WO1999006559A1 - Procedes d'identification des recepteurs des facteurs de differenciation de la croissance - Google Patents
Procedes d'identification des recepteurs des facteurs de differenciation de la croissance Download PDFInfo
- Publication number
- WO1999006559A1 WO1999006559A1 PCT/US1998/015598 US9815598W WO9906559A1 WO 1999006559 A1 WO1999006559 A1 WO 1999006559A1 US 9815598 W US9815598 W US 9815598W WO 9906559 A1 WO9906559 A1 WO 9906559A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gdf
- receptor
- binding
- antibody
- receptors
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 109
- 230000004069 differentiation Effects 0.000 title abstract description 9
- 230000012010 growth Effects 0.000 title abstract description 9
- 108020003175 receptors Proteins 0.000 claims abstract description 230
- 102000005962 receptors Human genes 0.000 claims abstract description 225
- 230000027455 binding Effects 0.000 claims abstract description 56
- 241000282414 Homo sapiens Species 0.000 claims abstract description 25
- 239000012634 fragment Substances 0.000 claims abstract description 18
- 239000011230 binding agent Substances 0.000 claims abstract description 14
- 210000004027 cell Anatomy 0.000 claims description 112
- 101000893545 Homo sapiens Growth/differentiation factor 11 Proteins 0.000 claims description 86
- 102100040898 Growth/differentiation factor 11 Human genes 0.000 claims description 83
- 108010056852 Myostatin Proteins 0.000 claims description 82
- 102100039939 Growth/differentiation factor 8 Human genes 0.000 claims description 81
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 71
- 241001465754 Metazoa Species 0.000 claims description 63
- 230000014509 gene expression Effects 0.000 claims description 61
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 54
- 108020004414 DNA Proteins 0.000 claims description 46
- 229920001184 polypeptide Polymers 0.000 claims description 37
- 239000013598 vector Substances 0.000 claims description 32
- 230000009261 transgenic effect Effects 0.000 claims description 28
- 239000013604 expression vector Substances 0.000 claims description 26
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 25
- 108700019146 Transgenes Proteins 0.000 claims description 25
- 102000040430 polynucleotide Human genes 0.000 claims description 20
- 108091033319 polynucleotide Proteins 0.000 claims description 20
- 239000002157 polynucleotide Substances 0.000 claims description 20
- 150000001875 compounds Chemical class 0.000 claims description 16
- 210000001161 mammalian embryo Anatomy 0.000 claims description 16
- 150000007523 nucleic acids Chemical group 0.000 claims description 12
- 241001529936 Murinae Species 0.000 claims description 10
- 210000004602 germ cell Anatomy 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 108020004999 messenger RNA Proteins 0.000 claims description 8
- 241000283690 Bos taurus Species 0.000 claims description 7
- 241000894007 species Species 0.000 claims description 7
- 241000271566 Aves Species 0.000 claims description 6
- 230000000692 anti-sense effect Effects 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 6
- 108091034117 Oligonucleotide Proteins 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 238000001727 in vivo Methods 0.000 claims description 5
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 4
- 108010033276 Peptide Fragments Proteins 0.000 claims description 3
- 102000007079 Peptide Fragments Human genes 0.000 claims description 3
- 210000001082 somatic cell Anatomy 0.000 claims description 3
- 241001430294 unidentified retrovirus Species 0.000 claims description 3
- 239000000074 antisense oligonucleotide Substances 0.000 claims description 2
- 238000012230 antisense oligonucleotides Methods 0.000 claims description 2
- 239000003124 biologic agent Substances 0.000 claims description 2
- 101150004578 gdf-8 gene Proteins 0.000 claims description 2
- 238000010255 intramuscular injection Methods 0.000 claims description 2
- 239000007927 intramuscular injection Substances 0.000 claims description 2
- 238000010253 intravenous injection Methods 0.000 claims description 2
- 239000000816 peptidomimetic Substances 0.000 claims description 2
- 230000000392 somatic effect Effects 0.000 claims description 2
- 238000010254 subcutaneous injection Methods 0.000 claims description 2
- 239000007929 subcutaneous injection Substances 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 claims 1
- 239000002738 chelating agent Substances 0.000 claims 1
- 239000003937 drug carrier Substances 0.000 claims 1
- 239000007850 fluorescent dye Substances 0.000 claims 1
- 230000002452 interceptive effect Effects 0.000 claims 1
- 239000002184 metal Substances 0.000 claims 1
- 230000000903 blocking effect Effects 0.000 abstract description 4
- 239000000556 agonist Substances 0.000 abstract description 3
- 239000005557 antagonist Substances 0.000 abstract description 3
- 239000003102 growth factor Substances 0.000 abstract description 3
- 230000001225 therapeutic effect Effects 0.000 abstract description 3
- 102000014187 peptide receptors Human genes 0.000 abstract 1
- 108010011903 peptide receptors Proteins 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 68
- 241000699670 Mus sp. Species 0.000 description 44
- 239000002299 complementary DNA Substances 0.000 description 28
- 235000018102 proteins Nutrition 0.000 description 28
- 102000004169 proteins and genes Human genes 0.000 description 28
- 239000000203 mixture Substances 0.000 description 26
- 230000009466 transformation Effects 0.000 description 23
- 210000002257 embryonic structure Anatomy 0.000 description 21
- 239000003446 ligand Substances 0.000 description 20
- 210000000115 thoracic cavity Anatomy 0.000 description 20
- 238000000844 transformation Methods 0.000 description 16
- 238000011813 knockout mouse model Methods 0.000 description 14
- 238000003752 polymerase chain reaction Methods 0.000 description 14
- 230000002068 genetic effect Effects 0.000 description 13
- 238000009396 hybridization Methods 0.000 description 13
- 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 13
- 108091026890 Coding region Proteins 0.000 description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 12
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 12
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 12
- 125000003275 alpha amino acid group Chemical group 0.000 description 12
- 238000013459 approach Methods 0.000 description 12
- 238000010367 cloning Methods 0.000 description 12
- 210000003205 muscle Anatomy 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 238000013518 transcription Methods 0.000 description 11
- 230000035897 transcription Effects 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 238000012216 screening Methods 0.000 description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 241000700605 Viruses Species 0.000 description 9
- 210000000349 chromosome Anatomy 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 210000003098 myoblast Anatomy 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 102000053602 DNA Human genes 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 8
- 230000007547 defect Effects 0.000 description 8
- 230000001965 increasing effect Effects 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 230000008685 targeting Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 230000005856 abnormality Effects 0.000 description 7
- 229940024606 amino acid Drugs 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 230000001939 inductive effect Effects 0.000 description 7
- 210000003734 kidney Anatomy 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 230000010076 replication Effects 0.000 description 7
- 210000001562 sternum Anatomy 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 6
- 230000000875 corresponding effect Effects 0.000 description 6
- 208000035475 disorder Diseases 0.000 description 6
- 238000009826 distribution Methods 0.000 description 6
- 210000004408 hybridoma Anatomy 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 5
- 108700005087 Homeobox Genes Proteins 0.000 description 5
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 5
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 206010028289 Muscle atrophy Diseases 0.000 description 5
- 102000043168 TGF-beta family Human genes 0.000 description 5
- 108091085018 TGF-beta family Proteins 0.000 description 5
- 239000000488 activin Substances 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 201000000585 muscular atrophy Diseases 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 238000012552 review Methods 0.000 description 5
- 241000701161 unidentified adenovirus Species 0.000 description 5
- 108010059616 Activins Proteins 0.000 description 4
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 description 4
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 description 4
- 101150004541 HOXC8 gene Proteins 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 4
- 102000009331 Homeodomain Proteins Human genes 0.000 description 4
- 108010048671 Homeodomain Proteins Proteins 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 4
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 4
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 4
- 241000700618 Vaccinia virus Species 0.000 description 4
- 230000004075 alteration Effects 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 4
- 102000018358 immunoglobulin Human genes 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- 229960000310 isoleucine Drugs 0.000 description 4
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 4
- 210000003141 lower extremity Anatomy 0.000 description 4
- 238000000059 patterning Methods 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 210000002023 somite Anatomy 0.000 description 4
- 238000010561 standard procedure Methods 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- UBWXUGDQUBIEIZ-UHFFFAOYSA-N (13-methyl-3-oxo-2,6,7,8,9,10,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-17-yl) 3-phenylpropanoate Chemical compound CC12CCC(C3CCC(=O)C=C3CC3)C3C1CCC2OC(=O)CCC1=CC=CC=C1 UBWXUGDQUBIEIZ-UHFFFAOYSA-N 0.000 description 3
- 102000005606 Activins Human genes 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 3
- 241000701822 Bovine papillomavirus Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102100040897 Embryonic growth/differentiation factor 1 Human genes 0.000 description 3
- 108010090296 Growth Differentiation Factor 1 Proteins 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 101100232268 Mus musculus Hoxc11 gene Proteins 0.000 description 3
- 101100507933 Mus musculus Hoxc6 gene Proteins 0.000 description 3
- 102000057297 Pepsin A Human genes 0.000 description 3
- 108090000284 Pepsin A Proteins 0.000 description 3
- 108010001648 Proto-Oncogene Proteins c-ret Proteins 0.000 description 3
- 102000000813 Proto-Oncogene Proteins c-ret Human genes 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 108091005735 TGF-beta receptors Proteins 0.000 description 3
- 102000016715 Transforming Growth Factor beta Receptors Human genes 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 3
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 229960001230 asparagine Drugs 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- 238000000376 autoradiography Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000009402 cross-breeding Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000003209 gene knockout Methods 0.000 description 3
- 229930195712 glutamate Natural products 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 238000007901 in situ hybridization Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 229940111202 pepsin Drugs 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 210000001811 primitive streak Anatomy 0.000 description 3
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000003362 replicative effect Effects 0.000 description 3
- 210000002027 skeletal muscle Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000010473 stable expression Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 238000011830 transgenic mouse model Methods 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 239000004474 valine Substances 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 108010005853 Anti-Mullerian Hormone Proteins 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 241001579853 Cossus Species 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 2
- 201000009051 Embryonal Carcinoma Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101000886562 Homo sapiens Growth/differentiation factor 8 Proteins 0.000 description 2
- 101100321817 Human parvovirus B19 (strain HV) 7.5K gene Proteins 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- 102100026818 Inhibin beta E chain Human genes 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 102000003792 Metallothionein Human genes 0.000 description 2
- 108090000157 Metallothionein Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 2
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 108020004440 Thymidine kinase Proteins 0.000 description 2
- 241000723873 Tobacco mosaic virus Species 0.000 description 2
- 229910052770 Uranium Inorganic materials 0.000 description 2
- 241000269370 Xenopus <genus> Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 239000000868 anti-mullerian hormone Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 229940009098 aspartate Drugs 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 239000003139 biocide Substances 0.000 description 2
- 239000002981 blocking agent Substances 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 210000000845 cartilage Anatomy 0.000 description 2
- 230000022159 cartilage development Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- VLCYCQAOQCDTCN-UHFFFAOYSA-N eflornithine Chemical compound NCCCC(N)(C(F)F)C(O)=O VLCYCQAOQCDTCN-UHFFFAOYSA-N 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000001400 expression cloning Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229910001385 heavy metal Inorganic materials 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000001096 hypoplastic effect Effects 0.000 description 2
- 229940027941 immunoglobulin g Drugs 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000005830 kidney abnormality Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 210000003716 mesoderm Anatomy 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 230000003278 mimic effect Effects 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 210000003101 oviduct Anatomy 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 2
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 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 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- 101150084750 1 gene Proteins 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 101150094949 APRT gene Proteins 0.000 description 1
- 108010052946 Activin Receptors Proteins 0.000 description 1
- 102100029457 Adenine phosphoribosyltransferase Human genes 0.000 description 1
- 108010024223 Adenine phosphoribosyltransferase Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 101150061927 BMP2 gene Proteins 0.000 description 1
- 241000750676 Bacillus phage Gamma Species 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 108010049951 Bone Morphogenetic Protein 3 Proteins 0.000 description 1
- 108010049955 Bone Morphogenetic Protein 4 Proteins 0.000 description 1
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 1
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 1
- 102100024504 Bone morphogenetic protein 3 Human genes 0.000 description 1
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 241000701489 Cauliflower mosaic virus Species 0.000 description 1
- 101710104316 Cell surface-binding protein Proteins 0.000 description 1
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 1
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 108700003483 Drosophila dpp Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 101000824878 Gallus gallus Somatotropin Proteins 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101710194452 Growth/differentiation factor 11 Proteins 0.000 description 1
- 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 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 1
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 1
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 108010004250 Inhibins Proteins 0.000 description 1
- 102000002746 Inhibins Human genes 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 1
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- 206010023435 Kidney small Diseases 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
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101100261636 Methanothermobacter marburgensis (strain ATCC BAA-927 / DSM 2133 / JCM 14651 / NBRC 100331 / OCM 82 / Marburg) trpB2 gene Proteins 0.000 description 1
- 102000003505 Myosin Human genes 0.000 description 1
- 108060008487 Myosin Proteins 0.000 description 1
- 241001045988 Neogene Species 0.000 description 1
- 108020004485 Nonsense Codon Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 208000011623 Obstructive Lung disease Diseases 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 229940122060 Ornithine decarboxylase inhibitor Drugs 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 241000282520 Papio Species 0.000 description 1
- 241001504519 Papio ursinus Species 0.000 description 1
- 206010034016 Paronychia Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 101000851110 Periplaneta americana Vitellogenin-1 Proteins 0.000 description 1
- 101100124346 Photorhabdus laumondii subsp. laumondii (strain DSM 15139 / CIP 105565 / TT01) hisCD gene Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 101710182846 Polyhedrin Proteins 0.000 description 1
- 241001505332 Polyomavirus sp. Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108020004518 RNA Probes Proteins 0.000 description 1
- 239000003391 RNA probe Substances 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 102000004060 Transforming Growth Factor-beta Type II Receptor Human genes 0.000 description 1
- 108010082684 Transforming Growth Factor-beta Type II Receptor Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 108010027570 Xanthine phosphoribosyltransferase Proteins 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 238000007259 addition reaction Methods 0.000 description 1
- 230000011759 adipose tissue development Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 229940126575 aminoglycoside Drugs 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 102000025171 antigen binding proteins Human genes 0.000 description 1
- 108091000831 antigen binding proteins Proteins 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 1
- 238000000211 autoradiogram Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 101150067309 bmp4 gene Proteins 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 229940112869 bone morphogenetic protein Drugs 0.000 description 1
- 210000003557 bones of lower extremity Anatomy 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- 210000004900 c-terminal fragment Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- VDQQXEISLMTGAB-UHFFFAOYSA-N chloramine T Chemical compound [Na+].CC1=CC=C(S(=O)(=O)[N-]Cl)C=C1 VDQQXEISLMTGAB-UHFFFAOYSA-N 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 229940047120 colony stimulating factors Drugs 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 208000028831 congenital heart disease Diseases 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229920001577 copolymer Chemical compound 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000013058 crude material Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- ZWIBGKZDAWNIFC-UHFFFAOYSA-N disuccinimidyl suberate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCCC(=O)ON1C(=O)CCC1=O ZWIBGKZDAWNIFC-UHFFFAOYSA-N 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000009786 epithelial differentiation Effects 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 1
- 229960002963 ganciclovir Drugs 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 231100000118 genetic alteration Toxicity 0.000 description 1
- 210000002980 germ line cell Anatomy 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 239000008241 heterogeneous mixture Substances 0.000 description 1
- 101150113423 hisD gene Proteins 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 239000013029 homogenous suspension Substances 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000000893 inhibin Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 150000002614 leucines Chemical class 0.000 description 1
- 238000000670 ligand binding assay Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004705 lumbosacral region Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000011206 morphological examination Methods 0.000 description 1
- 230000020763 muscle atrophy Effects 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 201000006938 muscular dystrophy Diseases 0.000 description 1
- 229960000951 mycophenolic acid Drugs 0.000 description 1
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 230000001114 myogenic effect Effects 0.000 description 1
- 101150091879 neo gene Proteins 0.000 description 1
- 208000018360 neuromuscular disease Diseases 0.000 description 1
- 230000006959 non-competitive inhibition Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- -1 or other enzymatic Substances 0.000 description 1
- 239000002818 ornithine decarboxylase inhibitor Substances 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 235000020004 porter Nutrition 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 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 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000000734 protein sequencing Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 239000002464 receptor antagonist Substances 0.000 description 1
- 229940044551 receptor antagonist Drugs 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 238000013391 scatchard analysis Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013606 secretion vector Substances 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000008142 sex development Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 231100001055 skeletal defect Toxicity 0.000 description 1
- 210000002363 skeletal muscle cell Anatomy 0.000 description 1
- 230000022379 skeletal muscle tissue development Effects 0.000 description 1
- 210000001057 smooth muscle myoblast Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000033451 somitogenesis Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 101150081616 trpB gene Proteins 0.000 description 1
- 101150111232 trpB-1 gene Proteins 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000006967 uncompetitive inhibition Effects 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 210000001364 upper extremity Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- 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
- 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
- A01K67/0276—Knock-out vertebrates
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- 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
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- 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
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/495—Transforming growth factor [TGF]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- This invention relates generally to ligand-receptor interactions and more specifically to growth differentiation factor receptor proteins and the ligands that bind to such receptors and methods of use therefor.
- TGF- ⁇ transforming growth factor ⁇
- the transforming growth factor ⁇ (TGF- ⁇ ) superfamily encompasses a group of structurally-related proteins which affect a wide range of differentiation processes during embryonic development.
- the family includes, Mullerian inhibiting substance (MIS), which is required for normal male sex development (Behringer, et al, Nature, 345:167.
- MIS Mullerian inhibiting substance
- Drosophila decapentaplegic (DPP) gene product, which is required for dorsal -ventral axis formation and morphogenesis of the imaginal disks (Padgett, et al, Nature, 325:81 -84, 1987), the Xenopus Vg-1 gene product, which localizes to the vegetal pole of eggs ((Weeks, et al, Cell, 51:861-867, 1987), the activins (Mason, et al, Biochem, Biophys. Res. Commun., 135:957-964.
- DPP Drosophila decapentaplegic
- TGF- ⁇ s can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, hematopolesis, and epithelial cell differentiation (for review, see Massague, Cell 49:437, 1987).
- the proteins of the TGF- ⁇ family are initially synthesized as a large precursor protein which subsequently undergoes proteolytic cleavage at a cluster of basic residues approximately 110-140 amino acids from the C-terminus.
- the C-terminal regions, or mature regions, of the proteins are all structurally related and the different family members can be classified into distinct subgroups based on the extent of their homology. Although the homologies within particular subgroups range from 70% to 90% amino acid sequence identity, the homologies between subgroups are significantly lower, generally ranging from only 20% to 50%. In each case, the active species appears to be a disulfide-linked dimer of C-terminal fragments.
- the homodimeric species has been found to be biologically active, but for other family members, like the inhibins (Ling, et al, Nature, 321 :779, 1986) and the TGF- ⁇ s (Cheifetz, et al, Cell, 48:409, 1987), heterodimers have also been detected, and these appear to have different biological properties than the respective homodimers.
- the present invention provides receptors for the growth differentiation factor (GDF) growth factor family. These receptors are useful for identifying antagonists and agonists for agricultural and human therapeutic purposes.
- GDF growth differentiation factor
- the invention provides a recombinant cell line that expresses growth differentiation factor-8 (GDF-8) or growth differentiation factor- 11 (GDF-11) receptor polypeptide. Also included are antibodies that bind to GDF receptors, polynucleotides encoding the receptors and the GDF receptor proteins themselves. Peptide fragments of GDF receptors, such as the GDF-8 or GDF-11 receptors, are also included. Such peptides may be useful in inhibiting binding of GDF-8 or GDF-11 to either its own receptor or another GDF-receptor (e.g., GDF-8 and -11 may bind the same receptor).
- GDF-8 growth differentiation factor-8
- GDF-11 growth differentiation factor- 11
- the invention provides a substantially purified GDF-8-binding agent, wherein the binding agent inhibits GDF-8 binding to GDF-8 receptor.
- the binding agent inhibits GDF-8 binding to GDF-8 receptor.
- agents that inhibit GDF-11 binding are also included.
- the invention provides a method for identifying a GDF receptor polypeptide including incubating components such as GDF polypeptide and a cell expressing a receptor or a soluble receptor under conditions sufficient to allow the GDF to bind to the receptor; measuring the binding of the GDF polypeptide to the receptor; and isolating the receptor.
- the invention also includes a method for identifying a compound that binds to GDF receptor polypeptide including incubating components comprising the compound and GDFpolypeptide under conditions sufficient to allow the components to interact and measuring the binding or effect of binding of the compound to GDF receptor polypeptide.
- the invention also provides non-human transgenic animals that have a phenotype characterized by expression of GDF-receptor polypeptide, the phenotype being conferred by a transgene contained in the somatic and germ cells of the animal, the transgene comprising a nucleic acid sequence which encodes GDF- receptor polypeptide. Methods of producing such transgenic animals are also included.
- the invention includes a method for inhibiting the expression of
- GDF-receptor in a cell including contacting GDF-receptor with an inhibiting effective amount of an antisense oligonucleotide that binds to a segment of an mRNA transcribed from a GDF-receptor gene, whereby the binding of the antisense to the mRNA segment inhibits GDF-receptor expression.
- Figure la and lb are the nucleotide and amino acid sequence of murine GDF-8.
- Figure lc and Id are the nucleotide and amino acid sequence of human GDF-8.
- Figures 2a-2e are the nucleotide and amino acid sequence of baboon, bovine, chicken, rat, and turkey GDF-8.
- Figures 3a and 3b are Northern blots showing expression of GDF-8 in muscle and in various species, respectively.
- Figures 4a and 4b show the nucleotide and amino acid sequence of murine GDF-11 and expression of GDF-11, respectively.
- Figure 5 shows an autoradiogram showing GDF-8.
- Figures 6 and 7 show binding studies for GDF-8.
- Figures 8-11 show 4 myoblast cell lines that do not bind GDF-8.
- Figure 12 shows the construction of GDF-11 null mice by homologous targeting, a) is a map of the GDF-11 locus (top line) and targeting construct (second line). The black and stippled boxes represent coding sequences for the pro-and C-terminal regions, respectively.
- the targeting construct contains a total of 11 kb of homology with the GDF-11 gene.
- a probe derived from the region upstream of the 3' homology fragment and downstream of the first EcoRI site shown hybridizes to a 6.5 kb EcoRI fragment in the GDF-1 1 gene and a 4.8 kb fragment in a homologously targeted gene.
- FIG. 13 shows kidney abnormalities in GDF-11 knockout mice. Kidneys of newborn animals were examined and classified according to the number of normal sized or small kidneys as shown at the top. Numbers in the table indicate number of animals falling into each classification according to genotype.
- Figure 14 shows homeotic transformations in GDF-11 mutant mice, a) Newborn pups with missing (first and second from left) and normal looking tails, b-j) Skeleton preparations for newborn wild-type (b, e, h), heterozygous (c, f, I) and homozygous (d, g, j) mutant mice.
- Whole skeleton preparations (b-d), vertebral columns (e-g), vertebrosternal ribs (h-j) showing transformations and defects in homozygous and heterozygous mutant mice. Numbers indicate thoracic segments.
- Figure 15 is a table summarizing anterior transformations in wild-type, heterozygous and homozygous GDF-11 mice.
- the invention provides an isolated polynucleotide sequence encoding the receptors of the invention.
- isolated includes polynucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated.
- Polynucleotide sequences of the invention include DNA, cDNA and RNA sequences which encode GDF receptors. It is understood that all polynucleotides encoding all or a portion of GDF receptors are also included herein, as long as they encode a polypeptide with GDF receptors activity (e.g., bind to GDF).
- Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides.
- portions of the mRNA sequence may be altered due to alternate RNA splicing patterns or the use of alternate promoters for RNA transcription.
- GDF receptor polynucleotide may be subjected to site-directed mutagenesis.
- the polynucleotide sequence for GDF receptors also includes antisense sequences.
- the polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of GDF receptors polypeptide encoded by the nucleotide sequence is functionally unchanged.
- nucleotide sequences which encode GDF receptors polypeptide.
- the polynucleotide encoding GDF receptors for GDFs such as GDF-8 or 11 (shown in the figures).
- the sequence is RNA
- the deoxyribonucleotides A, G, C, and T are replaced by ribonucleotides A, G, C, and U, respectively.
- fragments (portions) of the above-described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encodes the GDF receptor.
- “Selective hybridization” as used herein refers to hybridization under moderately stringent or highly stringent physiological conditions (See, for example, the techniques described in Maniatis et al., 1989 Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., incorporated herein by reference), which distinguishes related from unrelated nucleotide sequences.
- nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
- An example of progressively higher stringency conditions is as follows: 2 x SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1 % SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68 °C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
- SEQ ID NO:3 represents the wild-type sequence
- SEQ ID NO: 1 represents a cDNA which encodes GDF receptors having a conservative substitution of Leucine for Alanine at amino acid residue 127. The result of this conservative variation should not affect biological activity of GDF receptors polypetide or peptides containing the variation (see Example 5).
- DNA sequences of the invention can be obtained by several methods.
- the DNA can be isolated using hybridization or computer-based techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features; 3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of interest; 4) computer searches of sequence databases for similar sequences; and 5) differential screening of a subtracted DNA library.
- hybridization or computer-based techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features; 3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of
- the GDF receptor polynucleotide of the invention is derived from avian, bovine, ovine, piscine, murine, human or porcine. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of amino acid sequence must be known. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate.
- hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA.
- Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present.
- stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization of the target DNA to that single probe in the mixture which is its complete complement (Wallace, et al, Nucl Acid Res., 9:879, 1981).
- a subtractive library as illustrated herein is useful for elimination of non-specific cDNA clones.
- the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al, Nucl. Acid Res., ⁇ :2325, 1983).
- a cDNA expression library such as lambda gtl 1
- Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of GDF receptors cDNA.
- Alterations in GDF receptors nucleic acid include intragenic mutations (e.g., point mutation, nonsense (stop), missense, splice site and frameshift) and heterozygous or homozygous deletions. Detection of such alterations can be done by standard methods known to those of skill in the art including sequence analysis, Southern blot analysis, PCR based analyses (e.g., multiplex PCR, sequence tagged sites (STSs)) and in situ hybridization. Such proteins can be analyzed by standard SDS-PAGE and/or immuno- precipitation analysis and/or Western blot analysis, for example.
- DNA sequences encoding GDF receptors can be expressed in vitro by DNA transfer into a suitable host cell.
- "Host cells” are cells in which a vector can be propagated and its DNA expressed.
- the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
- the GDF receptor polynucleotide sequences may be inserted into a recombinant expression vector.
- recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the GDF receptors genetic sequences.
- Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
- the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
- Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al., Gene ,56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells.
- the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., Tl, metallothionein I, or polyhedrin promoters).
- Polynucleotide sequences encoding GDF receptors can be expressed in either prokaryotes or eukaryotes.
- Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
- Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
- a variety of host-expression vector systems may be utilized to express the GDF receptors coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the GDF receptors coding sequence; yeast transformed with recombinant yeast expression vectors containing the GDF receptors coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the GDF receptors coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the GDF receptors coding sequence; or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus) containing the
- any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al., 1987, Methods in Enzymology j_53_:516-544).
- inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
- promoters derived from the genome of mammalian cells e.g., metallothionein promoter
- mammalian viruses e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
- Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted GDF receptors coding sequence.
- yeast a number of vectors containing constitutive or inducible promoters may be used.
- Current Protocols in Molecular Biology Vol. 2, 1988, Ed. Ausubel et al, Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch.
- yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol.l 1, A Practical Approach, Ed. DM Glover, 1986, IRL Press, Wash., D.C.).
- vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
- Eukaryotic systems and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur.
- Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and advantageously, plasma membrane insertion of the gene product may be used as host cells for the expression of GDF receptors.
- Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression may be engineered.
- the GDF receptors coding sequence may be ligated to an adenovirus transcription/- translation control complex, e.g., the late promoter and tripartite leader sequence.
- the vaccinia virus 7.5K promoter may be used, (e.g., see, Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7415-7419; Mackett et al, 1984, J. Virol. 49: 857- 864; Panicali et al, 1982, Proc. Natl. Acad. Sci. USA 79: 4927-4931).
- vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements (Sarver, et al, 1981, Mol. Cell. Biol. 1 : 486).
- the plasmid Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression.
- These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, the neo gene.
- the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the GDF receptors gene in host cells (Cone & Mulligan, 1984, Proc. Natl Acad. Sci. USA 81:6349-6353). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters.
- host cells can be transformed with the GDF receptors cDNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
- appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
- the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
- engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
- a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler,et al, 1977, Cell I k 223), hypoxanthine-guanine phospho- ribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci.
- adenine phosphoribosyltransferase genes can be employed in tk-, hgprt " or aprt " cells respectively.
- antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al, 1980, Natl. Acad. Sci. USA 77: 3567; O ⁇ are, et al, 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl.
- neo which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al, 1981, J. Mol. Biol. 150: 1); and hygro, which confers resistance to hygromycin (Santerre, et al, 1984, Gene 30: 147) genes.
- trpB which allows cells to utilize indole in place of tryptophan
- hisD which allows cells to utilize histinol in place of histidine
- ODC ornithine decarboxylase
- Eukaryotic cells can also be cotransformed with DNA sequences encoding the GDF receptors of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
- Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein, (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed, 1982).
- a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
- the present invention relates to stable recombinant cell lines, the cells of which express GDF receptor polypeptides and contain DNA that encodes GDF receptors.
- Suitable cell types include but are not limited to cells of the following types: NIH 3T3 (Murine), C2C12, L6, and P19.
- C2C12 and L6 myoblasts will differentiate spontaneously in culture and form myotubes depending on the particular growth conditions (Yaffe and Saxel, 1977; Yaffe, 1968).
- PI 9 is an embryonal carcinoma cell line.
- Such cells are described, for example, in the Cell Line Catalog of the American Type Culture Collection (ATCC). These cells can be stably transformed by a method known to the skilled artisan. See, for example, Ausubel et al.
- GDF receptors can be expressed using inducible or constituitive regulatory elements for such expression.
- constituitive or inducible promoters for example, are known in the art.
- the desired protein encoding sequence and an operably linked promoter may be introduced into a recipient cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the desired molecule may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sequence into the host chromosome. Therefore the cells can be transformed stably or transiently.
- An example of a vector that may be employed is one which is capable of integrating the desired gene sequences into the host cell chromosome.
- Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
- the marker may complement an auxotrophy in the host (such as leu2, or ura3, which are common yeast auxotrophic markers), biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
- the selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection.
- the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
- a plasmid or viral vector capable of autonomous replication in the recipient host.
- Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
- vectors For a mammalian host, several possible vector systems are available for expression.
- One class of vectors utilize DNA elements which provide autonomously replicating extra-chromosomal plasmids, derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, or SV40 virus.
- a second class of vectors include vaccinia virus expression vectors.
- a third class of vectors relies upon the integration of the desired gene sequences into the host chromosome. Cells which have stably integrated the introduced DNA into their chromosomes may be selected by also introducing one or more markers (e.g., an exogenous gene) which allow selection of host cells which contain the expression vector.
- the marker may provide for prototropy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper or the like.
- the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
- the cDNA expression vectors incorporating such elements include those described by Okayama, H, Mol. Cell. Biol, 3:280 (1983), and others.
- the DNA construct may be introduced (transformed) into an appropriate host.
- Various techniques may be employed, such as protoplast fusion, calcium phosphate precipitation, electroporation or other conventional techniques.
- the present invention relates to transgenic animals having cells that express GDF receptors.
- Such transgenic animals may have decreased fat content and increased muscle mass.
- the subject invention provides non-human transgenic animals which are useful as a source of food products with high muscle and protein content, and reduced fat and cholesterol content.
- the animals have been altered chromosomally in their germ cells and somatic cells so that the production of GDF-8 may be at "normal" levels, however, the GDF-8 receptor is produced in reduced amounts, or is completely disrupted, resulting in animals with decreased binding of GDF-8 and higher than normal levels of muscle tissue, preferably without increased fat and/or cholesterol levels.
- the present invention also includes food products provided by the animals. Such food products have increased nutritional value because of the increase in muscle tissue.
- the transgenic non-human animals of the invention include bovine, porcine, ovine and avian animals, for example.
- the subject invention also provides a method of producing animal food products having increased muscle content.
- the method includes modifying the genetic makeup of the germ cells of apronuclear embryo of the animal, implanting the embryo into the oviduct of a pseudopregnant female thereby allowing the embryo to mature to full term progeny, testing the progeny for presence of the transgene to identify transgene-positive progeny, cross-breeding transgene-positive progeny to obtain further transgene-positive progeny and processing the progeny to obtain foodstuff.
- the modification of the germ cell comprises altering the genetic composition so as to disrupt or reduce the expression of the naturally occurring gene encoding for production of GDF-8 receptor protein.
- the transgene comprises antisense polynucleotide sequences to the GDF-8 receptor protein.
- the transgene may comprise a non-functional sequence which replaces or intervenes in the native GDF-8 receptor gene or the transgene may encode a GDF-8 receptor antagonist.
- the subject invention also provides a method of producing avian food products having improved muscle content.
- the method includes modifying the genetic makeup of the germ cells of a pronuclear embryo of the avian animal, implanting the embryo into the oviduct of a pseudopregnant female into an embryo of a chicken, culturing the embryo under conditions whereby progeny are hatched, testing the progeny for presence of the genetic alteration to identify transgene-positive progeny, cross-breeding transgene- positive progeny and processing the progeny to obtain foodstuff.
- animal here denotes all mammalian species except human. It also includes an individual animal in all stages of development, including embryonic and fetal stages. Farm animals (pigs, goats, sheep, cows, horses, rabbits and the like), rodents (such as mice), and domestic pets (for example, cats and dogs) are included within the scope of the present invention.
- transgenic animal is any animal containing cells that bear genetic information received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by microinjection or infection with recombinant virus. "Transgenic” in the present context does not encompass classical crossbreeding or in vitro fertilization, but rather denotes animals in which one or more cells receive a recombinant DNA molecule.
- transgenic animal also includes a "germ cell line” transgenic animal.
- a germ cell line transgenic animal is a transgenic animal in which the genetic information has been taken up and incorporated into a germ line cell, therefore conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information, then they, too, are transgenic animals.
- the cDNA that encodes GDF receptors can be fused in proper reading frame under the transcriptional and translational control of a vector to produce a genetic construct that is then amplified, for example, by preparation in a bacterial vector, according to conventional methods. See, for example, the standard work: Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Press 1989), the contents of which are incorporated by reference. The amplified construct is thereafter excised from the vector and purified for use in producing transgenic animals.
- transgenic as used herein additionally includes any organism whose genome has been altered by in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to induce a specific gene knockout.
- gene knockout refers to the targeted disruption of a gene in vivo with complete loss of function that has been achieved by any transgenic technology familiar to those in the art.
- transgenic animals having gene knockouts are those in which the target gene has been rendered nonfunctional by an insertion targeted to the gene to be rendered non-functional by homologous recombination.
- transgenic includes any transgenic technology familiar to those in the art which can produce an organism carrying an introduced transgene or one in which an endogenous gene has been rendered non-functional or "knocked out.”
- the transgene to be used in the practice of the subject invention may be a DNA sequence comprising a modified GDF receptors coding sequence.
- the transgene to be used in the practice of the subject invention may be a DNA sequence comprising a modified GDF receptors coding sequence.
- the GDF receptor gene is disrupted by homologous targeting in embryonic stem cells.
- the entire mature C-terminal region of the GDF receptors gene may be deleted as described in the examples below.
- the GDF receptors disruption or deletion may be accompanied by insertion of or replacement with other DNA sequences, such as a non-functional GDF receptors sequence.
- the transgene comprises DNA antisense to the coding sequence for GDF receptors.
- the transgene comprises DNA encoding an antibody or receptor peptide sequence which is able to bind to GDF receptors.
- DNA sequences that encode proteins having GDF receptors activity but differ in nucleic acid sequence due to the degeneracy of the genetic code may also be used herein, as may truncated forms, allelic variants and interspecies homologues.
- the present invention relates to antibodies that bind GDF receptors that block GDF binding to the receptor.
- such antibodies may be useful for ameliorating disorders associated with muscle tissue.
- a monoclonal antibody which binds to GDF-8 receptor may have the effect of increasing the development of skeletal muscles.
- the GDF-8 receptor monoclonal antibody, polypeptide, or polynucleotide is administered to a patient suffering from a disorder selected from the group consisting of muscle wasting disease, neuromuscular disorder, muscle atrophy or aging.
- the GDF-8 receptor antibody may also be administered to a patient suffering from a disorder selected from the group consisting of muscular dystrophy, spinal cord injury, traumatic injury, congestive obstructive pulmonary disease (COPD), AIDS or cachechia.
- COPD congestive obstructive pulmonary disease
- the GDF-8 antibody is administered to a patient with muscle wasting disease or disorder by intravenous, intramuscular or subcutaneous injection; preferably, a monoclonal antibody is administered within a dose range between about 0.1 mg/kg to about 100 mg/kg; more preferably between about 1 ug/kg to 75 mg/kg; most preferably from about 10 mg/kg to 50 mg/kg.
- the antibody may be administered, for example, by bolus injunction or by slow infusion. Slow infusion over a period of 30 minutes to 2 hours is preferred.
- the GDF-8 antibody may be formulated in a formulation suitable for administration to a patient. Such formulations are known in the art.
- the dosage regimen will be determined by the attending physician considering various factors which modify the action of the GDF-8 receptor protein, e.g. amount of tissue desired to be formed, the site of tissue damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue, the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
- the dosage may vary with the type of matrix used in the reconstitution and the types of agent, such as anti- GDF-8 receptor antibodies, to be used in the composition.
- systemic or injectable administration such as intravenous (IV), intramuscular (IM) or subcutaneous (Sub-Q) injection.
- Administration will generally be initiated at a dose which is minimally effective, and the dose will be increased over a preselected time course until a positive effect is observed. Subsequently, incremental increases in dosage will be made limiting such incremental increases to such levels that produce a corresponding increase in effect, while taking into account any adverse affects that may appear.
- growth factors such as IGF I (insulin like growth factor I), human, bovine, or chicken growth hormone which may aid in increasing muscle mass, to the final composition, may also affect the dosage.
- the anti-GDF-8 antibody is generally administered within a dose range of about 0.1 ug/kg to about 100 mg/kg.; more preferably between about 10 mg/kg to 50 mg/kg.
- polyclonal antibodies The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al , Production of Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al, Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS N IMMUNOLOGY, section 2.4.1 (1992), which are hereby incorporated by reference. The preparation of monoclonal antibodies likewise is conventional.
- monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
- Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion- exchange chromatography.
- Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
- suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium
- a mammalian serum such as fetal calf serum or trace elements
- growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
- Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies.
- Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture.
- Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g. , osyngeneic mice, to cause growth of antibody- producing tumors.
- the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.
- pristane tetramethylpentadecane
- the desired monoclonal antibody is recovered from the body fluid of the animal.
- Therapeutic applications for antibodies disclosed herein are also part of the present invention.
- antibodies of the present invention may also be derived from subhuman primate antibody.
- a therapeutically useful anti-GDF receptors antibody may be derived from a "humanized" monoclonal antibody.
- Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts.
- the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
- General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Nat'l Acad. Sci. USA 86:3833 (1989), which is hereby incorporated in its entirety by reference.
- Antibodies of the invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al, METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 119 (1991); Winter etal, Ann. Rev. Immunol 12: 433 (1994), which are hereby incorporated by reference.
- Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).
- antibodies of the present invention may be derived from a human monoclonal antibody.
- Such antibodies are obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
- elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci.
- the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
- Methods for obtaining human antibodies from transgenic mice are described by Green et al, Nature Genet. 7:13 (1994); Lonberg et al, Nature 368:856 (1994); and Taylor et al, Int. Immunol. 6:579 (1994), which are hereby incorporated by reference.
- Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
- Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
- antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
- This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
- cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
- Fv fragments comprise an association of V H and Y chains. This association may be noncovalent, as described in Inbar et al, Proc. Nat'l Acad. Sci. USA 69:2659 (1972).
- the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu, supra.
- the Fv fragments comprise V H and V L chains connected by a peptide linker.
- These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains connected by an oligonucleotide.
- the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
- the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
- Methods for producing sFvs are described, for example, by Whitlow et al, METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97 (1991); Bird etal, Science 242:423-426 (1988); Ladner et al, U.S. patent No. 4,946,778; Pack et al, Bio/Technology 11 : 1271-77 (1993); and Sandhu, supra.
- CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al, METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).
- the invention provides a method for identifying a GDF receptor polypeptide comprising incubating components comprising GDF polypeptide and a cell expressing a receptor or a soluble receptor under conditions sufficient to allow the GDF to bind to the receptor; measuring the binding of the GDF polypeptide to the receptor; and isolating the receptor.
- the GDF may be any of the known GDFs (e.g., GDF-1-16), and preferably is GDF-8 or GDF-11. Methods of isolating the receptors are described in more detail in the Examples section below.
- GDF receptors variant means a molecule that simulates at least part of the structure of GDF receptors. GDF receptor variants may also be useful in preventing GDF binding, thereby ameliorating symptoms of disorders described above.
- the present invention relates to peptides and peptide derivatives that have fewer amino acid residues than GDF receptors.
- Such peptides and peptide derivatives could represent research and diagnostic tools in the study of muscle wasting diseases and the development of more effective therapeutics.
- the invention relates not only to peptides and peptide derivatives of naturally-occurring GDF receptors, but also to GDF receptor mutants and chemically synthesized derivatives of GDF receptors that bind GDFs.
- changes in the amino acid sequence of GDF receptors are contemplated in the present invention.
- GDF receptors can be altered by changing the DNA encoding the protein. Preferably, only conservative amino acid alterations are undertaken, using amino acids that have the same or similar properties.
- Illustrative amino acid substitutions include the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.
- Variants useful for the present invention comprise analogs, homologs, muteins and mimetics of GDF receptors that retain the ability to bind to their respective GDFs.
- Peptides of the GDF receptors refer to portions of the amino acid sequence of GDF receptors that also retain this ability.
- the variants can be generated directly from GDF receptors itself by chemical modification, by proteolytic enzyme digestion, or by combinations thereof. Additionally, genetic engineering techniques, as well as methods of synthesizing polypeptides directly from amino acid residues, can be employed.
- Peptides of the invention can be synthesized by such commonly used methods as t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C terminus of the peptide (See, Coligan, et al, Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). Peptides of the invention can also be synthesized by the well known solid phase peptide synthesis methods described Merrifield, J. Am. Chem. Soc, 85:2149.
- This can normally be purified by such techniques as gel filtration on Sephadex G-15 using 5% acetic acid as a solvent. Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid phase Edman degradation.
- peptides can be produced by recombinant methods as described below.
- substantially purified refers to a molecule, such as a peptide that is substantially free of other proteins, lipids, carbohydrates, nucleic acids, and other biological materials with which it is naturally associated.
- a substantially pure molecule such as a polypeptide, can be at least 60%, by dry weight, the molecule of interest.
- GDF receptors peptides can be purified using standard protein purification methods and the purity of the polypeptides can be determined using standard methods including, e.g., polyacrylamide gel electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., high performance liquid chromatography (HPLC)), and amino-terminal amino acid sequence analysis.
- polyacrylamide gel electrophoresis e.g., SDS-PAGE
- column chromatography e.g., high performance liquid chromatography (HPLC)
- amino-terminal amino acid sequence analysis e.g., amino-terminal amino acid sequence analysis.
- Non-peptide compounds that mimic the binding and function of GDF receptors can be produced by the approach outlined in Saragovi et al, Science 253: 792-95 (1991).
- Mimetics are molecules which mimic elements of protein secondary structure. See, for example, Johnson et ⁇ /,”Peptide Turn Mimetics,” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al, Eds, (Chapman and Hall, New York 1993).
- the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions.
- appropriate mimetics can be considered to be the equivalent of GDF receptors itself.
- Longer peptides can be produced by the "native chemical" ligation technique which links together peptides (Dawson, et al, Science, 266:776. 1994). Variants can be created by recombinant techniques employing genomic or cDNA cloning methods. Site-specific and region-directed mutagenesis techniques can be employed. See CURRENT PROTOCOLS IN MOLECULAR BIOLOGY vol. 1, ch. 8 (Ausubel et al eds, J. Wiley & Sons 1989 & Supp. 1990-93); PROTEIN ENGINEERING (Oxender & Fox eds, A. Liss, Inc. 1987).
- linker-scanning and PCR-mediated techniques can be employed for mutagenesis. See PCR TECHNOLOGY (Erlich ed, Stockton Press 1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vols. 1 & 2, supra. Protein sequencing, structure and modeling approaches for use with any of the above techniques are disclosed in PROTEIN ENGINEERING, loc. cit, and CURRENT PROTOCOLS PN MOLECULAR BIOLOGY, vols. 1 & 2, supra.
- the present invention relates to GDF receptor-binding agents that block binding of GDFs to their receptors.
- GDF receptor-binding agents may represent effective therapeutics.
- GDF receptor-binding agent denotes a naturally occurring ligand of GDF receptors such as, for example: GDF-1 -16; a synthetic ligand of GDF receptors, or appropriate derivatives of the natural or synthetic ligands. The determination and isolation of ligands is well described in the art. See, e.g., Lerner, Trends NeuroSci. 17:142-146 (1994) which is hereby incorporated in its entirety by reference.
- the present invention relates to GDF receptor-binding agents that interfere with binding between GDF receptor and a GDF.
- binding agents may interfere by competitive inhibition, by non-competitive inhibition or by uncompetitive inhibition. Interference with normal binding between GDF receptors and one or more GDF can result in a useful pharmacological effect.
- the invention provides a method for identifying a composition which binds to GDF receptors.
- the method includes incubating components comprising the composition and GDF receptors under conditions sufficient to allow the components to interact and measuring the binding of the composition to GDF receptors.
- Compositions that bind to GDF receptors include peptides, peptidomimetics, polypeptides, chemical compounds and biologic agents as described above.
- Incubating includes conditions which allow contact between the test composition and GDF receptors.
- Contacting includes in solution and in solid phase.
- the test ligand(s)/composition may optionally be a combinatorial library for screening a plurality of compositions.
- compositions identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki, et al, Bio/Technology, 3_: 1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner, et al, Proc. Natl. Acad. Sci. USA, 80:278, 1983), oligonucleotide ligation assays (OLAs) (Landegren, et al, Science, 241: 1077, 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren, et al, Science, 242:229-237. 1988).
- compositions can functionally complex with the receptor protein
- induction of the exogenous gene is monitored by monitoring changes in the protein levels of the protein encoded for by the exogenous gene, for example.
- this composition(s) can bind to the receptor protein coded for by the nucleic acid encoding the initial sample test composition(s).
- exogenous gene can be monitored by a functional assay or assay for a protein product, for example.
- the exogenous gene is therefore a gene which will provide an assayable/measurable expression product in order to allow detection of expression of the exogenous gene.
- exogenous genes include, but are not limited to, reporter genes such as chloramphenicol acetyltransferase gene, an alkaline phosphatase gene, beta-galactosidase,a luciferase gene, a green fluorescent protein gene, guanine xanthine phosphoribosyltransferase, alkaline phosphatase, and antibiotic resistance genes (e.g., neomycin phosphotransferase).
- reporter genes such as chloramphenicol acetyltransferase gene, an alkaline phosphatase gene, beta-galactosidase,a luciferase gene, a green fluorescent protein gene, guanine x
- compositions of the present invention can be extracted and purified from the culture media or a cell by using known protein purification techniques commonly employed, such as extraction, precipitation, ion exchange chromatography, affinity chromatography, gel filtration and the like.
- Compositions can be isolated by affinity chromatography using the modified receptor protein extracellular domain bound to a column matrix or by heparin chromatography.
- screening method of the invention is combinatorial chemistry methods for identifying chemical compounds that bind to GDF receptors.
- the screening method is also useful for identifying variants, binding or blocking agents, etc, which functionally, if not physically (e.g., sterically) act as antagonists or agonists, as desired.
- the purified GDF-8 and GDF-11 proteins will be used primarily to assay for biological activities. In order to identify potential target cells for GDF-8 and GDF-11 action cells expressing their receptors will be searched. For this purpose, the purified protein will be radioiodinated using the chloramine T method, which has been used successfully to label other members of this superfamily, like TGF- ⁇ (Cheifetz et al, 1987), activins (Sugino et al, 1988), and BMPs (Paralkar et al, 1991), for receptor-binding studies. The mature processed forms of GDF-8 and GDF-11 each contain multiple tyrosine residues. Two different approaches will then be taken to attempt to identify receptors for these proteins.
- One approach will be taken to determine the number, affinity, and distribution of receptors. Either whole cells grown in culture, frozen sections of embryos or adult tissues, or total membrane fractions prepared from tissues or cultured cells will be incubated with the labeled protein, and the amount or distribution of bound protein will be determined. For experiments involving cell lines or membranes, the amount of binding will be determined by measuring either the amount of radioactivity bound to cells on the dish after several washes or, in the case of membranes, the amount of radioactivity sedimented with the membranes after centrifugation or retained with the membranes on a filter. For experiments involving primary cultures, where the number of cells may be more limited, binding sites will be visualized directly by overlaying with photographic emulsion.
- a second approach will also be taken to begin to characterize the receptor biochemically.
- Membrane preparations or potential target cells grown in culture will be incubated with labeled ligand, and receptor/ligand complexes will be covalently cross-linked using disuccinimidyl suberate, which has been commonly used to identify receptors for a variety of ligands, including members of the TGF- ⁇ superfamily (for example, see Massague and Like, 1985).
- Cross-linked complexes will then be electrophoresed on SDS polyacrylamide gels to look for bands labeled in the absence but not in the presence of excess unlabeled protein.
- the molecular weight of the putative receptor will be estimated by subtracting the molecular weight of the ligand.
- An important question that these experiments will address is whether GDF-8 and GDF-11 signal through type I and type II receptors like many other members of the TGF- ⁇ superfamily (for review, see Massague, 1996).
- binding studies on these primary cells after various days in culture will be performed and binding sites localized by autoradiography so that the binding sites can be co-localized with various myogenic markers, such as muscle myosin (Vivarelli et al, 1988), and correlate binding with the differentiation state of the cells, such as formation of multinucleated myotubes.
- myogenic markers such as muscle myosin (Vivarelli et al, 1988)
- cell lines will be utilized to look for receptors.
- the initial focus will be on three cells lines, C2C12, L6, and PI 9.
- C2C12 and L6 myoblasts will differentiate spontaneously in culture and form myotubes depending on the particular growth conditions (Yaffe and Saxel, 1977; Yaffe, 1968).
- PI 9 embryonal carcinoma cells can be induced to differentiate into various cell types, including skeletal muscle cells in the presence of DMSO (Rudnicki and McBurney, 1987). Receptor binding studies will be carried out on these cell lines under various growth conditions and at various stages of differentiation.
- the dissociation constant (K d ) is 370 pM and L6 myoblasts have a high number (5,000 receptors/cell) of cell surface binding proteins (Figure 6).
- GDF-11 also called BMP-11
- BMP-11 BMP-11
- Receptor binding studies were performed to determine if GDF-11 also binds the GDF-8 receptor.
- Figure 6 shows that GDF-8 and GDF-11 do bind to the same binding proteins on L6 myoblasts. It is important to establish whether or not GDF-8 binds to the known TGF- ⁇ receptor. As shown in Figure 6, TGF- ⁇ does not compete the binding of GDF-8, indicating that the GDF-8 receptor is distinct from the TGF- ⁇ receptor.
- the GDF-8 receptor is not expressed on all myoblast cell lines.
- Figures 8-11 are examples of four myoblast cell lines (C2C12, G7, MLB13MYC cl4 and BC3H1) which do not bind GDF-8.
- the first approach will be to use an expression cloning strategy. In fact, this was the strategy that was orginally used by Mathews and Vale (1991) and Lin et al. (1992) to clone the first activin and TGF- ⁇ receptors.
- We will begin by preparing poly A-selected RNA from the tissue or cell type that expresses the highest relative number of high affinity binding sites.
- We will then use this RNA to prepare a cDNA library in the mammalian expression vector pcDNA-1. This vector contains a CMV promoter and an SV40 origin of replication.
- the library will be plated, and cells from each plate will be pooled into broth and frozen. Aliquots from each pool will then be grown for preparation of DNA.
- Each individual pool will be transiently transfected into COS cells in chamber slides, and transfected cells will be incubated with iodinated GDF-8 or GDF-11. After washing away the unbound protein, the sites of ligand binding will be visualized by autoradiography. Once a positive pool is identified, the cells from that pool will be replated at lower density, and the process will be repeated. Positive pools will then be plated, and individual colonies will be picked into grids and re-analyzed as described (Wong et al, 1985).
- TGF- ⁇ and a cloned type II receptor The coding sequence for the TGF- ⁇ type II receptor will be cloned into the pcDNA-1 vector, and bacteria transformed with this construct will be mixed with bacteria from our library at various ratios, including 1 : 1500.
- the general strategy will be to design degenerate primers corresponding to conserved regions of the known receptors, to use these primers for PCR on cDNA prepared from the appropriate RNA samples (most likely from skeletal muscle), to subclone the PCR products, and finally to sequence individual subclones. As sequences are identified, they will be used as hybridization probes to eliminate duplicate clones from further analysis. We will then test the receptors that we identify for their ability to bind purified GDF-8 and GDF-11.
- this screen will yield only small PCR products, we will obtain full-length cDNA clones for each receptor from cDNA libraries prepared from the appropriate tissue, insert these cDNA clones into the pcDNA-1 vector, transfect these constructs into COS cells, and assay the transfected cells for their ability to bind iodinated GDF-8 or GDF-11. Ideally, we would like to test every receptor that we identify in this screen for their ability to bind these ligands. However, the number of receptors that we identify may be large, and isolating all of the full-length cDNAs and testing them may require considerable effort.
- GDF-8 and GDF-11 receptors are further complicated by the fact at least one member of the TGF- ⁇ superfamily, namely, GDNF, is capable of signalling through a completely different type of receptor complex involving a GPI-linked component (GDNFR-alpha) and a receptor tyrosine kinase (c-ret) (Trupp et al, 1996; Durbec et al, 1996; Treanor et al, 1996; Jing et al, 1996).
- GDNFR-alpha GPI-linked component
- c-ret receptor tyrosine kinase
- GDF-8 and GDF-11 do signal through a similar receptor complex
- our expression screening approach should be able to identify at least the GPI-linked component (indeed GDNFR-alpha was identified using an expression screening approach) of this complex.
- identifying the analogous receptor tyrosine kinase would probably require a substantial amount of additional work, such as biochemical purification of the complex.
- the similar phenotypes of GDNF- and c-ret-deficient mice suggested c-ret as a potential receptor for GDNF.
- the phenotype of GDF-11 knockout mice in several respects resembles the phenotype of mice carrying a deletion of a receptor for some members of the TGF- ⁇ superfamily, the activintype HB receptor (ActRIIB).
- ActRIIB activintype HB receptor
- RI ES cells were transfected with the targeting construct, selected with gancyclovir (2 ⁇ M) and G418 (250 ⁇ g/ml), and analyzed by Southern analysis. Homologous targeting of the GDF-11 gene was seen in 8/155 g- ancyclovir/G418 doubly resistant ES cell clones. Following injection of several targeted clones into C57BL/6J blastocysts, we obtained chimeras from one ES clone that produced heterozygous pups when crossed to both C57BL/6J and 129/SvJ females.
- Crosses of C57BL/6J/129/SvJ hybrid FI heterozygotes produced 49 wild-type (34%), 94 heterozygous (66%) and no homozygous mutant adult offspring. Similarly, there were no adult homozygous null animals seen in the 129/SvJ background (32 wild-type (36%>) and 56 heterozygous mutant (64%) animals).
- thoracic segments T8, T9, T10, and in some cases even Tl 1, which all have free ribs in wild-type animals, were transformed in mutant animals to have a characteristic typical of more anterior thoracic segments, namely, the presence of ribs attached to the sternum. Consistent with this finding, the transitional spinous process and transitional articular processes which are normally found on T10 in wild-type animals were instead found on T13 in homozygous mutants (data not shown). Additional transformations within the thoracic region were also noted in certain mutant animals. For example, in wild-type mice, the ribs derived from Tl normally touch the top of the sternum.
- T2 appeared to have been transformed to have a morphology resembling that of T 1 ; that is, in these animals, the ribs derived from T2 extended to touch the top of the sternum. In these cases, the ribs derived from Tl appeared to fuse to the second pair of ribs. Finally, in 82% of homozygous mutants, the long spinous process normally present on T2 was shifted to the position of T3. In certain other homozygous mutants, asymmetric fusion of a pair of vertebrosternal ribs was seen at other thoracic levels.
- the anterior transformations were not restricted to the thoracic region. The anterior most transformation that we observed was at the level of the 6th cervical vertebra (C6).
- C6 6th cervical vertebra
- C6 is readily identifiable by the presence of two anterior tuberculi on the ventral side.
- C7 appeared to have been partially transformed to have a morphology resembling that of C6.
- One other homozygous mutant had 2 anterior tuberculi on C7 but retained one on C6 for a complete C7 to C6 transformation but a partial C6 to C5 transformation.
- Transformations of the axial skeleton also extended into the lumbar region.
- wild-type animals normally have only 6 lumbar vertebrae
- homozygous mutants had 8-9. At least 6 of the lumbar vertebrae in the mutants must have derived from segments that would normally have given rise to sacral and caudal vertebrae as the data described above suggest that 4 to 5 lumbar segments were transformed into thoracic segments.
- homozygous mutant mice had a total of 33-34 presacral vertebrae compared to 26 presacral vertebrae normally present in wild-type mice.
- Heterozygous mice also showed abnormalities in the axial skeleton although the phenotype was much milder than in homozygous mice.
- the most obvious abnormality in heterozygous mice was the presence of an additional thoracic segment with an associated pair of ribs (Figure 14(c,f)). This transformation was present in every heterozygous animal examined, and in every case, the additional pair of ribs was attached to the sternum ( Figure 14(i)).
- T8 whose associated rib normally does not touch the sternum, appeared to have been transformed to a morphology characteristic of a more anterior thoracic vertebra, and LI appeared to have been transformed to a morphology characteristic of a posterior thoracic vertebra.
- mutant embryos isolated at various stages of development were not readily distinguishable from corresponding wild-type embryos.
- the number of somites present at any given developmental age was identical between mutant and wild-type embryos, suggesting that the rate of somite formation was unaltered in the mutants.
- day 10.5-11.5 p.c mutant embryos could be easily distinguished from wild-type embryos by the posterior displacement of the hindlimb by 7-8 somites.
- the abnormalities in tail development were also readily apparent at this stage.
- Alterations in expression of homeobox containing genes are known to cause transformations in Drosophila and in vertebrates.
- Hox genes the vertebrate homeobox containing genes
- GDF-11 null mutants we determined the expression pattern of 3 representative Hox genes, Hoxc-6, Hoxc-8 and Hoxc-11, in day 12.5 p.c. wild-type, heterozygous and homozygous mutant embryos by whole mount in situ hybridization.
- the expression pattern of Hoxc-6 in wild-type embryos spanned prevertebrae 8-15 which correspond to thoracic segments T1-T8.
- Hoxc-6 expression pattern was shifted posteriorly and expanded to prevertebrae 9-18 (T2-T11). A similar shift was seen with the Hoxc-8 probe.
- Hoxc-8 was expressed in prevertebrae 13-18 (T6-T11) but, in homozygous mutant embryos, Hoxc-8 was expressed in prevertebrae 14-22 (T7-T15).
- Hoxc-11 expression was also shifted posteriorly in that the anterior boundary of expression changed from prevertebrae 28 tin wild-type embryos to prevertebrae 36 in mutant embryos.
- GDF-11 acts early during embryogenesis as a global regulator of axial patterning.
- GDF-1 1 acts early during embryogenesis as a global regulator of axial patterning.
- GDF-11 acts early during embryogenesis as a global regulator of axial patterning.
- the phenotype of GDF-11 knockout mice in several respects resembles the phenotype of mice carrying a deletion of a receptor for some members of the TGF- ⁇ superfamily, the activin type IIB receptor (ActRIIB).
- ActRIIB activin type IIB receptor
- the ActRHB knockout mice have extra pairs of ribs and a spectrum of kidney defects ranging from hypoplastic kidneys to complete absence of kidneys.
- the similarity in the phenotypes of these mice raises the possibility that ActRIIB may be a receptor for GDF-11.
- Act RJJB may not be the sole receptor for GDF-11 because the phenotype of GDF-11 knockout mice is more severe than the phenotype of ActRIIB mice.
- the ActRIIB knockout animals have only 3 extra pairs of ribs and do not show transformations at other axial levels.
- the data indicate that the kidney defects in the GDF-11 knockout mice are also more severe than those in ActRIIB knockout mice.
- the ActRHB knockout mice show defects in left/right axis formation, such as lung isomerixm and a range of heart defects that we have not yet observed in GDF- 11 knockout mice.
- ActRIIB can bind the activins and certain BMPs, although none of the knockout mice generated for these ligands show defects in left/right axis formation.
- GDF-11 does act directly on mesodermal cells to establish positional identity, the data presented here would be consistent with either short range or morphogen models for GDF-11 action. That is, GDF-11 may act on mesodermal precursors to establish patterns of Hox gene expression as these cells are being generated at the site of GDF-11 expression, or alternatively, GDF-11 produced at the posterior end of the embryo may diffuse to form a morphogen gradient. Whatever the mechanism of action of GDF-11 may be, the fact that gross anterior/posterior patterning still does occur in GDF-11 knockout animals suggests that GDF-11 may not be the sole regulator of anterior/posterior specification. Nevertheless, it is clear that GDF-11 plays an important role as a global regulator of axial patterning and that further study of this molecule will lead to important new insights into how positional identity along the anterior/posterior axis is established in the vertebrate embryo.
- GDF-8 knockout animals Similar phenotypes are expected in GDF-8 knockout animals.
- GDF-8 knockout animals are expected to have increased number of ribs, kidney defects and anatomical differences when compared to wild-type.
- TGF ⁇ signaling Receptors, transducers, and Mad proteins. Cell 85: 947-950.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Environmental Sciences (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/485,046 US6696260B1 (en) | 1997-08-01 | 1997-08-01 | Methods to identify growth differentiation factor (GDF) binding proteins |
AU86663/98A AU8666398A (en) | 1997-08-01 | 1998-07-28 | Methods to identify growth differentiation factor (gdf) receptors |
US09/841,730 US6891082B2 (en) | 1997-08-01 | 2001-04-24 | Transgenic non-human animals expressing a truncated activintype II receptor |
US11/051,267 US20050257278A1 (en) | 1997-08-01 | 2005-02-03 | Transgenic non-human animals expressing a truncated activin type II receptor |
US12/360,093 US20090186806A1 (en) | 1997-08-01 | 2009-01-26 | Truncated Activin Type II Receptor and Methods of Use |
US12/361,512 US8124830B2 (en) | 1997-08-01 | 2009-01-28 | Transgenic non-human animals expressing a truncated activin type II receptor |
US12/861,738 US8822411B2 (en) | 1997-08-01 | 2010-08-23 | Truncated activin type II receptor and methods of use |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US5446197P | 1997-08-01 | 1997-08-01 | |
US60/054,461 | 1997-08-01 |
Related Child Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/485,046 A-371-Of-International US6696260B1 (en) | 1997-08-01 | 1997-08-01 | Methods to identify growth differentiation factor (GDF) binding proteins |
US09485046 A-371-Of-International | 1998-07-28 | ||
US09/626,896 Continuation-In-Part US6656475B1 (en) | 1997-08-01 | 2000-07-27 | Growth differentiation factor receptors, agonists and antagonists thereof, and methods of using same |
US10/456,852 Division US20040077053A1 (en) | 1997-08-01 | 2003-06-06 | Methods to identify growth differentiation factor (GDF) receptors |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999006559A1 true WO1999006559A1 (fr) | 1999-02-11 |
Family
ID=21991236
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1998/015598 WO1999006559A1 (fr) | 1997-08-01 | 1998-07-28 | Procedes d'identification des recepteurs des facteurs de differenciation de la croissance |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU8666398A (fr) |
WO (1) | WO1999006559A1 (fr) |
Cited By (51)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2333706A (en) * | 1998-02-02 | 1999-08-04 | Merck & Co Inc | Method for increasing muscle mass in animals |
WO2000043781A2 (fr) * | 1999-01-21 | 2000-07-27 | Metamorphix, Inc. | Inhibiteurs de facteurs de differenciation de la croissance et leurs utilisations |
US6103466A (en) * | 1997-07-14 | 2000-08-15 | University Of Liege | Double-muscling in mammals |
US6369201B1 (en) | 1998-02-19 | 2002-04-09 | Metamorphix International, Inc. | Myostatin multimers |
JP2002513768A (ja) * | 1998-05-06 | 2002-05-14 | メタモーフイクス・インコーポレーテツド | Gdf−8の阻害による糖尿病の処置法 |
US6656475B1 (en) | 1997-08-01 | 2003-12-02 | The Johns Hopkins University School Of Medicine | Growth differentiation factor receptors, agonists and antagonists thereof, and methods of using same |
US6696260B1 (en) | 1997-08-01 | 2004-02-24 | The Johns Hopkins University School Of Medicine | Methods to identify growth differentiation factor (GDF) binding proteins |
US6891082B2 (en) | 1997-08-01 | 2005-05-10 | The Johns Hopkins University School Of Medicine | Transgenic non-human animals expressing a truncated activintype II receptor |
EP1593689A2 (fr) * | 2000-01-18 | 2005-11-09 | Ovita Limited | myostatine et mimetiques de ces derniers |
US7037501B2 (en) | 2001-01-04 | 2006-05-02 | Regents Of The University Of Minnesota | Myostatin immnoconjugate |
WO2006083182A1 (fr) * | 2005-02-07 | 2006-08-10 | Orico Limited | Utilisation d'antagonistes de myostatine (gdf-8) pour ameliorer la cicatrisation et prevenir les maladies fibreuses |
WO2006107611A2 (fr) * | 2005-03-23 | 2006-10-12 | Wyeth | Detection d'une reponse immunitaire contre les agents de modulation gdf-8 |
US7384753B2 (en) | 1994-07-08 | 2008-06-10 | The Johns Hopkins University School Of Medicine | Growth differentiation factor-11 |
US7393682B1 (en) | 1993-03-19 | 2008-07-01 | The Johns Hopkins University School Of Medicine | Polynucleotides encoding promyostatin polypeptides |
US7566768B1 (en) | 1995-10-26 | 2009-07-28 | The Johns Hopkins University School Of Medicine | Promyostatin peptides and methods of using same |
US7632499B2 (en) | 2005-10-12 | 2009-12-15 | Eli Lilly And Company | Anti-myostatin antibodies |
US7635760B2 (en) | 2005-10-06 | 2009-12-22 | Eli Lilly And Company | Anti-myostatin antibodies |
US7655763B2 (en) | 2002-10-22 | 2010-02-02 | Wyeth | Neutralizing antibodies against GDF-8 and uses therefor |
US7709605B2 (en) | 2004-07-23 | 2010-05-04 | Acceleron Pharma Inc. | ActRII receptor polypeptides, methods and compositions |
US7731961B1 (en) | 2001-09-26 | 2010-06-08 | Wyeth | Methods of increasing muscle mass or muscle strength using antibody inhibitors of GDF-8 |
US7737116B2 (en) | 2001-02-08 | 2010-06-15 | Wyeth | Modified and stabilized GDF propeptides and uses thereof |
US7785587B2 (en) | 2003-06-02 | 2010-08-31 | Wyeth | Therapeutic methods for muscular or neuromuscular disorders |
US7842663B2 (en) | 2007-02-02 | 2010-11-30 | Acceleron Pharma Inc. | Variants derived from ActRIIB and uses therefor |
US7888486B2 (en) | 2005-08-19 | 2011-02-15 | Wyeth Llc | Antagonist antibodies against GDF-8 |
US7951771B2 (en) | 2005-11-23 | 2011-05-31 | Acceleron Pharma Inc. | Activin-ActRIIa antagonists and uses for promoting bone growth |
US7960343B2 (en) | 2007-09-18 | 2011-06-14 | Acceleron Pharma Inc. | Activin-ActRIIa antagonists and uses for decreasing or inhibiting FSH secretion |
US7988973B2 (en) | 2006-12-18 | 2011-08-02 | Acceleron Pharma Inc. | Activin-ActRII antagonists and uses for increasing red blood cell levels |
US8058229B2 (en) | 2008-08-14 | 2011-11-15 | Acceleron Pharma Inc. | Method of increasing red blood cell levels or treating anemia in a patient |
US8063188B2 (en) | 2006-09-05 | 2011-11-22 | Eli Lilly And Company | Anti-myostatin antibodies |
US8128933B2 (en) | 2005-11-23 | 2012-03-06 | Acceleron Pharma, Inc. | Method of promoting bone growth by an anti-activin B antibody |
US8138142B2 (en) | 2009-01-13 | 2012-03-20 | Acceleron Pharma Inc. | Methods for increasing adiponectin in a patient in need thereof |
US8173601B2 (en) | 2007-02-09 | 2012-05-08 | Acceleron Pharma, Inc. | Activin-ActRIIa antagonists and uses for treating multiple myeloma |
US8178488B2 (en) | 2009-06-08 | 2012-05-15 | Acceleron Pharma, Inc. | Methods for increasing thermogenic adipocytes |
US8216997B2 (en) | 2008-08-14 | 2012-07-10 | Acceleron Pharma, Inc. | Methods for increasing red blood cell levels and treating anemia using a combination of GDF traps and erythropoietin receptor activators |
US8293881B2 (en) | 2009-06-12 | 2012-10-23 | Acceleron Pharma Inc. | Isolated nucleic acid encoding a truncated ActRIIB fusion protein |
US8388968B2 (en) | 2009-04-27 | 2013-03-05 | Novartis Ag | Compositions and methods for increasing muscle growth |
US8895016B2 (en) | 2006-12-18 | 2014-11-25 | Acceleron Pharma, Inc. | Antagonists of activin-actriia and uses for increasing red blood cell levels |
US8992913B2 (en) | 2012-06-15 | 2015-03-31 | Pfizer Inc. | Antagonist antibodies against GDF-8 and uses therefor |
US9493556B2 (en) | 2010-11-08 | 2016-11-15 | Acceleron Pharma Inc. | Actriia binding agents and uses thereof |
US9526759B2 (en) | 2007-02-01 | 2016-12-27 | Acceleron Pharma Inc. | Activin-actriia antagonists and uses for treating or preventing breast cancer |
US9617319B2 (en) | 2009-11-17 | 2017-04-11 | Acceleron Pharma Inc. | ActRIIB proteins and variants and uses therefore relating to utrophin induction for muscular dystrophy therapy |
US9850298B2 (en) | 2014-06-13 | 2017-12-26 | Acceleron Pharma Inc. | Methods for treating ulcers in thalassemia syndrome with an ActRIIB polypeptide |
US9919030B2 (en) | 2008-06-26 | 2018-03-20 | Acceleron Pharma Inc. | Follistatin fusion proteins and uses thereof |
US9975934B2 (en) | 2015-03-26 | 2018-05-22 | Acceleron Pharma Inc. | Follistatin-related fusion proteins |
US10010498B2 (en) | 2014-06-04 | 2018-07-03 | Acceleron Pharma Inc. | Methods for treatment of amyotrophic lateral sclerosis with follistatin fusion proteins |
US10023621B2 (en) | 2014-06-04 | 2018-07-17 | Acceleron Pharma Inc. | Follistatin-related fusion proteins |
US10195249B2 (en) | 2012-11-02 | 2019-02-05 | Celgene Corporation | Activin-ActRII antagonists and uses for treating bone and other disorders |
US10307455B2 (en) | 2016-03-10 | 2019-06-04 | Acceleron Pharma Inc. | Activin type 2 receptor antibodies |
RU2708170C2 (ru) * | 2014-11-24 | 2019-12-04 | Сомалоджик, Инк. | Соединения нуклеиновой кислоты для связывания ростового фактора дифференцировки 11 |
US11471510B2 (en) | 2014-12-03 | 2022-10-18 | Celgene Corporation | Activin-ActRII antagonists and uses for treating anemia |
US11813308B2 (en) | 2014-10-09 | 2023-11-14 | Celgene Corporation | Treatment of cardiovascular disease using ActRII ligand traps |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5639638A (en) * | 1993-05-12 | 1997-06-17 | Genetics Institute, Inc. | DNA molecules encoding bone morpogenetic protein-11 |
-
1998
- 1998-07-28 WO PCT/US1998/015598 patent/WO1999006559A1/fr active Application Filing
- 1998-07-28 AU AU86663/98A patent/AU8666398A/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5639638A (en) * | 1993-05-12 | 1997-06-17 | Genetics Institute, Inc. | DNA molecules encoding bone morpogenetic protein-11 |
Non-Patent Citations (3)
Title |
---|
BOUIZAR Z., ET AL.: "PURIFICATION AND CHARACTERIZATION OF CALCITONIN RECEPTORS IN RAT KIDNEY MEMBRANES BY COVALENT CROSS-LINKING TECHNIQUES.", EUROPEAN JOURNAL OF BIOCHEMISTRY, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 155., 1 January 1986 (1986-01-01), GB, pages 141 - 147., XP002913509, ISSN: 0014-2956, DOI: 10.1111/j.1432-1033.1986.tb09469.x * |
HANNON K., ET AL.: "DIFFERENTIALLY EXPRESSED FIBROBLAST GROWTH FACTORS REGULATE SKELETAL MUSCLE DEVELOPMENT THROUGH AUTOCRINE AND PARACRINE MECHANISMS.", THE JOURNAL OF CELL BIOLOGY : JCB, THE ROCKEFELLER UNIVERSITY PRESS, US, vol. 132., no. 06., 1 March 1996 (1996-03-01), US, pages 1151 - 1159., XP002913507, ISSN: 0021-9525, DOI: 10.1083/jcb.132.6.1151 * |
MCPHERRON A. C., LAWLER A. M., LEE S.-I.: "REGULATION OF SKELETAL MUSCLE MASS IN MICE BY A NEW TGF-BETA SUPERFAMILY MEMBER.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 387., no. 6628., 1 May 1997 (1997-05-01), United Kingdom, pages 83 - 90., XP002913508, ISSN: 0028-0836, DOI: 10.1038/387083a0 * |
Cited By (125)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8323964B2 (en) | 1993-03-19 | 2012-12-04 | The John Hopkins University School Of Medicine | Polynucleotides encoding promyostatin polypeptides |
US7393682B1 (en) | 1993-03-19 | 2008-07-01 | The Johns Hopkins University School Of Medicine | Polynucleotides encoding promyostatin polypeptides |
US7976839B2 (en) | 1994-07-08 | 2011-07-12 | Johns Hopkins University School Of Medicine | Growth differentiation factor-11 |
US7384753B2 (en) | 1994-07-08 | 2008-06-10 | The Johns Hopkins University School Of Medicine | Growth differentiation factor-11 |
US7566768B1 (en) | 1995-10-26 | 2009-07-28 | The Johns Hopkins University School Of Medicine | Promyostatin peptides and methods of using same |
US6103466A (en) * | 1997-07-14 | 2000-08-15 | University Of Liege | Double-muscling in mammals |
US6891082B2 (en) | 1997-08-01 | 2005-05-10 | The Johns Hopkins University School Of Medicine | Transgenic non-human animals expressing a truncated activintype II receptor |
US8124830B2 (en) | 1997-08-01 | 2012-02-28 | The Johns Hopkins University School Of Medicine | Transgenic non-human animals expressing a truncated activin type II receptor |
US6696260B1 (en) | 1997-08-01 | 2004-02-24 | The Johns Hopkins University School Of Medicine | Methods to identify growth differentiation factor (GDF) binding proteins |
US6656475B1 (en) | 1997-08-01 | 2003-12-02 | The Johns Hopkins University School Of Medicine | Growth differentiation factor receptors, agonists and antagonists thereof, and methods of using same |
US8822411B2 (en) | 1997-08-01 | 2014-09-02 | The Johns Hopkins University School Of Medicine | Truncated activin type II receptor and methods of use |
GB2333706A (en) * | 1998-02-02 | 1999-08-04 | Merck & Co Inc | Method for increasing muscle mass in animals |
US6369201B1 (en) | 1998-02-19 | 2002-04-09 | Metamorphix International, Inc. | Myostatin multimers |
JP2002513768A (ja) * | 1998-05-06 | 2002-05-14 | メタモーフイクス・インコーポレーテツド | Gdf−8の阻害による糖尿病の処置法 |
WO2000043781A3 (fr) * | 1999-01-21 | 2001-02-01 | Metamorphix Inc | Inhibiteurs de facteurs de differenciation de la croissance et leurs utilisations |
WO2000043781A2 (fr) * | 1999-01-21 | 2000-07-27 | Metamorphix, Inc. | Inhibiteurs de facteurs de differenciation de la croissance et leurs utilisations |
EP1593689A3 (fr) * | 2000-01-18 | 2006-04-05 | Ovita Limited | Myostatine et mimétiques de ces derniers |
US7368534B2 (en) | 2000-01-18 | 2008-05-06 | Orico Limited | Myostatin and mimetics thereof |
EP1593689A2 (fr) * | 2000-01-18 | 2005-11-09 | Ovita Limited | myostatine et mimetiques de ces derniers |
CN100422212C (zh) * | 2000-07-27 | 2008-10-01 | 约翰斯·霍普金斯大学医学院 | 生长分化因子受体、其激动剂和拮抗剂及其使用方法 |
EP1317485B1 (fr) * | 2000-07-27 | 2011-03-30 | The Johns Hopkins University School Of Medicine | Antagonistes de la myostatine et leurs utilisations |
EP2322199A1 (fr) | 2000-07-27 | 2011-05-18 | The Johns Hopkins University School Of Medicine | Récepteurs de facteur de différentiation de la croissance et procédés d'utilisation correspondants |
US7037501B2 (en) | 2001-01-04 | 2006-05-02 | Regents Of The University Of Minnesota | Myostatin immnoconjugate |
US7488480B2 (en) | 2001-01-04 | 2009-02-10 | Regents Of The University Of Minnesota | Use of passive myostatin immunization in egg laying vertebrates |
US8710025B2 (en) | 2001-02-08 | 2014-04-29 | Wyeth Llc | Modified and stabilized GDF propeptides and uses thereof |
US7737116B2 (en) | 2001-02-08 | 2010-06-15 | Wyeth | Modified and stabilized GDF propeptides and uses thereof |
US7731961B1 (en) | 2001-09-26 | 2010-06-08 | Wyeth | Methods of increasing muscle mass or muscle strength using antibody inhibitors of GDF-8 |
US8710202B2 (en) | 2001-09-26 | 2014-04-29 | Wyeth Llc | Isolated nucleic acid molecule encoding an antibody that reduces GDF-8 activity |
US8092798B2 (en) | 2001-09-26 | 2012-01-10 | Wyeth Llc | Method of increasing trabecular bone density in a patient in need thereof by an antibody against GDF-8 |
US9505831B2 (en) | 2001-09-26 | 2016-11-29 | Wyeth Llc | Isolated cell comprising a nucleic acid encoding antibody inhibitors of gdf-8 and uses thereof |
US7655763B2 (en) | 2002-10-22 | 2010-02-02 | Wyeth | Neutralizing antibodies against GDF-8 and uses therefor |
US8940874B2 (en) | 2002-10-22 | 2015-01-27 | Wyeth Llc | Neutralizing antibodies against GDF-8 and uses therefor |
US8420082B2 (en) | 2002-10-22 | 2013-04-16 | Wyeth Llc | Neutralizing antibodies against GDF-8 and uses therefor |
US7785587B2 (en) | 2003-06-02 | 2010-08-31 | Wyeth | Therapeutic methods for muscular or neuromuscular disorders |
US9138459B2 (en) | 2004-07-23 | 2015-09-22 | Acceleron Pharma Inc. | ACTRIIB-FC polynucleotides, polypeptides, and compositions |
US8252900B2 (en) | 2004-07-23 | 2012-08-28 | Acceleron Pharma Inc. | Actriib-Fc polynucleotides, polypeptides, and compositions |
US7709605B2 (en) | 2004-07-23 | 2010-05-04 | Acceleron Pharma Inc. | ActRII receptor polypeptides, methods and compositions |
WO2006083182A1 (fr) * | 2005-02-07 | 2006-08-10 | Orico Limited | Utilisation d'antagonistes de myostatine (gdf-8) pour ameliorer la cicatrisation et prevenir les maladies fibreuses |
WO2006107611A3 (fr) * | 2005-03-23 | 2007-04-26 | Wyeth Corp | Detection d'une reponse immunitaire contre les agents de modulation gdf-8 |
WO2006107611A2 (fr) * | 2005-03-23 | 2006-10-12 | Wyeth | Detection d'une reponse immunitaire contre les agents de modulation gdf-8 |
US7888486B2 (en) | 2005-08-19 | 2011-02-15 | Wyeth Llc | Antagonist antibodies against GDF-8 |
US9926368B2 (en) | 2005-08-19 | 2018-03-27 | Wyeth Llc | Method for treating muscle wasting syndrome using antagonist antibodies against GDF-8 |
US8956608B2 (en) | 2005-08-19 | 2015-02-17 | Wyeth Llc | Method for treating sarcopenia using antagonist antibodies against GDF-8 |
US7910107B2 (en) | 2005-08-19 | 2011-03-22 | Wyeth Llc | Antagonist antibodies against GDF-8 and uses in treatment of ALS and other GDF-8 associated disorders |
US8349327B2 (en) | 2005-08-19 | 2013-01-08 | Wyeth Llc | Method for treating muscular dystrophy using antagonist antibodies against GDF-8 |
US8372625B2 (en) | 2005-08-19 | 2013-02-12 | Wyeth Llc | Polynucleotides encoding antagonist antibodies against GDF-8 |
US7745583B2 (en) | 2005-10-06 | 2010-06-29 | Eli Lilly And Company | Anti-myostatin antibodies |
US7635760B2 (en) | 2005-10-06 | 2009-12-22 | Eli Lilly And Company | Anti-myostatin antibodies |
US8066995B2 (en) | 2005-10-12 | 2011-11-29 | Eli Lilly And Company | Anti-myostatin antibodies |
US7632499B2 (en) | 2005-10-12 | 2009-12-15 | Eli Lilly And Company | Anti-myostatin antibodies |
US8629109B2 (en) | 2005-11-23 | 2014-01-14 | Acceleron Pharma Inc. | Method for promoting bone growth using activin-actriia antagonists |
US8486403B2 (en) | 2005-11-23 | 2013-07-16 | Acceleron Pharma, Inc. | Method of promoting bone growth by an anti-activin A antibody |
US11129873B2 (en) | 2005-11-23 | 2021-09-28 | Acceleron Pharma Inc. | Method for promoting bone growth using activin-actriia antagonists |
US7951771B2 (en) | 2005-11-23 | 2011-05-31 | Acceleron Pharma Inc. | Activin-ActRIIa antagonists and uses for promoting bone growth |
US9163075B2 (en) | 2005-11-23 | 2015-10-20 | Acceleron Pharma Inc. | Isolated polynucleotide that encodes an ActRIIa-Fc fusion polypeptide |
US10239940B2 (en) | 2005-11-23 | 2019-03-26 | Acceleron Pharma Inc. | Method of promoting bone growth by an anti-actriia antibody |
US9480742B2 (en) | 2005-11-23 | 2016-11-01 | Acceleron Pharma Inc. | Method of promoting bone growth by an anti-actriia antibody |
US9572865B2 (en) | 2005-11-23 | 2017-02-21 | Acceleron Pharma Inc. | Activin-actriia antagonists and uses for treating multiple myeloma |
US8067360B2 (en) | 2005-11-23 | 2011-11-29 | Acceleron Pharma Inc. | Method for promoting bone growth using activin-ActRIIa antagonists |
US10071135B2 (en) | 2005-11-23 | 2018-09-11 | Acceleron Pharma Inc. | Method of identifying an agent that promotes bone growth or increases bone density |
US8128933B2 (en) | 2005-11-23 | 2012-03-06 | Acceleron Pharma, Inc. | Method of promoting bone growth by an anti-activin B antibody |
US8063188B2 (en) | 2006-09-05 | 2011-11-22 | Eli Lilly And Company | Anti-myostatin antibodies |
US8007809B2 (en) | 2006-12-18 | 2011-08-30 | Acceleron Pharma Inc. | Activin-actrii antagonists and uses for increasing red blood cell levels |
US8895016B2 (en) | 2006-12-18 | 2014-11-25 | Acceleron Pharma, Inc. | Antagonists of activin-actriia and uses for increasing red blood cell levels |
US10093707B2 (en) | 2006-12-18 | 2018-10-09 | Acceleron Pharma Inc. | Antagonists of activin-ActRIIa and uses for increasing red blood cell levels |
US7988973B2 (en) | 2006-12-18 | 2011-08-02 | Acceleron Pharma Inc. | Activin-ActRII antagonists and uses for increasing red blood cell levels |
US9526759B2 (en) | 2007-02-01 | 2016-12-27 | Acceleron Pharma Inc. | Activin-actriia antagonists and uses for treating or preventing breast cancer |
US7842663B2 (en) | 2007-02-02 | 2010-11-30 | Acceleron Pharma Inc. | Variants derived from ActRIIB and uses therefor |
US10259861B2 (en) | 2007-02-02 | 2019-04-16 | Acceleron Pharma Inc. | Variants derived from ActRIIB and uses therefor |
US8343933B2 (en) | 2007-02-02 | 2013-01-01 | Acceleron Pharma, Inc. | Variants derived from ActRIIB and uses therefor |
US9399669B2 (en) | 2007-02-02 | 2016-07-26 | Acceleron Pharma Inc. | Variants derived from ActRIIB |
US8173601B2 (en) | 2007-02-09 | 2012-05-08 | Acceleron Pharma, Inc. | Activin-ActRIIa antagonists and uses for treating multiple myeloma |
US8367611B2 (en) | 2007-09-18 | 2013-02-05 | Acceleron Pharma Inc. | Activin-actriia antagonists for inhibiting germ cell maturation |
US7960343B2 (en) | 2007-09-18 | 2011-06-14 | Acceleron Pharma Inc. | Activin-ActRIIa antagonists and uses for decreasing or inhibiting FSH secretion |
US9353356B2 (en) | 2007-09-18 | 2016-05-31 | Acceleron Pharma Inc. | Activin-actriia antagonists for treating a follicle-stimulating horomone-secreting pituitary tumor |
US9919030B2 (en) | 2008-06-26 | 2018-03-20 | Acceleron Pharma Inc. | Follistatin fusion proteins and uses thereof |
US8216997B2 (en) | 2008-08-14 | 2012-07-10 | Acceleron Pharma, Inc. | Methods for increasing red blood cell levels and treating anemia using a combination of GDF traps and erythropoietin receptor activators |
US10829532B2 (en) | 2008-08-14 | 2020-11-10 | Acceleron Pharma Inc. | Combined use of gdf traps and erythropoietin receptor activators to increase red blood cell levels |
US9505813B2 (en) | 2008-08-14 | 2016-11-29 | Acceleron Pharma Inc. | Use of GDF traps to treat anemia |
US11162085B2 (en) | 2008-08-14 | 2021-11-02 | Acceleron Pharma Inc. | Methods for treating anemia in a subject in need thereof |
US11155791B2 (en) | 2008-08-14 | 2021-10-26 | Acceleron Pharma Inc. | Methods for treating anemia in a subject in need thereof |
US8058229B2 (en) | 2008-08-14 | 2011-11-15 | Acceleron Pharma Inc. | Method of increasing red blood cell levels or treating anemia in a patient |
US11168311B2 (en) | 2008-08-14 | 2021-11-09 | Acceleron Pharma Inc. | Methods for treating anemia in a subject in need thereof |
US10889626B2 (en) | 2008-08-14 | 2021-01-12 | Acceleron Pharma Inc. | Combined use of GDF traps and erythropoietin receptor activators to increase red blood cell levels |
US8361957B2 (en) | 2008-08-14 | 2013-01-29 | Acceleron Pharma, Inc. | Isolated GDF trap polypeptide |
US10829533B2 (en) | 2008-08-14 | 2020-11-10 | Acceleron Pharma Inc. | Combined use of GDF traps and erythropoietin receptor activators to increase red blood cell levels |
US10689427B2 (en) | 2008-08-14 | 2020-06-23 | Acceleron Pharma Inc. | Combined use of GDF traps and erythropoietin receptor activators to increase red blood cell levels |
US10377996B2 (en) | 2008-08-14 | 2019-08-13 | Acceleron Pharma Inc. | Methods of identifying ActRIIB variants |
US9439945B2 (en) | 2008-08-14 | 2016-09-13 | Acceleron Pharma Inc. | Isolated nucleotide sequences encoding GDF traps |
US9932379B2 (en) | 2008-08-14 | 2018-04-03 | Acceleron Pharma Inc. | Isolated nucleotide sequences encoding GDF traps |
US8138142B2 (en) | 2009-01-13 | 2012-03-20 | Acceleron Pharma Inc. | Methods for increasing adiponectin in a patient in need thereof |
US8551482B2 (en) | 2009-04-27 | 2013-10-08 | Novartis Ag | Compositions and methods for increasing muscle growth |
US8388968B2 (en) | 2009-04-27 | 2013-03-05 | Novartis Ag | Compositions and methods for increasing muscle growth |
US8178488B2 (en) | 2009-06-08 | 2012-05-15 | Acceleron Pharma, Inc. | Methods for increasing thermogenic adipocytes |
US10968282B2 (en) | 2009-06-08 | 2021-04-06 | Acceleron Pharma Inc. | Methods for screening compounds for increasing thermogenic adipocytes |
US9790284B2 (en) | 2009-06-08 | 2017-10-17 | Acceleron Pharma Inc. | Methods for increasing thermogenic adipocytes |
US9745559B2 (en) | 2009-06-12 | 2017-08-29 | Acceleron Pharma Inc. | Method for decreasing the body fat content in a subject by administering an ActRIIB protein |
US9181533B2 (en) | 2009-06-12 | 2015-11-10 | Acceleron Pharma, Inc. | Truncated ACTRIIB-FC fusion protein |
US10358633B2 (en) | 2009-06-12 | 2019-07-23 | Acceleron Pharma Inc. | Method for producing an ActRIIB-Fc fusion polypeptide |
US8293881B2 (en) | 2009-06-12 | 2012-10-23 | Acceleron Pharma Inc. | Isolated nucleic acid encoding a truncated ActRIIB fusion protein |
US11066654B2 (en) | 2009-06-12 | 2021-07-20 | Acceleron Pharma Inc. | Methods and compositions for reducing serum lipids |
US10968262B2 (en) | 2009-11-17 | 2021-04-06 | Acceleron Pharma Inc. | Methods of increasing sarcolemmal utrophin |
US9617319B2 (en) | 2009-11-17 | 2017-04-11 | Acceleron Pharma Inc. | ActRIIB proteins and variants and uses therefore relating to utrophin induction for muscular dystrophy therapy |
US9493556B2 (en) | 2010-11-08 | 2016-11-15 | Acceleron Pharma Inc. | Actriia binding agents and uses thereof |
US8992913B2 (en) | 2012-06-15 | 2015-03-31 | Pfizer Inc. | Antagonist antibodies against GDF-8 and uses therefor |
US9751937B2 (en) | 2012-06-15 | 2017-09-05 | Pfizer Inc. | Antagonist antibodies against GDF-8 and uses therefor |
US10195249B2 (en) | 2012-11-02 | 2019-02-05 | Celgene Corporation | Activin-ActRII antagonists and uses for treating bone and other disorders |
US11497792B2 (en) | 2014-06-04 | 2022-11-15 | Acceleron Pharma Inc. | Methods for treatment of Duchenne muscular dystrophy with follistatin polypeptides |
US10023621B2 (en) | 2014-06-04 | 2018-07-17 | Acceleron Pharma Inc. | Follistatin-related fusion proteins |
US10954279B2 (en) | 2014-06-04 | 2021-03-23 | Acceleron Pharma Inc. | Methods and compositions for treatment of disorders with follistatin polypeptides |
US10010498B2 (en) | 2014-06-04 | 2018-07-03 | Acceleron Pharma Inc. | Methods for treatment of amyotrophic lateral sclerosis with follistatin fusion proteins |
US10765626B2 (en) | 2014-06-04 | 2020-09-08 | Acceleron Pharma Inc. | Methods for treatment of charcot-marie-tooth disease with follistatin polypeptides |
US11260107B2 (en) | 2014-06-13 | 2022-03-01 | Acceleron Pharma Inc. | Methods and compositions for treating ulcers |
US10487144B2 (en) | 2014-06-13 | 2019-11-26 | Acceleron Pharma Inc. | Methods for treating ulcers in a hemoglobinopathy anemia with a soluble actRIIB polypeptide |
US9850298B2 (en) | 2014-06-13 | 2017-12-26 | Acceleron Pharma Inc. | Methods for treating ulcers in thalassemia syndrome with an ActRIIB polypeptide |
US11813308B2 (en) | 2014-10-09 | 2023-11-14 | Celgene Corporation | Treatment of cardiovascular disease using ActRII ligand traps |
RU2708170C2 (ru) * | 2014-11-24 | 2019-12-04 | Сомалоджик, Инк. | Соединения нуклеиновой кислоты для связывания ростового фактора дифференцировки 11 |
US10808251B2 (en) | 2014-11-24 | 2020-10-20 | Somalogic, Inc. | Nucleic acid compounds for binding growth differentiation factor 11 |
US11535852B2 (en) | 2014-11-24 | 2022-12-27 | Somalogic Operating Co., Inc. | Nucleic acid compounds for binding growth differentiation factor 11 |
US11471510B2 (en) | 2014-12-03 | 2022-10-18 | Celgene Corporation | Activin-ActRII antagonists and uses for treating anemia |
US11001614B2 (en) | 2015-03-26 | 2021-05-11 | Acceleron Pharma Inc. | Method for treating a muscle-related disorder with follistatin-related fusion proteins |
US9975934B2 (en) | 2015-03-26 | 2018-05-22 | Acceleron Pharma Inc. | Follistatin-related fusion proteins |
US11000565B2 (en) | 2016-03-10 | 2021-05-11 | Acceleron Pharma Inc. | Methods of increasing muscle mass by administration of activin type 2 receptor antibodies |
US10307455B2 (en) | 2016-03-10 | 2019-06-04 | Acceleron Pharma Inc. | Activin type 2 receptor antibodies |
US12042524B2 (en) | 2016-03-10 | 2024-07-23 | Acceleron Pharma Inc. | Activin type 2 receptor binding proteins methods of making them |
Also Published As
Publication number | Publication date |
---|---|
AU8666398A (en) | 1999-02-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6696260B1 (en) | Methods to identify growth differentiation factor (GDF) binding proteins | |
WO1999006559A1 (fr) | Procedes d'identification des recepteurs des facteurs de differenciation de la croissance | |
US7976839B2 (en) | Growth differentiation factor-11 | |
US7399848B2 (en) | Polynucleotides encoding growth differentiation factor-8 | |
AU772694B2 (en) | Growth differentiation factor inhibitors and uses therefor | |
US6607884B1 (en) | Methods of detecting growth differentiation factor-8 | |
JP3786909B2 (ja) | ポリペプチド及び同じものをコードしている核酸 | |
US20080213426A1 (en) | Growth Differentiation Factor-8 | |
US20020150577A1 (en) | Use of antibodies specific for growth differentiation factor-11 | |
KR20180019521A (ko) | 인간화된 sirpa-il15 녹인 마우스 및 이의 이용 방법 | |
WO1998033887A1 (fr) | Facteur-8 de differenciation de croissance | |
US6517835B2 (en) | Growth differentiation factor-11 | |
JP2004504832A (ja) | 増殖分化因子受容体、そのアゴニスト、およびアンタゴニスト、ならびにそれらの使用方法 | |
US8236751B2 (en) | Methods of increasing muscle mass using follistatin-like related gene (FLRG) | |
CA2807478A1 (fr) | Utilisation d'expression specifique aux muscles de gene de recepteur d'activine de type ii pour augmenter la masse musculaire chez un animal non humain | |
WO1999040181A1 (fr) | Facteur-8 de differenciation de la croissance | |
AU726918B2 (en) | TGFbeta signal transduction proteins, genes, and uses related thereto | |
AU765832B2 (en) | Growth differentiation factor-11 | |
US6210964B1 (en) | Avian extracellular calcium-sensing receptor | |
JP2001505420A (ja) | 肝臓アクチビン/インヒビンのヌクレオチド配列およびタンパク質配列ならびにそれらに基づく方法 | |
AU2003262440A1 (en) | Growth differentiation factor-11 | |
Superfamily | A aturally Occurring Growth Hormone Receptor Mutation: in Vivo |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
NENP | Non-entry into the national phase |
Ref country code: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09485046 Country of ref document: US |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: CA |