Plasmids pUHD 10-3 and pUHD 15-1 were obtained from H. Bujard, Zentrum fur Molekulare Biologie der Universitat Heidelberg, Im Neuenheimer Feld 282, W- 6900 Heidelberg, Germany.
- To construct the tet-Tag plasmid the Xho I - Xba I fragment from pUHD 10-3 (13) , containing a tandem array of 7 copies of the Tc operator sequence and a CMV minimal promoter, was placed in front of the T antigen gene in pRIP-Tag (14) by deleting the Aat II-Xba I fragment containing the RIP from pRIP-Tag and converting the Aat II and Xho I sites into blunt ends.
- BTC-tet B-cell line
- All media supplies were from GIBCO.
- Cells were grown in DMEM containing 25 mM glucose and supplemented with 15% horse serum, 2.5% fetal bovine serum, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin. Tetracycline (United States Biochemical Corporation) and anhydrotetracycline (ATc) (Lederle) were included at the indicated concentrations.
- mice Eight mice were then injected I.P. with 2xl0 6 BTC-tet cells, while 4 mice were kept as diabetic controls. Mice were monitored weekly for blood glucose levels. When euglycemia was obtained in the cell- implanted group, 4 of the mice in this group were implanted subcutaneously with a slow-release Tc pellet (Innovative Research of America) designed to release 3.3 mg per day, and were further followed by weekly blood glucose checks. Glucose levels below 40 mg/dl were determined using a Beckman glucose analyzer. Immunohistochemistrv. Cells were plated in 16-well slides (Nunc) for the indicated period in the absence or presence of Tc or ATc.
- Tc slow-release Tc pellet
- BrdU incorporation assay cells were pulsed for 60 min with 10 ⁇ M BrdU (Sigma) and stained with an anti-BrdU monoclonal antibody (Becton-Dickinson) according to manufacturer's recommendations. The bound antibody was visualized with biotinated anti-mouse IgG and horseradish peroxidase-conjugated avidin (Vector, ABC kit) , and a diaminobenzidine (DAB) substrate. Cells in separate wells were stained with a rabbit-anti-Tag serum (17) . The bound antibody was visualized with a horseradish peroxidase-conjugated goat-anti-rabbit antibody and DAB.
- an anti-BrdU monoclonal antibody Becton-Dickinson
- mice with BTC-tet tumors were injected I.P. with 100 ⁇ g BrdU/g body weight. One hour later they were sacrificed, and the tumors were removed, fixed with 4% buffered formaldehyde, processed for paraffin embedding, and sectioned. Tumor sections were stained with anti-Tag and anti-BrdU antibodies as described above.
- Thymidine incorporation assay 2xl0 4 cells were seeded into 96-well plates. Following the indicated incubation, they were pulsed with 1 ⁇ Ci [methyl- 3 H]thymidine (Amersham, 78 Ci/mmol) for 6 h. The cells were then lysed in water using a cell harvester, and the DNA was retained on a glass fiber filter (Whittaker) . The filters were dried, and the radioactivity incorporated into DNA was quantitated with a scintillation counter. Each condition was assayed in quadruplicates.
- the tTa gene was placed under control of RIP ( Figure 1) , and the construct was used to generate transgenic mice, in which tTA would be constitutively expressed specifically in B cells.
- the Tag gene was introduced under the control of a minimal promoter and a tandem array of Tc operator sequences ( Figure 1) . This promoter does not allow expression of the gene by itself. Therefore, as expected, these transgenic mice did not develop tumors.
- the two lines of mice were crossed to generate double- transgenic mice. In these mice the tetR part of the tTA protein is expected to bind to the target Tc operator sequences in B cells and allow the VP16 part of the molecule to activate transcription of the Tag gene. This resulted in the development of multiple B-cell tumors by 5-6 months of age. No tumors were detected in other organs, demonstrating the need for the tTA-induced expression of Tag for the B-cell specific tumor development.
- BTC-tet a stable cell line
- BTC-tet cells I.P.
- BTC cell lines are tumorigenic and form benign tumors at the site of injection (2) .
- Tumor development leads to hypoglycemia and can be detected by monitoring blood glucose levels.
- Mice in one group were maintained on drinking water containing Tc. None of them developed tumors within 14 weeks, as judged by blood glucose measurements and a careful autopsy. No abnormalities were observed as a result of the prolonged Tc treatment. Mice maintained in the absence of Tc developed hypoglycemia and tumors within 8-13 weeks. When hypoglycemia was detected, one sub-group continued to drink regular water.
- mice were then pulsed with BrdU, sacrificed, and the tumors were removed and processed for immunohistochemical analyses.
- Tumors from mice that were not treated with Tc contained numerous cells that stained for BrdU and Tag ( Figures 4A and 4C) .
- tumors from mice treated for 7 days with Tc showed no BrdU and Tag staining ( Figures 4B and 4D) .
- the BTC-tet cells demonstrate correct responsiveness to glucose in the physiological concentration range (data not shown) .
- BTC-tet cells were implanted into diabetic recipients ( Figure 5) .
- the cell implantation led to correction of hyperglycemia within two weeks, demonstrating the ability of BTC-tet cells to function as normal B cells in vivo.
- the implanted cells continued to proliferate in mice not treated with Tc, which resulted in hypoglycemia and premature death.
- blood glucose levels were stabilized in the normal range.
- mice will allow the derivation of conditionally-transformed cell lines from other cell types, by targeting the expression of the tTA fusion protein with the appropriate cell-specific promoters.
- mice expressing the tTA protein in B cells can be used to obtain reversible expression of other genes of interest in these cells by crossing them with mice expressing such genes under control of the Tc operator minimal promoter.
- the BTC-tet cell line will allow studies on the effect of cell proliferation on the expression of differentiated functions in B cells.
- the results obtained with cells transplanted into diabetic mice demonstrate that insulin secretion from the growth-arrested BTC-tet cells remains correctly-regulated, which enables them to maintain blood glucose levels in the physiological range.
- To determine the effect of cell proliferation on glucose- induced insulin synthesis and secretion in these cells cells propagated in culture and induced to undergo growth- arrest in the presence of Tc will be studied in comparison with actively proliferating cells cultured in the absence of Tc.
- the strategy described here will contribute to the development of B-cell lines for cell therapy of diabetes, as well as to generation of conditionally-transformed cell lines from other cell types with therapeutic potential.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Diabetes (AREA)
- Animal Husbandry (AREA)
- Endocrinology (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Public Health (AREA)
- Hematology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Emergency Medicine (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU55375/96A AU720662B2 (en) | 1995-04-07 | 1996-04-03 | Recombinant B-cell and uses thereof |
EP96912616A EP0822834A4 (en) | 1995-04-07 | 1996-04-03 | Recombinant (beta)-cell and uses thereof |
JP8530530A JPH11505411A (en) | 1995-04-07 | 1996-04-03 | Recombinant beta cells and uses thereof |
MXPA/A/1997/007727A MXPA97007727A (en) | 1995-04-07 | 1997-10-07 | Recombinant cellular and uses of the mi |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US41841695A | 1995-04-07 | 1995-04-07 | |
US08/418,416 | 1995-04-07 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1996031242A1 true WO1996031242A1 (en) | 1996-10-10 |
Family
ID=23658036
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1996/004792 WO1996031242A1 (en) | 1995-04-07 | 1996-04-03 | RECOMBINANT β-CELL AND USES THEREOF |
Country Status (6)
Country | Link |
---|---|
US (2) | US6114599A (en) |
EP (1) | EP0822834A4 (en) |
JP (1) | JPH11505411A (en) |
AU (1) | AU720662B2 (en) |
CA (1) | CA2217652A1 (en) |
WO (1) | WO1996031242A1 (en) |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998037185A2 (en) * | 1997-02-20 | 1998-08-27 | The Board Of Regents Of The University Of Texas System | Vectors for controlled gene expression |
FR2761576A1 (en) * | 1997-04-04 | 1998-10-09 | Inst Nat Sante Rech Med | NON-HUMAN TRANSGENIC ANIMAL IN WHICH EXPRESSION OF THE GENE CODING FOR INSULIN IS SUPPRESSED |
WO1998058658A1 (en) * | 1997-06-23 | 1998-12-30 | The Research Foundation Of State University Of New York | Therapeutic method for management of diabetes mellitus |
WO1998058536A1 (en) * | 1997-06-23 | 1998-12-30 | J. David Gladstone Institutes | Transgenic rabbits expressing cd4 and chemokine receptor |
WO1999063101A2 (en) * | 1998-06-02 | 1999-12-09 | University Of Washington | Transfected cells and methods for treating diabetes |
US6087129A (en) * | 1996-01-19 | 2000-07-11 | Betagene, Inc. | Recombinant expression of proteins from secretory cell lines |
EP1935978A1 (en) * | 2005-09-15 | 2008-06-25 | Japan Science and Technology Agency | Method of controlling decomposition of protein by tetracycline antibiotic |
US10030229B2 (en) | 2013-06-11 | 2018-07-24 | President And Fellows Of Harvard College | SC-β cells and compositions and methods for generating the same |
US10190096B2 (en) | 2014-12-18 | 2019-01-29 | President And Fellows Of Harvard College | Methods for generating stem cell-derived β cells and uses thereof |
US10253298B2 (en) | 2014-12-18 | 2019-04-09 | President And Fellows Of Harvard College | Methods for generating stem cell-derived beta cells and methods of use thereof |
US10443042B2 (en) | 2014-12-18 | 2019-10-15 | President And Fellows Of Harvard College | Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof |
US11466256B2 (en) | 2018-08-10 | 2022-10-11 | Vertex Pharmaceuticals Incorporated | Stem cell derived islet differentiation |
US11945795B2 (en) | 2017-11-15 | 2024-04-02 | Vertex Pharmaceuticals Incorporated | Islet cell manufacturing compositions and methods of use |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7081343B2 (en) | 2001-06-18 | 2006-07-25 | The Regents Of The University Of California | Methods for identifying modulators of NF-KB activity |
US6642003B2 (en) | 2001-08-02 | 2003-11-04 | Cedars-Sinai Medical Center | Human glucose-dependent insulin-secreting cell line |
US7141240B2 (en) * | 2002-03-12 | 2006-11-28 | Cedars-Sinai Medical Center | Glucose-dependent insulin-secreting cells transfected with a nucleotide sequence encoding GLP-1 |
FR2931141B1 (en) * | 2008-05-13 | 2011-07-01 | Commissariat Energie Atomique | MICROFLUIDIC SYSTEM AND METHOD FOR THE SORTING OF AMAS FROM CELLS AND PREFERENCE FOR CONTINUOUS ENCAPSULATION THROUGH THEIR SORTING |
EP2804603A1 (en) | 2012-01-10 | 2014-11-26 | President and Fellows of Harvard College | Beta-cell replication promoting compounds and methods of their use |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5840576A (en) * | 1994-07-20 | 1998-11-24 | Cytotherapeutics, Inc. | Methods and compositions of growth control for cells encapsulated within bioartificial organs |
US5935849A (en) * | 1994-07-20 | 1999-08-10 | Cytotherapeutics, Inc. | Methods and compositions of growth control for cells encapsulated within bioartificial organs |
-
1996
- 1996-04-03 JP JP8530530A patent/JPH11505411A/en not_active Ceased
- 1996-04-03 EP EP96912616A patent/EP0822834A4/en not_active Withdrawn
- 1996-04-03 CA CA002217652A patent/CA2217652A1/en not_active Abandoned
- 1996-04-03 WO PCT/US1996/004792 patent/WO1996031242A1/en not_active Application Discontinuation
- 1996-04-03 AU AU55375/96A patent/AU720662B2/en not_active Ceased
-
1998
- 1998-03-19 US US09/044,297 patent/US6114599A/en not_active Expired - Fee Related
-
2000
- 2000-01-27 US US09/492,905 patent/US6242254B1/en not_active Expired - Fee Related
Non-Patent Citations (5)
Title |
---|
ENDOCRINOLOGY, Volume 126, Number 06, issued 1990, D'AMBRA et al., "Regulation of Insulin Secretion from B-Cell Lines Derived from Transgenic Mice Insulinomas Resembles that of Normal B-Cells", pages 2815-2822. * |
NATURE, Volume 315, issued 09 May 1985, HANAHAN, "Heritable Formation of Pancreatic B-Cell Tumours in Transgenic Mice Expressing Recombinant Insulin/Simian Virus 40 Oncogenes", pages 115-122. * |
PROC. NATL. ACAD. SCI. U.S.A., Volume 85, issued December 1988, "Beta-Cell Lines Derived from Transgenic Mice Expressing a Hybrid Insulin Gene-Oncogene", pages 9037-9041. * |
PROC. NATL. ACAD. SCI. U.S.A., Volume 91, issued September 1994, FURTH et al., "Temporal Control of Gene Expression in Transgenic Mice by a Tetracycline-Responsive Promoter", pages 9302-9306. * |
See also references of EP0822834A4 * |
Cited By (33)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6194176B1 (en) | 1996-01-19 | 2001-02-27 | Board Of Regents, The University Of Texas System | Recombinant expression of proteins from secretory cell lines |
US6087129A (en) * | 1996-01-19 | 2000-07-11 | Betagene, Inc. | Recombinant expression of proteins from secretory cell lines |
WO1998037185A3 (en) * | 1997-02-20 | 1998-11-26 | Univ Texas | Vectors for controlled gene expression |
WO1998037185A2 (en) * | 1997-02-20 | 1998-08-27 | The Board Of Regents Of The University Of Texas System | Vectors for controlled gene expression |
FR2761576A1 (en) * | 1997-04-04 | 1998-10-09 | Inst Nat Sante Rech Med | NON-HUMAN TRANSGENIC ANIMAL IN WHICH EXPRESSION OF THE GENE CODING FOR INSULIN IS SUPPRESSED |
WO1998045422A1 (en) * | 1997-04-04 | 1998-10-15 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Non human transgenic animal in which the expression of the gene coding for insulin is deleted |
WO1998058658A1 (en) * | 1997-06-23 | 1998-12-30 | The Research Foundation Of State University Of New York | Therapeutic method for management of diabetes mellitus |
WO1998058536A1 (en) * | 1997-06-23 | 1998-12-30 | J. David Gladstone Institutes | Transgenic rabbits expressing cd4 and chemokine receptor |
US5929055A (en) * | 1997-06-23 | 1999-07-27 | The Research Foundation Of State University Of New York | Therapeutic method for management of diabetes mellitus |
AU744846B2 (en) * | 1997-06-23 | 2002-03-07 | Research Foundation Of The State University Of New York, The | Therapeutic method for management of diabetes mellitus |
WO1999063101A3 (en) * | 1998-06-02 | 2000-02-17 | Univ Washington | Transfected cells and methods for treating diabetes |
WO1999063101A2 (en) * | 1998-06-02 | 1999-12-09 | University Of Washington | Transfected cells and methods for treating diabetes |
US6537806B1 (en) | 1998-06-02 | 2003-03-25 | University Of Washington | Compositions and methods for treating diabetes |
US7341869B2 (en) | 1998-06-02 | 2008-03-11 | The University Of Washington | Compositions and methods for treating diabetes |
EP1935978A1 (en) * | 2005-09-15 | 2008-06-25 | Japan Science and Technology Agency | Method of controlling decomposition of protein by tetracycline antibiotic |
EP1935978A4 (en) * | 2005-09-15 | 2009-01-07 | Japan Science & Tech Agency | Method of controlling decomposition of protein by tetracycline antibiotic |
US10030229B2 (en) | 2013-06-11 | 2018-07-24 | President And Fellows Of Harvard College | SC-β cells and compositions and methods for generating the same |
US11078463B2 (en) | 2013-06-11 | 2021-08-03 | President And Fellows Of Harvard College | SC-beta cells and compositions and methods for generating the same |
US11827905B2 (en) | 2013-06-11 | 2023-11-28 | President And Fellows Of Harvard College | SC-beta cells and compositions and methods for generating the same |
US11162078B2 (en) | 2013-06-11 | 2021-11-02 | President And Fellows Of Harvard College | SC-beta cells and compositions and methods for generating the same |
US10655106B2 (en) | 2013-06-11 | 2020-05-19 | President And Fellows Of Harvard College | SC-beta cells and compositions and methods for generating the same |
US11104883B2 (en) | 2013-06-11 | 2021-08-31 | President And Fellows Of Harvard College | SC-beta cells and compositions and methods for generating the same |
US11085025B2 (en) | 2014-12-18 | 2021-08-10 | President And Fellows Of Harvard College | Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof |
US11085027B2 (en) | 2014-12-18 | 2021-08-10 | President And Fellows Of Harvard College | Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof |
US11085026B2 (en) | 2014-12-18 | 2021-08-10 | President And Fellows Of Harvard College | Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof |
US10190096B2 (en) | 2014-12-18 | 2019-01-29 | President And Fellows Of Harvard College | Methods for generating stem cell-derived β cells and uses thereof |
US10927350B2 (en) | 2014-12-18 | 2021-02-23 | President And Fellows Of Harvard College | Methods for generating stem cell-derived beta cells and uses thereof |
US11155787B2 (en) | 2014-12-18 | 2021-10-26 | President And Fellows Of Harvard College | Methods for generating stem cell-derived beta cells and methods of use thereof |
US10443042B2 (en) | 2014-12-18 | 2019-10-15 | President And Fellows Of Harvard College | Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof |
US10253298B2 (en) | 2014-12-18 | 2019-04-09 | President And Fellows Of Harvard College | Methods for generating stem cell-derived beta cells and methods of use thereof |
US11945795B2 (en) | 2017-11-15 | 2024-04-02 | Vertex Pharmaceuticals Incorporated | Islet cell manufacturing compositions and methods of use |
US11466256B2 (en) | 2018-08-10 | 2022-10-11 | Vertex Pharmaceuticals Incorporated | Stem cell derived islet differentiation |
US11525120B2 (en) | 2018-08-10 | 2022-12-13 | Vertex Pharmaceuticals Incorporated | Stem cell derived islet differentiation |
Also Published As
Publication number | Publication date |
---|---|
CA2217652A1 (en) | 1996-10-10 |
US6242254B1 (en) | 2001-06-05 |
MX9707727A (en) | 1998-03-31 |
AU5537596A (en) | 1996-10-23 |
EP0822834A1 (en) | 1998-02-11 |
EP0822834A4 (en) | 2002-09-18 |
US6114599A (en) | 2000-09-05 |
JPH11505411A (en) | 1999-05-21 |
AU720662B2 (en) | 2000-06-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU720662B2 (en) | Recombinant B-cell and uses thereof | |
Efrat et al. | Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene. | |
Landesman-Milo et al. | Correction of hyperglycemia in diabetic mice transplanted with reversibly immortalized pancreatic β cells controlled by the tet-on regulatory system | |
Sosa-Pineda et al. | The Pax4 gene is essential for differentiation of insulin-producing β cells in the mammalian pancreas | |
Mitanchez et al. | Glucose-stimulated genes and prospects of gene therapy for type I diabetes | |
US20210040459A1 (en) | Cells genetically modified to comprise pancreatic islet glucokinase and uses thereof | |
Mallol et al. | AAV-mediated pancreatic overexpression of Igf1 counteracts progression to autoimmune diabetes in mice | |
Gray et al. | Islet cell transplantation for insulin-dependent diabetes mellitus: perspectives from the present and prospects for the future | |
US20030157071A1 (en) | Treatment or replacement therapy using transgenic stem cells delivered to the gut | |
US20080267928A1 (en) | Compositions and Methods for Making Insulin-Producing Cells | |
US20060110377A1 (en) | Immunologically privileged cells and uses thereof | |
HRP20040446A2 (en) | Method of producing human beta cell lines | |
US6677311B1 (en) | Inducible HSV-TK in transformed cell populations | |
MXPA97007727A (en) | Recombinant cellular and uses of the mi | |
Efrat | Development of engineered pancreatic β-cell lines for cell therapy of diabetes | |
EP0958357B1 (en) | Novel method for testing the differentiation status in pancreatic cells of a mammal | |
Ferber et al. | Surrogate beta cells | |
US20090214482A1 (en) | Transgenic Mammals Expressing Human Preproinsulin | |
US20220022433A1 (en) | Method for developing organ that lacks specific functional cell | |
AU783594B2 (en) | Method of prophylaxis and treatment of diabetes | |
CA2284833A1 (en) | Non human transgenic animal in which the expression of the gene coding for insulin is deleted | |
Milo-Landesman et al. | Correction of Hyperglycemia in Diabetic Mice Transplanted With Reversibly Immortalized Pancreatic ß Cells Controlled by the tet-on Regulatory System | |
EP1288311A2 (en) | Method for testing small pharmaceutically active molecules in the activation of Pax4 and production of insulin producing beta-cells | |
CA2311282A1 (en) | Yac vectors | |
FLORIDA | In vitro growth of functional islets of Langerhans and in vivo uses thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW MX NO NZ PL PT RO RU SD SE SI SK TJ TT UA UZ VN |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2217652 Country of ref document: CA Ref country code: CA Ref document number: 2217652 Kind code of ref document: A Format of ref document f/p: F Ref country code: JP Ref document number: 1996 530530 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: PA/a/1997/007727 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1996912616 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1996912616 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1996912616 Country of ref document: EP |