WO1999001468A2 - Gene smoothened de vertebre, produits geniques et utilisations associees - Google Patents
Gene smoothened de vertebre, produits geniques et utilisations associees Download PDFInfo
- Publication number
- WO1999001468A2 WO1999001468A2 PCT/US1998/013793 US9813793W WO9901468A2 WO 1999001468 A2 WO1999001468 A2 WO 1999001468A2 US 9813793 W US9813793 W US 9813793W WO 9901468 A2 WO9901468 A2 WO 9901468A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- smoothened
- gene
- polypeptide
- protein
- sequence
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 354
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 115
- 241000251539 Vertebrata <Metazoa> Species 0.000 title description 26
- 230000019491 signal transduction Effects 0.000 claims abstract description 22
- 230000001404 mediated effect Effects 0.000 claims abstract description 12
- 241000289669 Erinaceus europaeus Species 0.000 claims abstract 4
- 210000004027 cell Anatomy 0.000 claims description 269
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 238
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 222
- 229920001184 polypeptide Polymers 0.000 claims description 211
- 108010090739 Smoothened Receptor Proteins 0.000 claims description 189
- 102000013380 Smoothened Receptor Human genes 0.000 claims description 189
- 238000000034 method Methods 0.000 claims description 161
- 230000014509 gene expression Effects 0.000 claims description 122
- 150000007523 nucleic acids Chemical class 0.000 claims description 102
- 102000039446 nucleic acids Human genes 0.000 claims description 94
- 108020004707 nucleic acids Proteins 0.000 claims description 94
- 241001465754 Metazoa Species 0.000 claims description 72
- 239000002773 nucleotide Substances 0.000 claims description 57
- 125000003729 nucleotide group Chemical group 0.000 claims description 57
- 150000001875 compounds Chemical class 0.000 claims description 55
- 239000012634 fragment Substances 0.000 claims description 55
- 230000001105 regulatory effect Effects 0.000 claims description 49
- 108010069873 Patched Receptors Proteins 0.000 claims description 45
- 239000000523 sample Substances 0.000 claims description 45
- 102000000017 Patched Receptors Human genes 0.000 claims description 43
- 108700019146 Transgenes Proteins 0.000 claims description 43
- 230000004069 differentiation Effects 0.000 claims description 41
- 239000000203 mixture Substances 0.000 claims description 40
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 39
- 238000003556 assay Methods 0.000 claims description 37
- 230000009261 transgenic effect Effects 0.000 claims description 33
- 238000012360 testing method Methods 0.000 claims description 30
- 230000002103 transcriptional effect Effects 0.000 claims description 30
- 230000000692 anti-sense effect Effects 0.000 claims description 29
- 230000003993 interaction Effects 0.000 claims description 29
- 238000002360 preparation method Methods 0.000 claims description 29
- 210000002569 neuron Anatomy 0.000 claims description 27
- 239000013604 expression vector Substances 0.000 claims description 26
- 108020004999 messenger RNA Proteins 0.000 claims description 26
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 25
- 239000002299 complementary DNA Substances 0.000 claims description 23
- 239000002502 liposome Substances 0.000 claims description 23
- 230000003902 lesion Effects 0.000 claims description 21
- -1 poly-cationic nucleic acid Chemical class 0.000 claims description 20
- 238000012216 screening Methods 0.000 claims description 20
- 230000004913 activation Effects 0.000 claims description 18
- 230000001419 dependent effect Effects 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 18
- 239000003446 ligand Substances 0.000 claims description 17
- 108091026890 Coding region Proteins 0.000 claims description 16
- 230000002068 genetic effect Effects 0.000 claims description 16
- 230000035772 mutation Effects 0.000 claims description 16
- 108091034117 Oligonucleotide Proteins 0.000 claims description 14
- 238000001476 gene delivery Methods 0.000 claims description 14
- 230000002163 immunogen Effects 0.000 claims description 14
- 230000003834 intracellular effect Effects 0.000 claims description 14
- 230000004083 survival effect Effects 0.000 claims description 14
- 230000008685 targeting Effects 0.000 claims description 12
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 11
- 238000002744 homologous recombination Methods 0.000 claims description 11
- 230000006801 homologous recombination Effects 0.000 claims description 11
- 238000009396 hybridization Methods 0.000 claims description 11
- 230000035755 proliferation Effects 0.000 claims description 11
- 230000001594 aberrant effect Effects 0.000 claims description 10
- 238000012217 deletion Methods 0.000 claims description 10
- 230000037430 deletion Effects 0.000 claims description 10
- 230000008859 change Effects 0.000 claims description 9
- 238000001890 transfection Methods 0.000 claims description 9
- 108700026244 Open Reading Frames Proteins 0.000 claims description 8
- 230000004075 alteration Effects 0.000 claims description 8
- 230000003042 antagnostic effect Effects 0.000 claims description 8
- 238000004113 cell culture Methods 0.000 claims description 8
- 230000000295 complement effect Effects 0.000 claims description 8
- 238000006467 substitution reaction Methods 0.000 claims description 8
- 230000024245 cell differentiation Effects 0.000 claims description 7
- 239000000816 peptidomimetic Substances 0.000 claims description 7
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 7
- 230000011987 methylation Effects 0.000 claims description 6
- 238000007069 methylation reaction Methods 0.000 claims description 6
- 108020005029 5' Flanking Region Proteins 0.000 claims description 5
- 230000010261 cell growth Effects 0.000 claims description 5
- 230000004663 cell proliferation Effects 0.000 claims description 5
- 230000026731 phosphorylation Effects 0.000 claims description 5
- 238000006366 phosphorylation reaction Methods 0.000 claims description 5
- 230000002381 testicular Effects 0.000 claims description 5
- 230000003612 virological effect Effects 0.000 claims description 5
- 230000004988 N-glycosylation Effects 0.000 claims description 4
- 230000030833 cell death Effects 0.000 claims description 4
- 108020005065 3' Flanking Region Proteins 0.000 claims description 3
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims description 3
- 108091000080 Phosphotransferase Proteins 0.000 claims description 3
- 239000006143 cell culture medium Substances 0.000 claims description 3
- 230000008711 chromosomal rearrangement Effects 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000002853 nucleic acid probe Substances 0.000 claims description 3
- 102000020233 phosphotransferase Human genes 0.000 claims description 3
- 239000011230 binding agent Substances 0.000 claims description 2
- 210000001124 body fluid Anatomy 0.000 claims description 2
- 230000028993 immune response Effects 0.000 claims description 2
- 230000002188 osteogenic effect Effects 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 230000003362 replicative effect Effects 0.000 claims description 2
- 230000031146 intracellular signal transduction Effects 0.000 claims 1
- 102000005962 receptors Human genes 0.000 abstract description 18
- 108020003175 receptors Proteins 0.000 abstract description 18
- 230000006698 induction Effects 0.000 abstract description 16
- WYTGDNHDOZPMIW-RCBQFDQVSA-N alstonine Natural products C1=CC2=C3C=CC=CC3=NC2=C2N1C[C@H]1[C@H](C)OC=C(C(=O)OC)[C@H]1C2 WYTGDNHDOZPMIW-RCBQFDQVSA-N 0.000 abstract description 5
- 235000018102 proteins Nutrition 0.000 description 99
- 210000001519 tissue Anatomy 0.000 description 96
- 230000000694 effects Effects 0.000 description 80
- 102000053602 DNA Human genes 0.000 description 66
- 108020004414 DNA Proteins 0.000 description 66
- 244000060234 Gmelina philippensis Species 0.000 description 53
- 230000001225 therapeutic effect Effects 0.000 description 43
- 239000003814 drug Substances 0.000 description 36
- 238000011282 treatment Methods 0.000 description 33
- 239000013598 vector Substances 0.000 description 33
- 241000282414 Homo sapiens Species 0.000 description 31
- 239000000556 agonist Substances 0.000 description 31
- 238000013518 transcription Methods 0.000 description 31
- 230000035897 transcription Effects 0.000 description 31
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 30
- 210000001161 mammalian embryo Anatomy 0.000 description 29
- 238000011161 development Methods 0.000 description 28
- 230000018109 developmental process Effects 0.000 description 28
- 108700008625 Reporter Genes Proteins 0.000 description 27
- 230000011664 signaling Effects 0.000 description 27
- 108020001507 fusion proteins Proteins 0.000 description 26
- 102000037865 fusion proteins Human genes 0.000 description 26
- 230000027455 binding Effects 0.000 description 25
- 102000003693 Hedgehog Proteins Human genes 0.000 description 24
- 108090000031 Hedgehog Proteins Proteins 0.000 description 24
- 239000005557 antagonist Substances 0.000 description 23
- 208000035475 disorder Diseases 0.000 description 23
- 239000000047 product Substances 0.000 description 23
- 210000002257 embryonic structure Anatomy 0.000 description 22
- 210000000845 cartilage Anatomy 0.000 description 21
- 230000006870 function Effects 0.000 description 21
- 238000001727 in vivo Methods 0.000 description 21
- 238000000338 in vitro Methods 0.000 description 20
- 108700028369 Alleles Proteins 0.000 description 18
- 102000008130 Cyclic AMP-Dependent Protein Kinases Human genes 0.000 description 18
- 108010049894 Cyclic AMP-Dependent Protein Kinases Proteins 0.000 description 18
- 235000001014 amino acid Nutrition 0.000 description 18
- 230000001939 inductive effect Effects 0.000 description 18
- 229940024606 amino acid Drugs 0.000 description 17
- 238000002560 therapeutic procedure Methods 0.000 description 17
- 230000004071 biological effect Effects 0.000 description 16
- 238000012423 maintenance Methods 0.000 description 16
- 239000013612 plasmid Substances 0.000 description 16
- 238000000059 patterning Methods 0.000 description 15
- 238000003752 polymerase chain reaction Methods 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 14
- 150000001413 amino acids Chemical class 0.000 description 14
- 230000001413 cellular effect Effects 0.000 description 14
- 239000003795 chemical substances by application Substances 0.000 description 14
- 229940088598 enzyme Drugs 0.000 description 14
- 210000004602 germ cell Anatomy 0.000 description 14
- 238000003780 insertion Methods 0.000 description 14
- 230000037431 insertion Effects 0.000 description 14
- 229920002477 rna polymer Polymers 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 12
- 150000002632 lipids Chemical class 0.000 description 12
- 241000894007 species Species 0.000 description 12
- 229940079593 drug Drugs 0.000 description 11
- 230000006798 recombination Effects 0.000 description 11
- 238000005215 recombination Methods 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 230000014616 translation Effects 0.000 description 11
- 239000003623 enhancer Substances 0.000 description 10
- 238000003018 immunoassay Methods 0.000 description 10
- 230000002401 inhibitory effect Effects 0.000 description 10
- 239000000463 material Substances 0.000 description 10
- 210000000276 neural tube Anatomy 0.000 description 10
- 230000004044 response Effects 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 9
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 9
- 102100039556 Galectin-4 Human genes 0.000 description 9
- 241000287828 Gallus gallus Species 0.000 description 9
- 101000608765 Homo sapiens Galectin-4 Proteins 0.000 description 9
- 241000700605 Viruses Species 0.000 description 9
- 235000013330 chicken meat Nutrition 0.000 description 9
- 230000007850 degeneration Effects 0.000 description 9
- 230000012010 growth Effects 0.000 description 9
- 239000007943 implant Substances 0.000 description 9
- 239000003550 marker Substances 0.000 description 9
- 238000000520 microinjection Methods 0.000 description 9
- 210000000653 nervous system Anatomy 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000013519 translation Methods 0.000 description 9
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 8
- 239000004480 active ingredient Substances 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 238000010367 cloning Methods 0.000 description 8
- 210000002808 connective tissue Anatomy 0.000 description 8
- 230000003412 degenerative effect Effects 0.000 description 8
- 210000003414 extremity Anatomy 0.000 description 8
- 230000004927 fusion Effects 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 239000011159 matrix material Substances 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 238000012986 modification Methods 0.000 description 8
- 230000001537 neural effect Effects 0.000 description 8
- 230000003389 potentiating effect Effects 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 230000008439 repair process Effects 0.000 description 8
- 238000010561 standard procedure Methods 0.000 description 8
- 239000013603 viral vector Substances 0.000 description 8
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 7
- 108020004511 Recombinant DNA Proteins 0.000 description 7
- 238000002105 Southern blotting Methods 0.000 description 7
- 108010016200 Zinc Finger Protein GLI1 Proteins 0.000 description 7
- 238000007792 addition Methods 0.000 description 7
- 230000000890 antigenic effect Effects 0.000 description 7
- 210000001612 chondrocyte Anatomy 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 238000002513 implantation Methods 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 208000014674 injury Diseases 0.000 description 7
- 210000004185 liver Anatomy 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 210000002161 motor neuron Anatomy 0.000 description 7
- 239000003921 oil Substances 0.000 description 7
- 235000019198 oils Nutrition 0.000 description 7
- 150000003904 phospholipids Chemical class 0.000 description 7
- 229920000642 polymer Polymers 0.000 description 7
- 239000003755 preservative agent Substances 0.000 description 7
- 210000000130 stem cell Anatomy 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 241000124008 Mammalia Species 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000013459 approach Methods 0.000 description 6
- 239000011324 bead Substances 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 210000000988 bone and bone Anatomy 0.000 description 6
- 210000004556 brain Anatomy 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 230000007812 deficiency Effects 0.000 description 6
- 238000004520 electroporation Methods 0.000 description 6
- 210000001671 embryonic stem cell Anatomy 0.000 description 6
- 230000002255 enzymatic effect Effects 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 210000000982 limb bud Anatomy 0.000 description 6
- 210000003716 mesoderm Anatomy 0.000 description 6
- 229930182817 methionine Natural products 0.000 description 6
- 210000000933 neural crest Anatomy 0.000 description 6
- 210000003458 notochord Anatomy 0.000 description 6
- 239000002751 oligonucleotide probe Substances 0.000 description 6
- 210000000056 organ Anatomy 0.000 description 6
- 230000011164 ossification Effects 0.000 description 6
- 230000037361 pathway Effects 0.000 description 6
- 239000000825 pharmaceutical preparation Substances 0.000 description 6
- 235000004400 serine Nutrition 0.000 description 6
- 230000021595 spermatogenesis Effects 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 239000004094 surface-active agent Substances 0.000 description 6
- 238000010361 transduction Methods 0.000 description 6
- 230000026683 transduction Effects 0.000 description 6
- 238000012546 transfer Methods 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 6
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical group OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 5
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 5
- 108700005472 Drosophila smo Proteins 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 5
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 5
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical group C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 5
- 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 5
- 101710181812 Methionine aminopeptidase Proteins 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 108010025020 Nerve Growth Factor Proteins 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Chemical group OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 5
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Chemical group CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 5
- 239000004473 Threonine Chemical group 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 230000001270 agonistic effect Effects 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- 230000022159 cartilage development Effects 0.000 description 5
- 210000003169 central nervous system Anatomy 0.000 description 5
- 239000003937 drug carrier Substances 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 238000001415 gene therapy Methods 0.000 description 5
- 230000009459 hedgehog signaling Effects 0.000 description 5
- 239000003906 humectant Substances 0.000 description 5
- 210000004408 hybridoma Anatomy 0.000 description 5
- 239000004615 ingredient Substances 0.000 description 5
- 239000003112 inhibitor Substances 0.000 description 5
- 210000003041 ligament Anatomy 0.000 description 5
- 229920002521 macromolecule Polymers 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 210000001020 neural plate Anatomy 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 239000003826 tablet Substances 0.000 description 5
- 239000002562 thickening agent Substances 0.000 description 5
- 235000008521 threonine Nutrition 0.000 description 5
- 239000003981 vehicle Substances 0.000 description 5
- 208000024827 Alzheimer disease Diseases 0.000 description 4
- 241000271566 Aves Species 0.000 description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 4
- 241000701022 Cytomegalovirus Species 0.000 description 4
- 206010012289 Dementia Diseases 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical group OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- 239000004166 Lanolin Substances 0.000 description 4
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 4
- 238000000636 Northern blotting Methods 0.000 description 4
- 108010064785 Phospholipases Proteins 0.000 description 4
- 102000015439 Phospholipases Human genes 0.000 description 4
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 206010002026 amyotrophic lateral sclerosis Diseases 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
- 230000006907 apoptotic process Effects 0.000 description 4
- 230000003376 axonal effect Effects 0.000 description 4
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 4
- 108010005774 beta-Galactosidase Proteins 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 210000001109 blastomere Anatomy 0.000 description 4
- 229910052791 calcium Inorganic materials 0.000 description 4
- 239000002775 capsule Substances 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 235000014633 carbohydrates Nutrition 0.000 description 4
- 210000003710 cerebral cortex Anatomy 0.000 description 4
- 230000002759 chromosomal effect Effects 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 230000001684 chronic effect Effects 0.000 description 4
- 230000009918 complex formation Effects 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 101150118300 cos gene Proteins 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 230000034994 death Effects 0.000 description 4
- 210000001647 gastrula Anatomy 0.000 description 4
- 229930195712 glutamate Natural products 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 230000013595 glycosylation Effects 0.000 description 4
- 238000006206 glycosylation reaction Methods 0.000 description 4
- 206010020718 hyperplasia Diseases 0.000 description 4
- 230000002390 hyperplastic effect Effects 0.000 description 4
- 238000011065 in-situ storage Methods 0.000 description 4
- 230000000977 initiatory effect Effects 0.000 description 4
- 235000019388 lanolin Nutrition 0.000 description 4
- 229940039717 lanolin Drugs 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
- 230000003278 mimic effect Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 230000001613 neoplastic effect Effects 0.000 description 4
- 210000005036 nerve Anatomy 0.000 description 4
- 208000015122 neurodegenerative disease Diseases 0.000 description 4
- 230000004031 neuronal differentiation Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000002674 ointment Substances 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 239000002304 perfume Substances 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 102000040430 polynucleotide Human genes 0.000 description 4
- 108091033319 polynucleotide Proteins 0.000 description 4
- 239000002157 polynucleotide Substances 0.000 description 4
- 230000004481 post-translational protein modification Effects 0.000 description 4
- 230000004850 protein–protein interaction Effects 0.000 description 4
- 230000008929 regeneration Effects 0.000 description 4
- 238000011069 regeneration method Methods 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 238000007423 screening assay Methods 0.000 description 4
- 210000003491 skin Anatomy 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 210000000278 spinal cord Anatomy 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 4
- 235000002374 tyrosine Nutrition 0.000 description 4
- 241001430294 unidentified retrovirus Species 0.000 description 4
- 108700026220 vif Genes Proteins 0.000 description 4
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 3
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 108020004635 Complementary DNA Proteins 0.000 description 3
- 241000255925 Diptera Species 0.000 description 3
- 108700024394 Exon Proteins 0.000 description 3
- 102000005720 Glutathione transferase Human genes 0.000 description 3
- 108010070675 Glutathione transferase Proteins 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-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
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-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
- 102000055008 Matrilin Proteins Human genes 0.000 description 3
- 108010072582 Matrilin Proteins Proteins 0.000 description 3
- 102000015336 Nerve Growth Factor Human genes 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- 241000051107 Paraechinus aethiopicus Species 0.000 description 3
- 108091093037 Peptide nucleic acid Proteins 0.000 description 3
- 206010038997 Retroviral infections Diseases 0.000 description 3
- 241000283984 Rodentia Species 0.000 description 3
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 3
- 241000700584 Simplexvirus Species 0.000 description 3
- 108091023040 Transcription factor Proteins 0.000 description 3
- 102000040945 Transcription factor Human genes 0.000 description 3
- 108700029229 Transcriptional Regulatory Elements Proteins 0.000 description 3
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 3
- 108090001012 Transforming Growth Factor beta Proteins 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
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Chemical class Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 150000001298 alcohols Chemical class 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940009098 aspartate Drugs 0.000 description 3
- 102000005936 beta-Galactosidase Human genes 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 210000002459 blastocyst Anatomy 0.000 description 3
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 3
- 238000000423 cell based assay Methods 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000002648 chondrogenic effect Effects 0.000 description 3
- 239000003593 chromogenic compound Substances 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000021615 conjugation Effects 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 230000001054 cortical effect Effects 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 239000006071 cream Substances 0.000 description 3
- 230000006735 deficit Effects 0.000 description 3
- 238000002405 diagnostic procedure Methods 0.000 description 3
- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 210000003981 ectoderm Anatomy 0.000 description 3
- 239000003792 electrolyte Substances 0.000 description 3
- 230000013020 embryo development Effects 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 239000003925 fat Substances 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- 239000010408 film Substances 0.000 description 3
- 238000010363 gene targeting Methods 0.000 description 3
- 210000003128 head Anatomy 0.000 description 3
- BXWNKGSJHAJOGX-UHFFFAOYSA-N hexadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCO BXWNKGSJHAJOGX-UHFFFAOYSA-N 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 229960000310 isoleucine Drugs 0.000 description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000005499 meniscus Effects 0.000 description 3
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 210000000478 neocortex Anatomy 0.000 description 3
- 229940053128 nerve growth factor Drugs 0.000 description 3
- 230000004770 neurodegeneration Effects 0.000 description 3
- 210000004940 nucleus Anatomy 0.000 description 3
- 210000000287 oocyte Anatomy 0.000 description 3
- 125000001151 peptidyl group Chemical group 0.000 description 3
- 210000001428 peripheral nervous system Anatomy 0.000 description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 3
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 230000002335 preservative effect Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 210000002023 somite Anatomy 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 210000002435 tendon Anatomy 0.000 description 3
- 230000001052 transient effect Effects 0.000 description 3
- 230000008733 trauma Effects 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 241000701447 unidentified baculovirus Species 0.000 description 3
- 239000004474 valine Substances 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 239000005711 Benzoic acid Substances 0.000 description 2
- 208000010392 Bone Fractures Diseases 0.000 description 2
- 241000701822 Bovine papillomavirus Species 0.000 description 2
- 239000004255 Butylated hydroxyanisole Substances 0.000 description 2
- 239000004322 Butylated hydroxytoluene Substances 0.000 description 2
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical compound CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- 102100023321 Ceruloplasmin Human genes 0.000 description 2
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 2
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 description 2
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 201000011180 Dental Pulp Calcification Diseases 0.000 description 2
- 208000012239 Developmental disease Diseases 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 101710181403 Frizzled Proteins 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- 208000023105 Huntington disease Diseases 0.000 description 2
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical group OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 241000270322 Lepidosauria Species 0.000 description 2
- 239000000232 Lipid Bilayer Substances 0.000 description 2
- 108060001084 Luciferase Proteins 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
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 241000713869 Moloney murine leukemia virus Species 0.000 description 2
- 241000713333 Mouse mammary tumor virus Species 0.000 description 2
- 102000007072 Nerve Growth Factors Human genes 0.000 description 2
- 241000906034 Orthops Species 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 208000018737 Parkinson disease Diseases 0.000 description 2
- 108010039918 Polylysine Proteins 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- ZTHYODDOHIVTJV-UHFFFAOYSA-N Propyl gallate Chemical compound CCCOC(=O)C1=CC(O)=C(O)C(O)=C1 ZTHYODDOHIVTJV-UHFFFAOYSA-N 0.000 description 2
- 101000708627 Rattus norvegicus Smoothened homolog Proteins 0.000 description 2
- 241000714474 Rous sarcoma virus Species 0.000 description 2
- 101150056682 Smo gene Proteins 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 102000030621 adenylate cyclase Human genes 0.000 description 2
- 108060000200 adenylate cyclase Proteins 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 210000001188 articular cartilage Anatomy 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 235000010233 benzoic acid Nutrition 0.000 description 2
- 229960004365 benzoic acid Drugs 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 229920000249 biocompatible polymer Polymers 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 230000008468 bone growth Effects 0.000 description 2
- 235000019282 butylated hydroxyanisole Nutrition 0.000 description 2
- CZBZUDVBLSSABA-UHFFFAOYSA-N butylated hydroxyanisole Chemical compound COC1=CC=C(O)C(C(C)(C)C)=C1.COC1=CC=C(O)C=C1C(C)(C)C CZBZUDVBLSSABA-UHFFFAOYSA-N 0.000 description 2
- 229940043253 butylated hydroxyanisole Drugs 0.000 description 2
- 235000010354 butylated hydroxytoluene Nutrition 0.000 description 2
- 229940095259 butylated hydroxytoluene Drugs 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000005266 casting Methods 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 230000008614 cellular interaction Effects 0.000 description 2
- 230000036755 cellular response Effects 0.000 description 2
- 210000001638 cerebellum Anatomy 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 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 2
- 210000002932 cholinergic neuron Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 235000015165 citric acid Nutrition 0.000 description 2
- 229960004106 citric acid Drugs 0.000 description 2
- 238000002742 combinatorial mutagenesis Methods 0.000 description 2
- 238000012875 competitive assay Methods 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- GVJHHUAWPYXKBD-UHFFFAOYSA-N d-alpha-tocopherol Natural products OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 210000005064 dopaminergic neuron Anatomy 0.000 description 2
- 238000007877 drug screening Methods 0.000 description 2
- 238000007878 drug screening assay Methods 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 230000005014 ectopic expression Effects 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- NUVBSKCKDOMJSU-UHFFFAOYSA-N ethylparaben Chemical compound CCOC(=O)C1=CC=C(O)C=C1 NUVBSKCKDOMJSU-UHFFFAOYSA-N 0.000 description 2
- 238000001400 expression cloning Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 230000003176 fibrotic effect Effects 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 108091006104 gene-regulatory proteins Proteins 0.000 description 2
- 102000034356 gene-regulatory proteins Human genes 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 102000054766 genetic haplotypes Human genes 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 235000004554 glutamine Nutrition 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 125000003147 glycosyl group Chemical group 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 2
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 2
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 2
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 2
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 2
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 2
- 210000003559 hypertrophic chondrocyte Anatomy 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 150000004001 inositols Chemical class 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- 210000003127 knee Anatomy 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 201000010901 lateral sclerosis Diseases 0.000 description 2
- 239000000787 lecithin Substances 0.000 description 2
- 235000010445 lecithin Nutrition 0.000 description 2
- 229940067606 lecithin Drugs 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 210000004779 membrane envelope Anatomy 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 2
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 2
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 2
- 229960002216 methylparaben Drugs 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 108091005601 modified peptides Proteins 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 230000000921 morphogenic effect Effects 0.000 description 2
- 208000005264 motor neuron disease Diseases 0.000 description 2
- 231100000219 mutagenic Toxicity 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 239000003900 neurotrophic factor Substances 0.000 description 2
- GLDOVTGHNKAZLK-UHFFFAOYSA-N octadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCCCO GLDOVTGHNKAZLK-UHFFFAOYSA-N 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000003239 periodontal effect Effects 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 229920000656 polylysine Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 201000002241 progressive bulbar palsy Diseases 0.000 description 2
- 230000000750 progressive effect Effects 0.000 description 2
- 201000008752 progressive muscular atrophy Diseases 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 238000001243 protein synthesis Methods 0.000 description 2
- 238000001525 receptor binding assay Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 230000007261 regionalization Effects 0.000 description 2
- 238000007634 remodeling Methods 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 210000001044 sensory neuron Anatomy 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 108091006024 signal transducing proteins Proteins 0.000 description 2
- 102000034285 signal transducing proteins Human genes 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000000392 somatic effect Effects 0.000 description 2
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N squalane Chemical compound CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 210000001550 testis Anatomy 0.000 description 2
- 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 2
- 235000010384 tocopherol Nutrition 0.000 description 2
- 239000011732 tocopherol Substances 0.000 description 2
- 229960001295 tocopherol Drugs 0.000 description 2
- 229930003799 tocopherol Natural products 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 230000008736 traumatic injury Effects 0.000 description 2
- 230000001228 trophic effect Effects 0.000 description 2
- 210000002845 virion Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 239000001993 wax Substances 0.000 description 2
- 239000000230 xanthan gum Substances 0.000 description 2
- 229920001285 xanthan gum Polymers 0.000 description 2
- 235000010493 xanthan gum Nutrition 0.000 description 2
- 229940082509 xanthan gum Drugs 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 2
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 1
- DSEKYWAQQVUQTP-XEWMWGOFSA-N (2r,4r,4as,6as,6as,6br,8ar,12ar,14as,14bs)-2-hydroxy-4,4a,6a,6b,8a,11,11,14a-octamethyl-2,4,5,6,6a,7,8,9,10,12,12a,13,14,14b-tetradecahydro-1h-picen-3-one Chemical compound C([C@H]1[C@]2(C)CC[C@@]34C)C(C)(C)CC[C@]1(C)CC[C@]2(C)[C@H]4CC[C@@]1(C)[C@H]3C[C@@H](O)C(=O)[C@@H]1C DSEKYWAQQVUQTP-XEWMWGOFSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- FFJCNSLCJOQHKM-CLFAGFIQSA-N (z)-1-[(z)-octadec-9-enoxy]octadec-9-ene Chemical compound CCCCCCCC\C=C/CCCCCCCCOCCCCCCCC\C=C/CCCCCCCC FFJCNSLCJOQHKM-CLFAGFIQSA-N 0.000 description 1
- QMMJWQMCMRUYTG-UHFFFAOYSA-N 1,2,4,5-tetrachloro-3-(trifluoromethyl)benzene Chemical compound FC(F)(F)C1=C(Cl)C(Cl)=CC(Cl)=C1Cl QMMJWQMCMRUYTG-UHFFFAOYSA-N 0.000 description 1
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 1
- 229940058015 1,3-butylene glycol Drugs 0.000 description 1
- UOTMYNBWXDUBNX-UHFFFAOYSA-N 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinolin-2-ium;chloride Chemical compound Cl.C1=C(OC)C(OC)=CC=C1CC1=NC=CC2=CC(OC)=C(OC)C=C12 UOTMYNBWXDUBNX-UHFFFAOYSA-N 0.000 description 1
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 1
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- SVUOLADPCWQTTE-UHFFFAOYSA-N 1h-1,2-benzodiazepine Chemical compound N1N=CC=CC2=CC=CC=C12 SVUOLADPCWQTTE-UHFFFAOYSA-N 0.000 description 1
- FKOKUHFZNIUSLW-UHFFFAOYSA-N 2-Hydroxypropyl stearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(C)O FKOKUHFZNIUSLW-UHFFFAOYSA-N 0.000 description 1
- LRYZPFWEZHSTHD-HEFFAWAOSA-O 2-[[(e,2s,3r)-2-formamido-3-hydroxyoctadec-4-enoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium Chemical class CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](NC=O)COP(O)(=O)OCC[N+](C)(C)C LRYZPFWEZHSTHD-HEFFAWAOSA-O 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- RFVNOJDQRGSOEL-UHFFFAOYSA-N 2-hydroxyethyl octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCCO RFVNOJDQRGSOEL-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- HIQIXEFWDLTDED-UHFFFAOYSA-N 4-hydroxy-1-piperidin-4-ylpyrrolidin-2-one Chemical compound O=C1CC(O)CN1C1CCNCC1 HIQIXEFWDLTDED-UHFFFAOYSA-N 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 108010013043 Acetylesterase Proteins 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 108010059616 Activins Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 241000269350 Anura Species 0.000 description 1
- 206010003591 Ataxia Diseases 0.000 description 1
- 208000025978 Athletic injury Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 101150061927 BMP2 gene Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108090000363 Bacterial Luciferases Proteins 0.000 description 1
- 229940122361 Bisphosphonate Drugs 0.000 description 1
- 108010049931 Bone Morphogenetic Protein 2 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
- 208000006386 Bone Resorption Diseases 0.000 description 1
- 206010065687 Bone loss Diseases 0.000 description 1
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 1
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 1
- 101100191768 Caenorhabditis elegans pbs-4 gene Proteins 0.000 description 1
- 102000055006 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 208000018380 Chemical injury Diseases 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 244000060011 Cocos nucifera Species 0.000 description 1
- 235000013162 Cocos nucifera Nutrition 0.000 description 1
- 108010051219 Cre recombinase Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241001559589 Cullen Species 0.000 description 1
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 239000004287 Dehydroacetic acid Substances 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 108700001637 Drosophila ci Proteins 0.000 description 1
- 108700002007 Drosophila fz Proteins 0.000 description 1
- 101000606317 Drosophila melanogaster Protein patched Proteins 0.000 description 1
- 101100084900 Drosophila melanogaster Rpn11 gene Proteins 0.000 description 1
- 108700039964 Duplicate Genes Proteins 0.000 description 1
- 208000012661 Dyskinesia Diseases 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241001144268 Echidna Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010014561 Emphysema Diseases 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000991587 Enterovirus C Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 239000004258 Ethoxyquin Substances 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 108010046276 FLP recombinase Proteins 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 102000005698 Frizzled receptors Human genes 0.000 description 1
- 108010045438 Frizzled receptors Proteins 0.000 description 1
- 201000011240 Frontotemporal dementia Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010008959 G-Protein-Coupled Receptor Kinases Proteins 0.000 description 1
- 102000006575 G-Protein-Coupled Receptor Kinases Human genes 0.000 description 1
- 229940125633 GPCR agonist Drugs 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 241001241365 Gehyra xenopus Species 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 208000035895 Guillain-Barré syndrome Diseases 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 102100035960 Hedgehog-interacting protein Human genes 0.000 description 1
- 101710164669 Hedgehog-interacting protein Proteins 0.000 description 1
- 208000028782 Hereditary disease Diseases 0.000 description 1
- 208000007514 Herpes zoster Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 206010021118 Hypotonia Diseases 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 102100026818 Inhibin beta E chain Human genes 0.000 description 1
- 208000015592 Involuntary movements Diseases 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- 239000011786 L-ascorbyl-6-palmitate Substances 0.000 description 1
- QAQJMLQRFWZOBN-LAUBAEHRSA-N L-ascorbyl-6-palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](O)[C@H]1OC(=O)C(O)=C1O QAQJMLQRFWZOBN-LAUBAEHRSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- 208000000172 Medulloblastoma Diseases 0.000 description 1
- 208000024556 Mendelian disease Diseases 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 206010049567 Miller Fisher syndrome Diseases 0.000 description 1
- 208000016285 Movement disease Diseases 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100446513 Mus musculus Fgf4 gene Proteins 0.000 description 1
- 101000708613 Mus musculus Smoothened homolog Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000007379 Muscle Hypotonia Diseases 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 206010056677 Nerve degeneration Diseases 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 108700015679 Nested Genes Proteins 0.000 description 1
- 208000009277 Neuroectodermal Tumors Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 230000004989 O-glycosylation Effects 0.000 description 1
- BZQFBWGGLXLEPQ-UHFFFAOYSA-N O-phosphoryl-L-serine Natural products OC(=O)C(N)COP(O)(O)=O BZQFBWGGLXLEPQ-UHFFFAOYSA-N 0.000 description 1
- REYJJPSVUYRZGE-UHFFFAOYSA-N Octadecylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 101150012394 PHO5 gene Proteins 0.000 description 1
- 208000034530 PLAA-associated neurodevelopmental disease Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 208000000609 Pick Disease of the Brain Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 208000032319 Primary lateral sclerosis Diseases 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 101001010097 Shigella phage SfV Bactoprenol-linked glucose translocase Proteins 0.000 description 1
- 101710090597 Smoothened homolog Proteins 0.000 description 1
- 102100032799 Smoothened homolog Human genes 0.000 description 1
- HVUMOYIDDBPOLL-XWVZOOPGSA-N Sorbitan monostearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O HVUMOYIDDBPOLL-XWVZOOPGSA-N 0.000 description 1
- 208000010112 Spinocerebellar Degenerations Diseases 0.000 description 1
- 206010041738 Sports injury Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- VBIIFPGSPJYLRR-UHFFFAOYSA-M Stearyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)C VBIIFPGSPJYLRR-UHFFFAOYSA-M 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 102000008983 Suppressor of fused Human genes 0.000 description 1
- 108050000968 Suppressor of fused Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 208000001871 Tachycardia Diseases 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 208000026062 Tissue disease Diseases 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- YPFDHNVEDLHUCE-UHFFFAOYSA-N Trimethylene glycol Natural products OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 description 1
- IVOMOUWHDPKRLL-UHFFFAOYSA-N UNPD107823 Natural products O1C2COP(O)(=O)OC2C(O)C1N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-UHFFFAOYSA-N 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 206010046298 Upper motor neurone lesion Diseases 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 108010087302 Viral Structural Proteins Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241001376713 Xenops Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- JPBAVLUULZJFFO-JENHRLMUSA-N [(2s)-2-[(2r)-3,4-dihydroxy-5-oxo-2h-furan-2-yl]-2-hydroxyethyl] (z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](O)[C@H]1OC(=O)C(O)=C1O JPBAVLUULZJFFO-JENHRLMUSA-N 0.000 description 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000000488 activin Substances 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 210000003423 ankle Anatomy 0.000 description 1
- 210000001264 anterior cruciate ligament Anatomy 0.000 description 1
- 239000000058 anti acne agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 230000002788 anti-peptide Effects 0.000 description 1
- 229940124340 antiacne agent Drugs 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000010385 ascorbyl palmitate Nutrition 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 230000001746 atrial effect Effects 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 230000002567 autonomic effect Effects 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- XYOVOXDWRFGKEX-UHFFFAOYSA-N azepine Chemical compound N1C=CC=CC=C1 XYOVOXDWRFGKEX-UHFFFAOYSA-N 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 1
- 239000013602 bacteriophage vector Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 210000004227 basal ganglia Anatomy 0.000 description 1
- 235000013871 bee wax Nutrition 0.000 description 1
- 239000012166 beeswax Substances 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 229940049706 benzodiazepine Drugs 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 238000010256 biochemical assay Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 150000004663 bisphosphonates Chemical class 0.000 description 1
- 230000014461 bone development Effects 0.000 description 1
- 230000024279 bone resorption Effects 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 235000010338 boric acid Nutrition 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 235000019437 butane-1,3-diol Nutrition 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000003185 calcium uptake Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 230000000453 cell autonomous effect Effects 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 210000004671 cell-free system Anatomy 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 229960000541 cetyl alcohol Drugs 0.000 description 1
- 239000012707 chemical precursor Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 229940107161 cholesterol Drugs 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000009693 chronic damage Effects 0.000 description 1
- 208000022371 chronic pain syndrome Diseases 0.000 description 1
- 230000001886 ciliary effect Effects 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 208000010877 cognitive disease Diseases 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000006854 communication Effects 0.000 description 1
- 230000006552 constitutive activation Effects 0.000 description 1
- 239000003433 contraceptive agent Substances 0.000 description 1
- 230000002254 contraceptive effect Effects 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000002316 cosmetic surgery Methods 0.000 description 1
- QTCANKDTWWSCMR-UHFFFAOYSA-N costic aldehyde Natural products C1CCC(=C)C2CC(C(=C)C=O)CCC21C QTCANKDTWWSCMR-UHFFFAOYSA-N 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 229940095074 cyclic amp Drugs 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 229920006237 degradable polymer Polymers 0.000 description 1
- PGRHXDWITVMQBC-UHFFFAOYSA-N dehydroacetic acid Natural products CC(=O)C1C(=O)OC(C)=CC1=O PGRHXDWITVMQBC-UHFFFAOYSA-N 0.000 description 1
- 235000019258 dehydroacetic acid Nutrition 0.000 description 1
- 229940061632 dehydroacetic acid Drugs 0.000 description 1
- JEQRBTDTEKWZBW-UHFFFAOYSA-N dehydroacetic acid Chemical compound CC(=O)C1=C(O)OC(C)=CC1=O JEQRBTDTEKWZBW-UHFFFAOYSA-N 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000005016 dendritic process Effects 0.000 description 1
- 239000005548 dental material Substances 0.000 description 1
- 238000000586 desensitisation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 229950006137 dexfosfoserine Drugs 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 230000024634 digestive tract morphogenesis Effects 0.000 description 1
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 1
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000001177 diphosphate Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000003291 dopaminomimetic effect Effects 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000002308 embryonic cell Anatomy 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000035194 endochondral ossification Effects 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 230000002121 endocytic effect Effects 0.000 description 1
- 210000001900 endoderm Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 150000002169 ethanolamines Chemical class 0.000 description 1
- 235000019285 ethoxyquin Nutrition 0.000 description 1
- DECIPOUIJURFOJ-UHFFFAOYSA-N ethoxyquin Chemical compound N1C(C)(C)C=C(C)C2=CC(OCC)=CC=C21 DECIPOUIJURFOJ-UHFFFAOYSA-N 0.000 description 1
- 229940093500 ethoxyquin Drugs 0.000 description 1
- 239000004403 ethyl p-hydroxybenzoate Substances 0.000 description 1
- 235000010228 ethyl p-hydroxybenzoate Nutrition 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000011512 eye pigmentation Effects 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 210000000968 fibrocartilage Anatomy 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- YFHXZQPUBCBNIP-UHFFFAOYSA-N fura-2 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=3OC(=CC=3C=2)C=2OC(=CN=2)C(O)=O)N(CC(O)=O)CC(O)=O)=C1 YFHXZQPUBCBNIP-UHFFFAOYSA-N 0.000 description 1
- IECPWNUMDGFDKC-MZJAQBGESA-N fusidic acid Chemical class O[C@@H]([C@@H]12)C[C@H]3\C(=C(/CCC=C(C)C)C(O)=O)[C@@H](OC(C)=O)C[C@]3(C)[C@@]2(C)CC[C@@H]2[C@]1(C)CC[C@@H](O)[C@H]2C IECPWNUMDGFDKC-MZJAQBGESA-N 0.000 description 1
- 210000001222 gaba-ergic neuron Anatomy 0.000 description 1
- 125000001965 gamma-lactamyl group Chemical group 0.000 description 1
- 230000000574 ganglionic effect Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000007045 gastrulation Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 230000004545 gene duplication Effects 0.000 description 1
- 238000003633 gene expression assay Methods 0.000 description 1
- 230000000762 glandular Effects 0.000 description 1
- 239000011491 glass wool Substances 0.000 description 1
- 101150033912 gli3 gene Proteins 0.000 description 1
- 108010087860 glutathione receptor Proteins 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 150000002321 glycerophosphoglycerophosphoglycerols Chemical class 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 230000036449 good health Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229960004198 guanidine Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000012203 high throughput assay Methods 0.000 description 1
- 238000007825 histological assay Methods 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000008348 humoral response Effects 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 238000002169 hydrotherapy Methods 0.000 description 1
- 230000001969 hypertrophic effect Effects 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000010249 in-situ analysis Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- PNDZEEPOYCVIIY-UHFFFAOYSA-N indo-1 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=C(C=2)C=2N=C3[CH]C(=CC=C3C=2)C(O)=O)N(CC(O)=O)CC(O)=O)=C1 PNDZEEPOYCVIIY-UHFFFAOYSA-N 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 230000030214 innervation Effects 0.000 description 1
- 238000012966 insertion method Methods 0.000 description 1
- 230000035990 intercellular signaling Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000010189 intracellular transport Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000002563 ionic surfactant Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000003447 ipsilateral effect Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- ISTFUJWTQAMRGA-UHFFFAOYSA-N iso-beta-costal Natural products C1C(C(=C)C=O)CCC2(C)CCCC(C)=C21 ISTFUJWTQAMRGA-UHFFFAOYSA-N 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- XUGNVMKQXJXZCD-UHFFFAOYSA-N isopropyl palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC(C)C XUGNVMKQXJXZCD-UHFFFAOYSA-N 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 230000007653 larval development Effects 0.000 description 1
- 210000002414 leg Anatomy 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000004973 liquid crystal related substance Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical class CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 210000004939 midgestation embryo Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 230000001002 morphogenetic effect Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 230000017311 musculoskeletal movement, spinal reflex action Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- JXTPJDDICSTXJX-UHFFFAOYSA-N n-Triacontane Natural products CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC JXTPJDDICSTXJX-UHFFFAOYSA-N 0.000 description 1
- GOQYKNQRPGWPLP-UHFFFAOYSA-N n-heptadecyl alcohol Natural products CCCCCCCCCCCCCCCCCO GOQYKNQRPGWPLP-UHFFFAOYSA-N 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 230000010309 neoplastic transformation Effects 0.000 description 1
- 210000001577 neostriatum Anatomy 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 210000001640 nerve ending Anatomy 0.000 description 1
- 230000003988 neural development Effects 0.000 description 1
- 230000001272 neurogenic effect Effects 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 208000018360 neuromuscular disease Diseases 0.000 description 1
- 208000014500 neuronal tumor Diseases 0.000 description 1
- 239000004090 neuroprotective agent Substances 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 235000018343 nutrient deficiency Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000034004 oogenesis Effects 0.000 description 1
- 239000006186 oral dosage form Substances 0.000 description 1
- 210000004789 organ system Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000000888 organogenic effect Effects 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 210000005009 osteogenic cell Anatomy 0.000 description 1
- 230000002138 osteoinductive effect Effects 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000012753 partial hepatectomy Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 239000003961 penetration enhancing agent Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 210000002951 peptidergic neuron Anatomy 0.000 description 1
- 210000002379 periodontal ligament Anatomy 0.000 description 1
- 210000000578 peripheral nerve Anatomy 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- RLZZZVKAURTHCP-UHFFFAOYSA-N phenanthrene-3,4-diol Chemical compound C1=CC=C2C3=C(O)C(O)=CC=C3C=CC2=C1 RLZZZVKAURTHCP-UHFFFAOYSA-N 0.000 description 1
- 150000008103 phosphatidic acids Chemical class 0.000 description 1
- 150000008105 phosphatidylcholines Chemical class 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 1
- 125000005642 phosphothioate group Chemical group 0.000 description 1
- USRGIUJOYOXOQJ-GBXIJSLDSA-N phosphothreonine Chemical compound OP(=O)(O)O[C@H](C)[C@H](N)C(O)=O USRGIUJOYOXOQJ-GBXIJSLDSA-N 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 239000000249 polyoxyethylene sorbitan monopalmitate Substances 0.000 description 1
- 235000010483 polyoxyethylene sorbitan monopalmitate Nutrition 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 239000000473 propyl gallate Substances 0.000 description 1
- 235000010388 propyl gallate Nutrition 0.000 description 1
- 229940075579 propyl gallate Drugs 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- 235000013772 propylene glycol Nutrition 0.000 description 1
- 229940093625 propylene glycol monostearate Drugs 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 210000001609 raphe nuclei Anatomy 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000002278 reconstructive surgery Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 150000003355 serines Chemical class 0.000 description 1
- 230000000862 serotonergic effect Effects 0.000 description 1
- 238000003567 signal transduction assay Methods 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 230000007781 signaling event Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 230000008410 smoothened signaling pathway Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 229940080236 sodium cetyl sulfate Drugs 0.000 description 1
- 235000011083 sodium citrates Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- 229960000414 sodium fluoride Drugs 0.000 description 1
- RYYKJJJTJZKILX-UHFFFAOYSA-M sodium octadecanoate Chemical compound [Na+].CCCCCCCCCCCCCCCCCC([O-])=O RYYKJJJTJZKILX-UHFFFAOYSA-M 0.000 description 1
- 239000004328 sodium tetraborate Substances 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- GGHPAKFFUZUEKL-UHFFFAOYSA-M sodium;hexadecyl sulfate Chemical compound [Na+].CCCCCCCCCCCCCCCCOS([O-])(=O)=O GGHPAKFFUZUEKL-UHFFFAOYSA-M 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002195 soluble material Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000001587 sorbitan monostearate Substances 0.000 description 1
- 235000011076 sorbitan monostearate Nutrition 0.000 description 1
- 229940035048 sorbitan monostearate Drugs 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000000920 spermatogeneic effect Effects 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 229940032094 squalane Drugs 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- SFVFIFLLYFPGHH-UHFFFAOYSA-M stearalkonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 SFVFIFLLYFPGHH-UHFFFAOYSA-M 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 229940012831 stearyl alcohol Drugs 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 210000001562 sternum Anatomy 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 210000003699 striated muscle Anatomy 0.000 description 1
- 230000002739 subcortical effect Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 230000000475 sunscreen effect Effects 0.000 description 1
- 239000000516 sunscreening agent Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 238000011477 surgical intervention Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000002889 sympathetic effect Effects 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 230000006794 tachycardia Effects 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- 238000010809 targeting technique Methods 0.000 description 1
- 210000003478 temporal lobe Anatomy 0.000 description 1
- 208000001608 teratocarcinoma Diseases 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000012956 testing procedure Methods 0.000 description 1
- 210000001103 thalamus Anatomy 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000009772 tissue formation Effects 0.000 description 1
- 230000030968 tissue homeostasis Effects 0.000 description 1
- 235000015961 tonic Nutrition 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 229960000716 tonics Drugs 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 230000036269 ulceration Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000008189 vertebrate development Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000012431 wafers Nutrition 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
- 210000000707 wrist Anatomy 0.000 description 1
- 101150061422 yip5 gene Proteins 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 210000004340 zona pellucida Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from 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
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
- C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- Pattern formation is the activity by which embryonic cells form ordered spatial arrangements of differentiated tissues.
- the physical complexity of higher organisms arises during embryogenesis through the interplay of cell-intrinsic lineage and cell-extrinsic signaling.
- Inductive interactions are essential to embryonic patterning in vertebrate development from the earliest establishment of the body plan, to the patterning of the organ systems, to the generation of diverse cell types during tissue differentiation (Davidson, E., (1990) Development 108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al., (1992) Cell 68: 257-270).
- the effects of developmental cell interactions are varied.
- responding cells are diverted from one route of cell differentiation to another by inducing cells that differ from both the uninduced and induced states of the responding cells (inductions).
- inductions usually cells induce their neighbors to differentiate like themselves (homoiogenetic induction); in other cases a cell inhibits its neighbors from differentiating like itself.
- Cell interactions in early development may be sequential, such that an initial induction between two cell types leads to a progressive amplification of diversity.
- inductive interactions occur not only in embryos, but in adult cells as well, and can act to establish and maintain morphogenetic patterns as well as induce differentiation (J.B. Gurdon (1992) Cell 68:185-199).
- the origin of the nervous system in all vertebrates can be traced to the end of gastrulation.
- the ectoderm in the dorsal side of the embryo changes its fate from epidermal to neural.
- the newly formed neuroectoderm thickens to form a flattened structure called the neural plate which is characterized, in some vertebrates, by a central groove (neural groove) and thickened lateral edges (neural folds).
- the neural plate already exhibits signs of regional differentiation along its anterior posterior (A-P) and mediolateral axis (M-L).
- the neural folds eventually fuse at the dorsal midline to form the neural tube which will differentiate into brain at its anterior end and spinal cord at its posterior end.
- the neural tube has a clear anterior-posterior (A-P), dorsal ventral (D-V) and mediolateral (M-L) polarities (see, for example, Principles in Neural Science (3rd), eds. Kandel, Schwartz and Jessell, Elsevier Science Publishing Company: NY, 1991 ; and Developmental Biology (3rd), ed. S.F. Gilbert, Sinauer Associates: Sunderland MA, 1991).
- A-P anterior-posterior
- D-V dorsal ventral
- M-L mediolateral
- the identify of cell types is controlled, in part, by signals from two midline cell groups, the notochord and floor plate, that induce neural plate cells to differentiate into floor plate, motor neurons, and other ventral neuronal types (van Straaten et al. (1988) Anat. Embryol. 177:317-324; Placzek et al. (1993) Development 117:205-218; Yamada et al. (1991) Cell 64:035-647; and Hatta et al. (1991) Nature 350:339-341).
- signals from the floor plate are responsible for the orientation and direction of commissural neuron outgrowth (Placzek, M. et al., (1990) Development 110: 19-30).
- the notochord and floorplate are also responsible for producing signals which control the patterning of the somites by inhibiting differentiation of dorsal somite derivatives in the ventral regions (Brand-Saberi, B. et al., (1993) Anat. Embryol. 188: 239-245; Porquie, O. et al., (1993) Proc. Natl. Acad. Sci. USA 90: 5242-5246).
- Zone of Polarizing Activity Another important signaling center exists in the posterior mesenchyme of developing limb buds, called the Zone of Polarizing Activity, or "ZPA".
- ZPA Zone of Polarizing Activity
- the ZPA has been hypothesized to function by releasing a signal, termed a "morphogen", which forms a gradient across the early embryonic bud.
- a signal termed a "morphogen”
- the fate of cells at different distances from the ZPA is determined by the local concentration of the morphogen, with specific thresholds of the morphogen inducing successive structures (Wolpert, (1969) Theor. Biol. 25:1-47). This is supported by the finding that the extent of digit duplication is proportional to the number of implanted ZPA cells (Tickle, (1981) Nature 254:199-202).
- the vertebrate hedgehog family of inducing molecules comprises three homologs named Sonic, Indian and Desert hedgehog (Riddle et al. (1993) supra).
- Desert hedgehog (Dhh) is expressed principally in the testes, both in mouse embryonic development and in the adult rodent and human; Indian hedgehog (Ihh) is involved in bone development during embryogenesis and in bone formation in the adult; and, Shh, which as described above, is primarily involved in morphogenic and neuroinductive activities.
- the present invention relates to the discovery of a new class of serpentine receptors, referred to herein as smoothened proteins.
- the smoothened proteins of the present invention include polypeptides which affect the transmission of signals by the products of the hedgehog gene family.
- Hedgehog family members are known for their broad involvement in the formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, both adult and embryonic, and can be used to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.
- the invention features isolated smoothened polypeptides, preferably substantially pure preparations of the subject smoothened polypeptides, such as liposomal preparations.
- the invention also provides recombinantly produced smoothened polypeptides.
- the polypeptide is identical with or similar to a smoothened polypeptide represented in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8.
- a smoothened polypeptide preferably has an amino acid sequence at least 65%, 70%, 75% or 80%) identical or similar to a polypeptide represented by SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8 though polypeptides with higher sequence homologies of, for example, 85, 90% and 95% or are also contemplated.
- the smoothened polypeptide is encoded by a nucleic acid which hybridizes under stringent conditions with a nucleic acid sequence represented in any one or more of SEQ ID Nos: 1-4 and 9.
- Homologs of the subject smoothened proteins also include versions of the protein which are resistant to post-translation modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with a smoothened ligand, e.g. a hedgehog polypeptide.
- the smoothened polypeptide can comprise a full length protein, such as represented in SEQ ID No: 5, SEQ ID No: 6 or SEQ ID No: 7, or it can comprise a fragment corresponding to one or more particular motifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150 or 200 amino acids in length.
- the smoothened polypeptide includes at least a portion of a smoothened protein corresponding to Met 13 - Ser 1035 of SEQ ID No. 5.
- the smoothened polypeptide includes a sufficient portion of the protein to be able to specifically bind to patched.
- Truncated forms of the protein include, but are not limited to. soluble extracellular and/or intracellular fragments, e.g., which bind to ligand or signal transduction proteins, respectively.
- the subject proteins can also be provided as chimeric molecules, such as in the form of fusion proteins.
- the smoothened protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the smoothened polypeptide.
- the invention features nucleic acids encoding smoothened polypeptides, which have the ability to modulate, e.g., either mimic or antagonize, at least a portion of the activity of a wild-type smoothened polypeptide.
- Exemplary smoothened-encoding nucleic acid sequences are represented by SEQ ID No: 1 , SEQ ID No: 2, SEQ ID No: 3 or SEQ ID No: 4.
- the nucleic acids of the present invention include coding sequences which hybridize under medium or high stringency conditions with all or a portion of the coding sequences designated in one or more of SEQ ID Nos: 1-4.
- the coding sequences of the nucleic acids can comprise sequences which are identical to coding sequences represented in SEQ ID Nos: 1-4, or it can merely be homologous to those sequences.
- the nucleic acids encode polypeptides which specifically modulate, by acting as either agonists or antagonists, one or more of the bioactivities of wild-type smoothened polypeptides.
- the subject smoothened nucleic acids will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the smoothened gene sequences.
- a transcriptional regulatory sequence e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the smoothened gene sequences.
- Such regulatory sequences can be used in to render the smoothened gene sequences suitable for use as an expression vector.
- This invention also contemplates the cells transfected with said expression vector whether prokaryotic or eukaryotic and a method for producing smoothened proteins by employing said expression vectors.
- the nucleic acid hybridizes under stringent conditions to nucleic acid probes corresponding to at least 12 consecutive nucleotides of either sense or antisense sequences of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 and SEQ ID No: 4; though preferably to at least 25 consecutive nucleotides; and more preferably to at least 40, 50 or 75 consecutive nucleotides of either sense or antisense sequence of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 and SEQ ID No: 4.
- Yet another aspect of the present invention concerns an immunogen comprising a smoothened polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a smoothened polypeptide; e.g.
- the immunogen comprising an antigenic determinant, e.g. a unique determinant, from a protein represented by one of SEQ ID No: 5, SEQ ID No: 6. SEQ ID No: 7 and/or SEQ ID No: 8.
- a still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the smoothened immunogen.
- the invention also features transgenic non-human animals, e.g. mice, rats, rabbits, chickens, frogs or pigs, having a transgene, e.g., animals which include (and preferably express) a heterologous form of a smoothened gene described herein, or which misexpress an endogenous smoothened gene, e.g., an animal in which expression of one or more of the subject smoothened proteins is disrupted.
- a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed smoothened alleles or for use in drug screening.
- the invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 12 consecutive nucleotides of sense or antisense sequences of any one or more of SEQ ID Nos: 1-4 and 9-14, or naturally occurring mutants thereof.
- the probe/primer further includes a label group attached thereto and able to be detected.
- the label group can be selected, e.g., from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co- factors.
- Probes of the invention can be used as a part of a diagnostic test kit for identifying dysfunctions associated with mis-expression of a smoothened protein, such as for detecting in a sample of cells isolated from a patient, a level of a nucleic acid encoding a smoothened protein; e.g. measuring a smoothened mRNA level in a cell, or determining whether a genomic smoothened gene has been mutated or deleted.
- probes/primers of the invention can also be used as a part of "antisense” therapy which refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g.
- the invention provides an assay for screening test compounds for inhibitors, or alternatively, potentiators, of an interaction between a patched protein and a smoothened polypeptide receptor.
- the step of detecting interaction of a target molecule, such as patched, and smoothened polypeptides is a competitive binding assay.
- the step of detecting interaction of the target molecule and smoothened polypeptides involves detecting, in a cell-based assay, change(s) in the level of an intracellular second messenger responsive to signaling mediated by the smoothened polypeptide.
- the ability to modulate the bioactivity of smoothened comprises detecting, in a cell-based assay, change(s) in the level of expression of a gene controlled by a transcriptional regulatory sequence responsive to signaling by the smoothened polypeptide.
- the steps of the assay are repeated for a variegated library of at least 100 different test compounds, more preferably at least 10 3 , 10 4 or 10 5 different test compounds.
- the test compound can be, e.g., a peptide, a nucleic acid, a carbohydrate, a small organic molecule, or natural product extract (or fraction thereof).
- Yet another aspect of the present invention concerns a method for modulating one or more of growth, differentiation, or survival of a cell by modulating smoothened bioactivity, e.g., by potentiating or disrupting certain protein-protein interactions.
- the method comprises treating the cell with an effective amount of a smoothened therapeutic so as to alter, relative to the cell in the absence of treatment, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell.
- the method can be carried out with smoothened therapeutics such as peptide and peptidomimetics or other molecules identified in the above-referenced drug screens which agonize or antagonize the effects of signaling from a smoothened protein.
- smoothened therapeutics include antisense constructs for inhibiting expression of smoothened proteins, and dominant negative mutants of smoothened proteins which competitively inhibit ligand interactions upstream and signal transduction downstream of the wild-type smoothened protein.
- the subject method of modulating smoothened bioactivity can be used in the treatment of testicular cells, so as to modulate spermatogenesis.
- the subject method is used to modulate osteogenesis, comprising the treatment of osteogenic cells with an agent that modulates smoothened bioactivity.
- the treated cell is a chondrogenic cell
- the present method is used to modulate chondrogenesis.
- the subject method can be used to modulate the differentiation of a neuronal cell, to maintain a neuronal cell in a differentiated state, and/or to enhance the survival of a neuronal cell, e.g., to prevent apoptosis or other forms of cell death.
- the present method can be used to affect the differentiation of neuronal cells such as motor neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, and peptidergic neurons.
- Another aspect of the present invention provides a method of determining if a subject, e.g. an animal patient, is at risk for a disorder characterized by unwanted cell proliferation or aberrant control of differentiation or apoptosis.
- the method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding a smoothened protein; or (ii) the mis- expression of a smoothened gene.
- detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from a smoothened gene; an addition of one or more nucleotides to the gene, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; a non-wild type level of the protein; and/or an aberrant level of soluble smoothened protein.
- detecting the genetic lesion can include (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of a smoothened gene or naturally occurring mutants thereof, or 5' or 3' flanking sequences naturally associated with the smoothened gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the smoothened gene and, optionally, of the flanking nucleic acid sequences.
- the probe/primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR).
- the level of a smoothened protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the smoothened protein.
- PCR polymerase chain reaction
- LCR ligation chain reaction
- the practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
- Figure 1 panels A-H are phase-contrast images of the ventral cuticle of pharate first instar larvae; anterior is to the top.
- a Wild type
- b smo ⁇ , raised at 25° C.
- Naked cuticle is deleted sporadically between adjacent denticle belts
- c smo ⁇ , raised at 18° C.
- Naked cuticle is replaced by denticle belts with reversed polarity, d, smo- embryo derived from smoDl ⁇ germline clone females.
- Germline clone females were generated by the dominant female sterile technique either by irradiating larvae with -rays or using flippase- induced mitotic recombination (Chou, T.B. & Perrimon, N. (1992) Genetics 131 :643-653). Ectopic expression of wg and hh was induced by the GAL4-UAS system using the hairy GAL4 enhancer trap, which expresses GAL4 in every other segment (Capdevilla, J. & Guerrero, 1.(1994) EMBO J. 13:4459-4468). /.GAL4 UASwg* 5 , UAS ⁇ , and /?GAL4 chromosomes have been described.
- Figure 2 show the pattern of expression of wg, hh, and En in wild-type and mutant embryos, a, Expression of wg in wild-type stage 10.
- the ectopic expression of hh induces the broadening of wg in all segments because of the overlap of endogenous wg and ectopic hh.
- En expression is maintained in alternate segments (arrowheads), corresponding to the segments where wg expression is driven by /zGAL4. In the intervening stripes, some En is rescued, presumably due to the paracrine action of the misexpressed wg.
- Embryos were collected for one hour and aged for the appropriate times. Fixations, in situ hybridizations and antibody stainings (for En, using monoclonal 4D9) have been described (Ingham, P.W. et al. (1991) supra; van den Huevel, M. et al. (1993) supra). Double-labeling with an hh flourescein-labeled and a wg digoxigenin probe was performed with two sequential alkaline phosphate substrates, BCIP NBT and Vector red; the enzyme was inactivated between the steps by treatment with 0.2M glycine, pH 2.5. Genotypes are described in Fig. 1 legend.
- Figure 3 Stainings of third instar imaginal discs, a, Dpp expression (green) anterior to the compartment boundary is shown, as marked by the expression of En (red) throughout the posterior compartment (as hh). b, Wing-blade area stained for Dpp (green) and Myc epitope (red). Arrow indicates a smo- clone in the dpp expression region, c-e, Details of disc in b. Similar clones within the dpp domain were found in six cases among 350 dissected progeny, of which 1/8 were of the correct genotype.
- Loss of Myc staining represents cells that have lost smo- activity, whereas nearby cells expressing high levels of Myc represent the sister clone carrying two copies of the myc construct.
- arrow points to a smo- clone within the posterior compartment.
- the absence of green staining (Dpp) corresponds cell for cell (e) with the absence of red staining smo-.
- f dpp expression g, as in /, except that clones lacking smo- and the catalytic subunit of PKA were induced; these express dpp ectopically in the compartment owing to the overexpression of dpp (Capdevilla J. & Guerrero I. (1994) EMBO J.
- CMOS complementary metal-oxide-semiconductor
- CMOS complementary metal-oxide-semiconductor
- Discs were stained with anti-En, anti-Dpp (Panganiban, G. et al. (1990) Mol. Cell. Biol. 10:2669- 2677), anti- -galactosidase (Promega) and anti-Myc epitope antibodies or stained for - galactosidase activity using X-gal, following standard procedures.
- Figure 4 show various steps in the cloning of smoothened.
- A Southern blot of genomic DNA (digested with BamW ⁇ ) from different smo alleles, hybridized with a subclone from a PI clone (see c). Six PI clones, covering the area of 21B7-8, the region to which smo maps (based on the exclusion from Df2L(al) and Df2L (PMF), were screened for the detection of aberrations in smo alleles.
- B Developmental northern blot hybridized with subclone as in C.
- C Restriction map of the smo locus. Complementary DNAs for all three transcripts were isolate.
- the genomic DNA used to make a transgenic fly is indicated.
- Bold bars indicate the smo gene exons.
- D The one-letter amino-acid sequence of the open reading frame in the smo gene is shown. Three more methionine codons are found in-frame before the indicated start of translation, two of which are not surrounded by residues creating a good translation start site ((Brown, N.H. & Kafatos, F.C. (1988) J. Mol. Biol. 203:425-437); a third methionine, just one codon before, is interrupted by an intron. Hydrophobic stretches are underlined, the first one probably representing a signal sequence. Arrow denotes a putative signal peptidase cleavage site. The homology with the Drosophila Fz protein is confined to the putative transmembrane region.
- PI phage DNA was prepared by the alkaline lysis method and digested; DNA fragments were isolated from agarose gels by spinning through glasswool and subcloned in BlueScript )Stratagene). Genomic fly DNA was prepared, digested with restriction enzymes, separated on agarose gels and transferred to Hybond N (Amersham). PI fragments were labeled with [ 32P]dCTP (Amersham protocol) and hybridized to the filters under standard conditions. Total RNA from different stages was prepared by guanidine-HCl extraction and acetic acid precipitation. RNA samples were run under identical conditions and the amount of RNA loaded was controlled by staining the blot with methylene blue after hybridization.
- the restriction fragment indicated in c was isolated and cloned into pCaSpeR for transformation.
- Transgenic flies were generated and identified among the FI progeny on the basis of their eye pigmentation, and balanced lines were established.
- a partial complementary DMA for smo was isolated from a 0-2 h cDNA library (Brown, N.H. and Kafatos, F.C. (1988) supra) by hybridization with a probe contained within the rescue fragment.
- the smo cDNA and genomic subclones completely covering the region of the rescue fragment were sequenced using the dideoxy method with Sequenase (USB); both strands of the DNA were sequenced at least twice.
- the start of transcription and intron- exon boundaries were confirmed by sequencing fragments generated by RT-PCR, using primers designed from the genomic sequence.
- induction a type of extracellular communication
- chemical signals secreted by one cell population influence the developmental fate of a second cell population.
- cells responding to the inductive signals are diverted from one cell fate to another, neither of which is the same as the fate of the signaling cells.
- Inductive signals are key regulatory proteins that function in vertebrate pattern formation, and are present in important signaling centers known to operate embryonically, for example, to define the organization of the vertebrate embryo.
- these signaling structures include the notochord, a transient structure which initiates the formation of the nervous system and helps to define the different types of neurons within it.
- the notochord also regulates mesodermal patterning along the body axis.
- Another distinct group of cells having apparent signaling activity is the floorplate of the neural tube (the precursor of the spinal cord and brain) which also signals the differentiation of different nerve cell types.
- Zone of Polarizing Activity operates as a signaling center by secreting a morphogen which ultimately produces the correct patterning of the developing limbs.
- hedgehog protein signaling is an important mechanism for developmental control.
- Members of the hedgehog family of secreted proteins control a number of important inductive interactions in the development of both vertebrates and Drosophila (Ingham, P.W. (1995) Opin. Gen. Dev. 5:492-498).
- hedgehog is one of the segment-polarity genes, mutations of which disrupt the pattern and polarity of individual embryonic segments (N ⁇ sslein-Volhard, C. & Wieschaus, E. (1980) Nature 287:795-801) and their adult derivatives (Williams, J.A. & Carroll, S.B. (1993) Bioessays 15:567-577).
- the present invention concerns the discovery of a new family of cell surface proteins, referred to herein as "smoothened” proteins.
- smoothened gene product is required for the response of cells to hedgehog signaling during the development.
- Sequence analysis of the smoothened transcription unit reveals a single open reading frame encoding a protein with seven putative transmembrane domains. This structure is typical of G-protein-coupled receptors.
- the vertebrate smoothened proteins exhibit spatially and temporally restricted expression domains indicative of important roles in hedgehog-mediated induction.
- the sequence of exemplary smoothened genes cloned from various metazoan organisms indicates it encodes a receptor-like serpentine protein that may be anchored at the cell membrane.
- Comparison of smoothened sequences from drosophila, chicken, rat and human clones suggests that smoothened is an integral membrane proteins with seven membrane spanning ⁇ helices, a long cytoplasmic tail, and a conserved signal peptide sequence.
- analysis of the protein sequences suggests potential sites for modification by N-linked glycosylation, as well as potential phosphorylation sites for G-protein coupled receptors and cAMP-dependent kinases (e.g., PKA).
- the vertebrate smoothened proteins also include potential protein-protein interaction modules such as leucine zippers and RGD sequences.
- the smoothened coding sequences has weak homology with the frizzled genes, particularly across the transmembrane domains. However, within the serpentine receptor family, the evidence provided herein suggests that these genes comprise a novel sub-family of receptors.
- the smoothened proteins through their ability to associate with patched and/or hedgehog proteins, are apparently capable of modulating hedgehog signaling.
- the smoothened proteins may function as a constitutively active signaling protein whose signal transduction capabilities are inhibited by interaction with patched, a hedgehog receptor (or subunit thereof). Binding of hedgehog to patched alters the interaction of smoothened and patched, and relieves the patched-mediated inhibition of smoothened signal transduction.
- the smoothened polypeptides of the present invention may affect a number of
- g-mediated biological activities including: an ability to modulate proliferation, survival and/or differentiation of mesodermally-derived tissue, such as tissue derived from dorsal mesoderm, cartilage and tissue involved in spermatogenesis; the ability to modulate proliferation, survival and/or differentiation of ectodermally-derived tissue, such as tissue derived from the epidermis, neural tube, neural crest, or head mesenchyme; the ability to modulate proliferation, survival and/or differentiation of endodermally-derived tissue, such as tissue derived from the primitive gut.
- mesodermally-derived tissue such as tissue derived from dorsal mesoderm, cartilage and tissue involved in spermatogenesis
- ectodermally-derived tissue such as tissue derived from the epidermis, neural tube, neural crest, or head mesenchyme
- endodermally-derived tissue such as tissue derived from the primitive gut.
- a drosophila smoothened orf open reading frame
- a chicken smoothened cDNA was isolated by hybridization under low to medium stringency conditions with the drosophila smoothened coding sequence.
- the art has recently reported cDNA clones from other vertebrates, including human and rodent smoothened genes.
- a drosophila smoothened polypeptide is encoded by SEQ ID No:l ; a human smoothened polypeptide is encoded by SEQ ID No:2; a rat smoothened polypeptide is encoded by SEQ ID No:3; and a chicken smoothened polypeptide is encoded by SEQ ID No:4.
- the vertebrate smoothened proteins are apparently present naturally in a number of different forms, including a pro-form.
- the pro-form includes an N-terminal signal peptide for directed secretion of at least the first extracellular domain of the protein, while the full- length mature form may lack this signal sequence. Further processing of the mature form may also occur in some instances to yield biologically active extracellular or intracellular fragments of the protein.
- the smoothened proteins may also be modified post- translationally, such as by O-, S- and/or N-linked glycosylation. Potential Asn- glycosylation sites are shown in figure 4.
- Smo mutants display phenotypes similar to the hh mutants, both in embryos and in clones of mutant cells in drosophila imaginal disks. Moreover, the effects of ectopic hedgehog become negligible when cells lack smoothened. When embryos lack both smoothened and patched, they have a similar phenotype to smo single mutants, indicating that patched is located genetically upstream of smoothened. It is postulated that secreted hedgehog protein binds to patched. This binding relieves the /. ⁇ tc/ze -dependent inhibition of smoothened, and may involve direct contact between smoothened and patched.
- smoothened activates the downstream genes wg (Wnts in vertebrates), dpp (TGF ⁇ proteins) and patched, through the signaling components fused, costal-2 and cubitus interruptus (Gli's).
- This pathway may also involve the inhibition of protein kinase A (PKA), though the latter may merely act in parallel.
- PKA protein kinase A
- smoothened would have a constitutive (hedgehog-m ⁇ e ⁇ endent) activity in the absence of any inhibition by patched.
- certain aspects of the present invention relate to nucleic acids encoding smoothened polypeptides, the smoothened polypeptides themselves (including various fragments), antibodies immunoreactive with smoothened proteins, and preparations of such compositions.
- the present invention provides diagnostic and therapeutic assays and reagents for detecting and treating disorders involving, for example, aberrant expression (or loss thereof) of smoothened, smoothened ligands, or signal transducers thereof.
- drug discovery assays are provided for identifying agents which can modulate the biological function of smoothened proteins, such as by altering the interaction of smoothened and patched proteins, or other extracellular/matrix factors, or the ability of smoothened proteins to transduce intracellular signals.
- agents can be useful therapeutically to alter the growth, maintenance and/or differentiation of a tissue, particularly a mesodermally-derived tissue, such cartilage, tissue involved in spermatogenesis and tissue derived from dorsal mesoderm; ectodermally-derived tissue, such as tissue derived from the epidermis, neural tube, neural crest, or head mesenchyme; endodermally-derived tissue, such as tissue derived from the primitive gut.
- a tissue particularly a mesodermally-derived tissue, such cartilage, tissue involved in spermatogenesis and tissue derived from dorsal mesoderm
- ectodermally-derived tissue such as tissue derived from the epidermis, neural tube, neural crest, or head me
- smoothened polypeptide refers to a family of polypeptides characterized at least in part by being identical or sharing a degree of sequence homology with all or a portion of the a smoothened polypeptide represented in any of SEQ ID Nos: 5-8.
- the smoothened polypeptides can be cloned or purified from any of a number of eukaryotic organisms, especially vertebrates, and particularly mammals.
- other smoothened polypeptides can be generated according to the present invention, which polypeptides do not ordinarily exist in nature, but rather are generated by non-natural mutagenic techniques.
- a “transmembrane” region refers to sequence of amino acids that is located in the cellular membrane, e.g., retained in the membrane at the cell surface.
- a "glycosylated” smoothened polypeptide is an smoothened polypeptide having a covalent linkage with a glycosyl group (e.g. a derivatized with a carbohydrate).
- the smoothened protein can be glycosylated on an existing residue, or can be mutated to preclude carbohydrate attachment, or can be mutated to provide new glycosylation sites, such as for N-linked or O-linked glycosylation.
- vertebrate hedgehog protein refers to vertebrate intercellular signaling molecules related to the Drosophilia hedgehog protein.
- Three of the vertebrate hedgehog proteins Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in all vertebrates, including amphibians, fish, birds, and mammals.
- Other members of this family such as Banded hedgehog, Cephalic hedgehog, tiggy-winkle hedgehog, and echidna hedgehog have been so far identified in fish and/or amphibians.
- Exemplary hedgehog polypeptides are described in PCT applications WO96/17924, WO96/16668, WO95/18856.
- nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
- the term "gene” or “recombinant gene” refers to a nucleic acid comprising an open reading frame encoding a smoothened polypeptide, including both exon and (optionally) intron sequences.
- a “recombinant gene” refers to nucleic acid encoding a smoothened polypeptide and comprising smoothened-encoding exon sequences, though it may optionally include intron sequences which are derived from, for example, a chromosomal smoothened gene (see SEQ ID NO. 9) or from an unrelated chromosomal gene.
- Exemplary recombinant genes encoding the subject smoothened polypeptide are represented in the appended Sequence Listing.
- intron refers to a DNA sequence present in a given smoothened gene which is not translated into protein and is generally found between exons.
- transfection means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
- Transformation refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of a smoothened polypeptide or, where anti- sense expression occurs from the transferred gene, the expression of a naturally-occurring form of the smoothened protein is disrupted.
- the term “specifically hybridizes” refers to the ability of a nucleic acid probe/primer of the invention to hybridize to at least 15 consecutive nucleotides of a smoothened gene, such as a smoothened sequence designated in any one or more of SEQ ID Nos: 1-4, or a sequence complementary thereto, or naturally occurring mutants thereof, such that it has less than 15%>, preferably less than 10%, and more preferably less than 5% background hybridization to a cellular nucleic acid (e.g., mRNA or genomic DNA) encoding a protein other than a smoothened protein, as defined herein.
- a smoothened gene such as a smoothened sequence designated in any one or more of SEQ ID Nos: 1-4, or a sequence complementary thereto, or naturally occurring mutants thereof, such that it has less than 15%>, preferably less than 10%, and more preferably less than 5% background hybridization to a cellular nucleic acid (e.g., mRNA or genomic DNA) encoding a protein other than
- an "effective amount" of a hedgehog polypeptide, or a bioactive fragment thereof, with respect to the subject method of treatment refers to an amount of agonist or antagonist in a preparation which, when applied as part of a desired dosage regimen, provides modulation of growth, differentiation or survival of cells, e.g., modulation of spermatogenesis, neuronal differentiation, or skeletogenesis, e.g., osteogenesis, chondrogenesis, or limb patterning.
- phenotype refers to the entire physical, biochemical, and physiological makeup of a cell, e.g., having any one trait or any group of traits.
- induction refers generally to the process or act of causing to occur a specific effect on the phenotype of cell.
- Such effect can be in the form of causing a change in the phenotype, e.g., differentiation to another cell phenotype, or can be in the form of maintaining the cell in a particular cell, e.g., preventing dedifferentation or promoting survival of a cell.
- a "patient” or “subject” to be treated can mean either a human or non-human animal.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
- Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked.
- Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
- expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
- plasmid and "vector” are used interchangeably as the plasmid is the most commonly used form of vector.
- vector is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
- Transcriptional regulatory sequence is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
- transcription of a recombinant smoothened gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring forms of smoothened genes.
- tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as cells of neuronal or hematopoietic origin.
- tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as cells of neuronal or hematopoietic origin.
- the term also covers so- called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but can cause at least low level expression in other tissues as well.
- target tissue refers to connective tissue, cartilage, bone tissue or limb tissue, which is either present in an animal, e.g., a mammal, e.g.. a human or is present in in vitro culture, e.g., a cell culture.
- a "transgenic animal” is any animal, preferably a non-human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
- the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
- the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
- transgenic animal the transgene causes cells to express a recombinant form of a smoothened protein, e.g. either agonistic or antagonistic forms.
- transgenic animals in which the recombinant smoothened gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below.
- transgenic animal also includes those recombinant animals in which gene disruption of one or more smoothened genes is caused by human intervention, including both recombination and antisense techniques.
- the "non-human animals" of the invention include vertebrates such as rodents, non- human primates, livestock, avian species, amphibians, reptiles, etc.
- chimeric animal is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant is expressed in some but not all cells of the animal.
- tissue-specific chimeric animal indicates that a recombinant smoothened gene is present and/or expressed or disrupted in some tissues but not others.
- transgene means a nucleic acid sequence (encoding, e.g., a smoothened polypeptide, or pending an antisense transcript thereto), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
- a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
- genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same activity.
- the term "DNA sequence encoding a smoothened polypeptide" may thus refer to one or more genes within a particular individual.
- certain differences in nucleotide sequences may exist between individuals of the same species, which are called alleles.
- allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.
- Homology refers to sequence similarity between two peptides or between two nucleic acid molecules.
- Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
- An "unrelated" or “non-homologous” sequence shares less than 40 percent identity, though preferably less than 25 percent identity, with a smoothened sequence of the present invention.
- the term “ortholog” refers to genes or proteins which are homologs via speciation, e.g., closely related and assumed to have common descent based on structural and functional considerations. Orthologous proteins function as recognizably the same activity in different species.
- paralog refers to genes or proteins which are homologs via gene duplication, e.g., duplicated variants of a gene within a genome. See also, Fritch, WM (1970) Syst Zool 19:99-
- Cells “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
- a “chimeric protein” or “fusion protein” is a fusion of a first amino acid sequence encoding a smoothened polypeptide with a second amino acid sequence defining a domain (e.g. polypeptide portion) foreign to and not substantially homologous with any domain of a smoothened protein.
- a chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", “intergenic”, etc. fusion of protein structures expressed by different kinds of organisms.
- a fusion protein can be represented by the general formula -smoothened-Y , wherein smoothened represents a portion of the fusion protein which is derived from a smoothened protein, and X and Y are, independently, absent or represent amino acid sequences which are not related to a smoothened sequences in an organism.
- a "reporter gene construct” is a nucleic acid that includes a “reporter gene” operatively linked to a transcriptional regulatory sequences. Transcription of the reporter gene is controlled by these sequences. The activity of at least one or more of these control sequences is directly or indirectly regulated by a signal transduction pathway involving a phospholipase, e.g., is directly or indirectly regulated by a second messenger produced by the phospholipase activity.
- the transcriptional regulatory sequences can include a promoter and other regulatory regions, such as enhancer sequences, that modulate the activity of the promoter, or regulatory sequences that modulate the activity or efficiency of the RNA polymerase that recognizes the promoter, or regulatory sequences that are recognized by effector molecules, including those that are specifically induced upon activation of a phospholipase.
- modulation of the activity of the promoter may be effected by altering the RNA polymerase binding to the promoter region, or, alternatively, by interfering with initiation of transcription or elongation of the mRNA.
- Such sequences are herein collectively referred to as transcriptional regulatory elements or sequences.
- the construct may include sequences of nucleotides that alter the stability or rate of translation of the resulting mRNA in response to second messages, thereby altering the amount of reporter gene product.
- an isolated nucleic acid encoding a smoothened polypeptide preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the smoothened gene in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequences, and most preferably less than 1.5kb of such naturally occurring flanking sequence.
- kb kilobases
- isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
- isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
- isolated nucleic acids comprising nucleotide sequences encoding smoothened polypeptides, and/or equivalents of such nucleic acids.
- nucleic acid as used herein is intended to include fragments as equivalents.
- equivalent is understood to include nucleotide sequences encoding functionally equivalent smoothened polypeptides or functionally equivalent peptides having an activity of a smoothened protein such as described herein.
- Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will, therefore, include sequences that differ from the nucleotide sequence of the smoothened coding sequences shown in any one or more of SEQ ID Nos: 1-4 due to the degeneracy of the genetic code.
- Equivalents will also include nucleotide sequences that hybridize under stringent conditions (i.e., equivalent to about 20-27°C below the melting temperature (T m ) of the DNA duplex formed in about 1M salt) to the nucleotide sequences represented in SEQ ID No: 1-4 or 9.
- equivalents will further include nucleic acid sequences derived from and evolutionarily related to, a nucleotide sequences shown in SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 and/or SEQ ID No: 4.
- homologs of a smoothened polypeptide which function in a limited capacity as one of either an agonist (e.g., mimics or potentiates a bioactivity of the wild-type smoothened protein) or an antagonist (e.g., inhibits a bioactivity of the wild-type smoothened protein), in order to promote or inhibit only a subset of the biological activities of the naturally-occurring form of the protein.
- an agonist e.g., mimics or potentiates a bioactivity of the wild-type smoothened protein
- an antagonist e.g., inhibits a bioactivity of the wild-type smoothened protein
- truncated forms of a smoothened protein e.g., soluble fragments of the extracellular domain
- Homologs of the subject smoothened protein can be generated by mutagenesis, such as by discrete point mutation(s), or by truncation. For instance, mutation can give rise to homologs which retain substantially the same, or merely a subset, of the biological activity of the smoothened protein from which it was derived.
- antagonistic forms of the protein can be generated which are able to inhibit the function of the naturally occurring form of the protein, such as by competitively binding to patched proteins and competing with wild-type smoothened, or binding to other smoothened interacting proteins.
- the smoothened protein and homologs thereof provided by the subject invention may be either positive or negative regulators of cell growth, death and/or differentiation.
- polypeptides referred to herein as having an activity of a smoothened protein are defined as polypeptides which include an amino acid sequence corresponding (e.g., identical or similar) to all or a portion of the amino acid sequences of the smoothened protein shown in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 8, and which agonize or antagonize all or a portion of the biological/biochemical activities of a naturally occurring smoothened protein.
- biological activity includes the ability to interact with patched, (optionally) interact with hedgehog, regulate fused, costal-2 or Gli activities.
- bioactivity of certain embodiments of the subject smoothened polypeptides can be characterized in terms of an ability to promote differentiation and/or maintenance of cells and tissue from mesodermally-derived tissue, such as tissue derived from dorsal mesoderm; ectodermally- origin, such as tissue derived from the neural tube, neural crest, or head mesenchyme; or endodermally-derived tissue, such as tissue derived from the primitive gut.
- mesodermally-derived tissue such as tissue derived from dorsal mesoderm
- ectodermally- origin such as tissue derived from the neural tube, neural crest, or head mesenchyme
- endodermally-derived tissue such as tissue derived from the primitive gut.
- a polypeptide has biological activity if it is a specific agonist or antagonist of a naturally- occurring form of a smoothened protein.
- Preferred nucleic acids encode a smoothened polypeptide comprising an amino acid sequence at least 60%>, 63%, 70% or 80% homologous, more preferably at least 85%o homologous and most preferably at least 93% or 95%) homologous with an amino acid sequence of a naturally occurring smoothened protein, e.g., such as represented in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 8.
- nucleic acids which encode polypeptides at least about 98-99% homology with an amino acid sequence represented in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 8 are of course also within the scope of the invention, as are nucleic acids identical in sequence with the enumerated smoothened sequence of the Sequence listing.
- the nucleic acid is a cDNA encoding a polypeptide having at least one activity of the subject smoothened polypeptide.
- the invention features a purified or recombinant smoothened polypeptide.
- the smoothened protein can include certain post-translational modifications, e.g., glycosylation, phosphorylation and the like, and cleavage of certain sequences, such as pro-sequences.
- nucleic acid which hybridizes under high or low stringency conditions to one or more of the nucleic acids represented by SEQ ID Nos: 1-4 and 9.
- Appropriate stringency conditions which promote DNA hybridization for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
- the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C.
- the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C.
- Nucleic acids having a sequence that differs from the nucleotide sequences shown in any of SEQ ID Nos: 1-4 due to degeneracy in the genetic code are also within the scope of the invention.
- Such nucleic acids encode functionally equivalent peptides (i.e., a peptide having a biological activity of a smoothened polypeptide) but differ in sequence from the sequence shown in the sequence listing due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC each encode histidine) may result in "silent" mutations which do not affect the amino acid sequence of a smoothened polypeptide.
- DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject smoothened polypeptides will exist among, for example, humans.
- these variations in one or more nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids encoding polypeptides having an activity of a smoothened polypeptide may exist among individuals of a given species due to natural allelic variation.
- a smoothened gene fragment refers to a nucleic acid having fewer nucleotides than the nucleotide sequence encoding the entire mature form of a smoothened protein yet which (preferably) encodes a polypeptide which retains some biological activity of the full length protein. Fragment sizes contemplated by the present invention include, for example, 5, 10, 25, 50, 75, 100, or 200 amino acids in length.
- the polypeptide will include all or a sufficient portion of a patched- interacting domain or, as appropriate, a ligand domain or intracellular domain involved in signal transduction.
- smoothened protein-encoding nucleic acids can be obtained from mRNA present in cells of metazoan organisms. As further illustrated in the examples, it is also possible to obtain nucleic acids encoding smoothened polypeptides of the present invention from genomic DNA from both adults and embryos.
- a gene encoding a smoothened protein can be cloned from either a cDNA or a genomic library in accordance with protocols described herein, as well as those generally known to persons skilled in the art.
- a cDNA encoding a smoothened protein can be obtained by isolating total mRNA from a cell, such as a mammalian cell, e.g. a human cell, as desired.
- Double stranded cDNAs can be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques.
- the gene encoding a smoothened protein can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention.
- the nucleic acid of the invention can be DNA or RNA.
- a preferred nucleic acid is a cDNA including a nucleotide sequence represented by any one of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, or SEQ ID No: 4.
- antisense therapy refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g. binds) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a subject smoothened protein so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation.
- the binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
- antisense refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.
- an antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a smoothened protein.
- the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of a smoothened gene.
- oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and are therefore stable in vivo.
- nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775), or peptide nucleic acids (PNAs). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668. Accordingly, the modified oligomers of the invention are useful in therapeutic, diagnostic, and research contexts.
- the oligomers are utilized in a manner appropriate for antisense therapy in general.
- the oligomers of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA.
- injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous.
- the oligomers of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
- the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
- Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally.
- penetrants appropriate to the barrier to be permeated are used in the formulation.
- penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
- detergents may be used to facilitate permeation.
- Transmucosal administration may be through nasal sprays or using suppositories.
- the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
- the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
- the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind. Such diagnostic tests are described in further detail below.
- the antisense constructs of the present invention by antagonizing the normal biological activity of a smoothened protein, e.g., by reducing the level of its expression, can be used in the manipulation of tissue, e.g. tissue maintenance, differentiation or growth, both in vivo and ex vivo.
- anti-sense techniques e.g. microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to a smoothened mRNA or gene sequence
- Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals (described infra).
- This invention also provides expression vectors containing a nucleic acid encoding a smoothened polypeptide, operably linked to at least one transcriptional regulatory sequence.
- Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence.
- Regulatory sequences are art-recognized and are selected to direct expression of the subject smoothened proteins. Accordingly, the term transcriptional regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
- any of a wide variety of expression control sequences, sequences that control the expression of a DNA sequence when operatively linked to it, may be used in these vectors to express DNA sequences encoding smoothened polypeptides of this invention.
- useful expression control sequences include, for example, a viral LTR, such as the LTR of the Moloney murine leukemia virus, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the tip system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage ⁇ , the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ -mating factors, the polyhedron promoter of the baculovirus system and other sequence
- the expression vector includes a recombinant gene encoding a polypeptide having an agonistic activity of a subject smoothened polypeptide, or alternatively, encoding a polypeptide which is an antagonistic form of the smoothened protein.
- Such expression vectors can be used to transfect cells and thereby produce polypeptides, including fusion proteins, encoded by nucleic acids as described herein.
- gene constructs of the present invention can also be used as a part of a gene therapy protocol to deliver nucleic acids, e.g., encoding either an agonistic or antagonistic form of a subject smoothened proteins or an antisense molecule described above.
- another aspect of the invention features expression vectors for in vivo or in vitro transfection and expression of a smoothened polypeptide or antisense molecule in particular cell types so as to reconstitute the function of, or alternatively, abrogate all or a portion of the biological function of .swoot/?e/.e_ -induced transcription in a tissue in which the naturally-occurring form of the protein is misexpressed (or has been disrupted); or to deliver a form of the protein which alters proliferation, maintenance or differentiation of tissue, or which inhibits neoplastic or hyperplastic proliferation.
- Expression constructs of the subject smoothened polypeptides, as well as antisense constructs, may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the recombinant gene to cells in vivo.
- Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids.
- Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g.
- transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e.g. locally or systemically.
- the particular gene construct provided for in vivo transduction of smoothened expression are also useful for in vitro transduction of cells, such as for use in the ex vivo tissue culture systems described below.
- a preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA encoding the particular smoothened polypeptide desired.
- a viral vector containing nucleic acid e.g. a cDNA encoding the particular smoothened polypeptide desired.
- Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid.
- molecules encoded within the viral vector e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
- Retrovirus vectors, adenovirus vectors, adeno-associated and herpes-based virus vectors are exemplary recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the
- non-viral methods can also be employed to cause expression of a subject smoothened polypeptide in the tissue of an animal.
- Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
- non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject smoothened genes by the targeted cell.
- Exemplary gene delivery systems of this type include liposomal derived systems, poly- lysine conjugates, and artificial viral envelopes.
- the gene delivery systems for the therapeutic smoothened gene can , be introduced into a patient-animal by any of a number of methods, each of which is familiar in the art.
- a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.
- initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
- the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g.
- a smoothened gene can be delivered in a gene therapy construct by electroporation using techniques described, for example, by Dev et al. ((1994) Cancer Treat Rev 20:105-115).
- the pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
- the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
- the subject invention provides a "gene activation" construct which, by homologous recombination with a genomic DNA, alters the transcriptional regulatory sequences of an endogenous smoothened gene.
- the gene activation construct can replace the endogenous promoter of a smoothened gene with a heterologous promoter, e.g., one which causes constitutive expression of the smoothened gene or which causes inducible expression of the gene under conditions different from the normal expression pattern of smoothened.
- a variety of different formats for the gene activation constructs are available. See, for example, the Transkaryotic Therapies, Inc PCT publications WO93/09222, WO95/31560, WO96/2941 1, WO95/31560 and WO94/12650.
- the nucleotide sequence used as the gene activation construct can be comprised of (1) DNA from some portion of the endogenous smoothened gene (exon sequence, intron sequence, promoter sequences, etc.) which direct recombination and (2) heterologous transcriptional regulatory sequence(s) which is to be operably linked to the coding sequence for the genomic smoothened gene upon recombination of the gene activation construct.
- the construct may further include a reporter gene to detect the presence of the knockout construct in the cell.
- the gene activation construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to provide the heterologous regulatory sequences in operative association with the native smoothened gene.
- Such insertion occurs by homologous recombination, i.e., recombination regions of the activation construct that are homologous to the endogenous smoothened gene sequence hybridize to the genomic DNA and recombine with the genomic sequences so that the construct is incorporated into the corresponding position of the genomic DNA.
- recombination region or "targeting sequence” refer to a segment (i.e., a portion) of a gene activation construct having a sequence that is substantially identical to or substantially complementary to a genomic gene sequence, e.g., including 5' flanking sequences of the genomic gene, and can facilitate homologous recombination between the genomic sequence and the targeting transgene construct.
- replacement region refers to a portion of a activation construct which becomes integrated into an endogenous chromosomal location following homologous recombination between a recombination region and a genomic sequence.
- the heterologous regulatory sequences can include one or more of a variety elements, including: promoters (such as constitutive or inducible promoters), enhancers, negative regulatory elements, locus control regions, transcription factor binding sites, or combinations thereof.
- Promoters/enhancers which may be used to control the expression of the targeted gene in vivo include, but are not limited to, the cytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989, J. Exp. Med, 169:13), the human ⁇ -actin promoter (Gunning et al.
- CMV cytomegalovirus
- MMTV LTR mouse mammary tumor virus long terminal repeat
- MoLV LTR Moloney murine leukemia virus
- SV40 early or late region promoter Bosset et al. (1981) Nature 290:304-310; Templeton et al. (1984) Mol. Cell Biol., 4:817; and Sprague et al.
- the replacement region merely deletes a negative transcriptional control element of the native gene, e.g., to activate expression, or ablates a positive control element, e.g., to inhibit expression of the targeted gene.
- Another aspect of the present invention concerns recombinant forms of the smoothened proteins.
- Recombinant polypeptides preferred by the present invention, in addition to native smoothened proteins, are at least 60%, 63%, 64% or 70% homologous, more preferably at least 80% homologous and most preferably at least 85% homologous with an amino acid sequence represented by one or more of SEQ ID Nos: 5, SEQ ID No: 6,
- Polypeptides which possess an activity of a smoothened protein i.e. either agonistic or antagonistic
- which are at least 90% more preferably at least 93% or 95%, and most preferably at least about 98-99%> homologous with SEQ ID No:
- SEQ ID No: 6 SEQ ID No: 7 and/or SEQ ID No: 8 are also within the scope of the invention.
- Such polypeptides, as described above, include various truncated forms of the protein.
- recombinant smoothened polypeptide refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding a smoothened polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
- the phrase "derived from”, with respect to a recombinant smoothened gene is meant to include within the meaning of "recombinant protein” those proteins having an amino acid sequence of a native smoothened protein, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions (including truncation) of a naturally occurring form of the protein.
- the present invention further pertains to recombinant forms of the subject smoothened polypeptides which are encoded by genes derived from a mammal (e.g. a human), reptile or amphibian and which have amino acid sequences evolutionarily related to the smoothened protein represented in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8.
- Such recombinant smoothened polypeptides preferably are capable of functioning in one of either role of an agonist or antagonist of at least one biological activity of a wild-type ("authentic") smoothened protein of the appended sequence listing.
- the present invention also provides methods of producing the subject smoothened polypeptides.
- a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding the subject polypeptides can be cultured under appropriate conditions to allow expression of the polypeptide to occur.
- a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
- the recombinant smoothened polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such peptide.
- the recombinant smoothened polypeptide is isolated in a membrane fraction of a liposome.
- the fusion protein can include a domain which facilitates its purification, such as GST fusion protein or poly(His) fusion protein.
- This invention also pertains to a host cell transfected to express recombinant forms of the subject smoothened polypeptides.
- the host cell may be any eukaryotic or prokaryotic cell, though eukaryotic cells are preferred, especially mammalian cells.
- a nucleotide sequence derived from the cloning of smoothened genes, encoding all or a selected portion of a full-length protein can be used to produce a recombinant form of a smoothened polypeptide via microbial or eukaryotic cellular processes.
- the recombinant smoothened genes can be produced by ligating nucleic acid encoding a smoothened polypeptide into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both.
- Expression vectors for production of recombinant forms of the subject smoothened polypeptides include plasmids and other vectors.
- suitable vectors for the expression of a smoothened polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX- derived plasmids, pBTac-derived plasmids and pUC -derived plasmids for expression in prokaryotic cells, such as E. coli.
- YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incorporated by reference herein).
- These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.
- drug resistance markers such as ampicillin can be used.
- a smoothened polypeptide is produced recombinantly utilizing an expression vector generated by sub-cloning the coding sequence of a smoothened gene represented in SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, or SEQ ID No: 9.
- the preferred mammalian expression vectors contain both prokaryotic sequences, to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
- the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
- vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
- derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
- BBV-1 bovine papillomavirus
- pHEBo Epstein-Barr virus
- the various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.
- suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.
- baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the ⁇ -gal containing pBlueBac III).
- a start codon AGT
- MAP methionine aminopeptidase
- the coding sequences for the polypeptide can be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide.
- This type of expression system can be useful under conditions where it is desirable to produce an immunogenic fragment of a smoothened protein.
- the VP6 capsid protein of rotavirus can be used as an immunologic carrier protein for portions of the smoothened polypeptide, either in the monomeric form or in the form of a viral particle.
- nucleic acid sequences corresponding to the portion of a subject smoothened protein to which antibodies are to be raised can be incorporated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising smoothened epitopes as part of the virion. It has been demonstrated with the use of immunogenic fusion proteins utilizing the Hepatitis B surface antigen fusion proteins that recombinant Hepatitis B virions can be utilized in this role as well.
- chimeric constructs coding for fusion proteins containing a portion of a smoothened protein and the poliovirus capsid protein can be created to enhance immunogenicity of the set of polypeptide antigens (see, for example, EP Publication No: 0259149; and Evans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol. 62:3855; and Schlienger et al. (1992) J. Virol. 66:2).
- the Multiple Antigen Peptide system for peptide-based immunization can also be utilized to generate an immunogen, wherein a desired portion of a smoothened polypeptide is obtained directly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al. (1988) JBC 263:1719 and Nardelli et al. (1992) J. Immunol. 148:914).
- Antigenic determinants of smoothened proteins can also be expressed and presented by bacterial cells.
- fusion proteins can also facilitate the expression of proteins, and accordingly, can be used in the expression of the smoothened polypeptides of the present invention, particularly truncated forms of the smoothened protein.
- soluble forms of smoothened polypeptides can be generated as glutathione-S-transferase (GST- fusion) proteins.
- GST-fusion proteins can enable easy purification of the smoothened polypeptide, as for example by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).
- fusion genes are known to those skilled in the art. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
- the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
- PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
- the smoothened polypeptides may also be chemically modified to create smoothened derivatives by forming covalent or aggregate conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like.
- Covalent derivatives of smoothened proteins can be prepared by linking the chemical moieties to functional groups on amino acid sidechains of the protein or at the N-terminus or at the C- terminus of the polypeptide.
- the present invention also makes available isolated smoothened polypeptides which are isolated from, or otherwise substantially free of other cellular proteins, especially receptors and/or other inductive polypeptides which may normally be associated with the smoothened polypeptide.
- the term "substantially free of other cellular proteins" (also referred to herein as "contaminating proteins") or “substantially pure or purified preparations” are defined as encompassing preparations of smoothened polypeptides having less than 20%) (by dry weight) contaminating protein, and preferably having less than 5%> contaminating protein.
- Functional forms of the subject polypeptides can be prepared, for the first time, as purified preparations by using a cloned gene as described herein.
- purified it is meant, when referring to a peptide or DNA or RNA sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins.
- the term “purified” as used herein preferably means at least 80%> by dry weight, more preferably in the range of 95-99%> by weight, and most preferably at least 99.8%o by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
- pure as used herein preferably has the same numerical limits as “purified” immediately above.
- isolated and purified do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g. lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e.g. acrylamide or agarose) substances or solutions.
- purified smoothened preparations will lack any contaminating proteins from the same animal from that smoothened is normally produced, as can be accomplished by recombinant expression of, for example, a mammalian smoothened protein in a yeast or bacterial cell.
- isolated smoothened polypeptides can include all or a portion of an amino acid sequences corresponding to a smoothened polypeptide represented in SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8 or homologous sequences thereto.
- Isolated peptidyl portions of smoothened proteins can also be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides.
- fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
- a smoothened polypeptide of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length.
- the fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of a wild-type (e.g., "authentic") smoothened protein, such as by binding to patched.
- a wild-type e.g., "authentic” smoothened protein
- Modification of the structure of the subject smoothened polypeptides can be for such purposes as enhancing therapeutic or prophylactic efficacy, stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo), or post-translational modifications.
- modified peptides when designed to retain at least one activity of the naturally-occurring form of the protein, or to produce specific antagonists thereof, are considered functional equivalents of the smoothened polypeptides (though they may be agonistic or antagonistic of the bioactivities of the authentic protein).
- Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.
- isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
- Whether a change in the amino acid sequence of a peptide results in a functional smoothened homolog can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type protein, or competitively inhibit such a response.
- Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.
- This invention further contemplates a method for generating sets of combinatorial point mutants of the subject smoothened proteins as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g. homologs) that are functional in modulating signal transduction and/or ligand binding.
- the purpose of screening such combinatorial libraries is to generate, for example, novel smoothened homologs which can act as either agonists or antagonist, or alternatively, possess novel activities all together.
- smoothened homologs can be engineered by the present method to provide selective, constitutive activation of hedgehog activity, or alternatively, to be dominant negative inhibitors of smoothened-dependent signal transduction.
- mutagenesis can provide smoothened homologs which are able to bind or signal through intracellular regulatory proteins.
- the amino acid sequences for a population of smoothened homologs from different species or other related proteins are aligned, preferably to promote the highest homology possible.
- a population of variants can include, for example, smoothened homologs from one or more species.
- Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences.
- the variegated library of smoothened variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential smoothened sequences are expressible as individual polypeptides, or as a library.
- a library of coding sequence fragments can be provided for a smoothened clone in order to generate a variegated population of smoothened fragments for screening and subsequent selection of bioactive fragments.
- a variety of techniques are known in the art for generating such libraries, including chemical synthesis.
- a library of coding sequence fragments can be generated by (i) treating a double stranded PCR fragment of a smoothened coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule; (ii) denaturing the double stranded DNA; (iii) renaturing the DNA to form double stranded DNA which can include sense/anti sense pairs from different nicked products; (iv) removing single stranded portions from reformed duplexes by treatment with SI nuclease; and (v) ligating the resulting fragment library into an expression vector.
- an expression library can be derived which codes for N-terminal, C-terminal and internal fragments of various sizes.
- a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of smoothened homologs.
- the most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
- the invention also provides for reduction of the smoothened protein to generate mimetics, e.g. peptide or non-peptide agents, which are able to disrupt a biological activity of a wild-type smoothened protein, e.g. as inhibitors of protein-protein interactions, such as with patched.
- mimetics e.g. peptide or non-peptide agents
- Such mutagenic techniques as described above are also useful to map the determinants of the smoothened proteins which participate in protein-protein interactions.
- a similar system can be used to derive fragments of a patched protein which bind to a smoothened protein and competitively inhibit binding of the full length patched protein.
- the critical residues of either a smoothened protein or a patched protein which are involved in molecular recognition of the other can be determined and used to generate smoothened-de ⁇ ve ⁇ or patched-de ⁇ ved peptidomimetics which competitively inhibit patched/ smoothened protein interactions.
- peptidomimetic compounds can be generated which mimic those residues which facilitate the interaction.
- mimetics may then be used to interfere with the normal function of a smoothened protein.
- non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al.
- anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
- a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., a smoothened polypeptide or an antigenic fragment which is capable of eliciting an antibody response).
- Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
- an immunogenic portion of a smoothened protein can be administered in the presence of adjuvant.
- the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
- Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
- the subject antibodies are immunospecific for antigenic determinants of a smoothened protein of a organism, such as a mammal, e.g. antigenic determinants of a protein represented by SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8 or closely related homologs (e.g. at least 70% identical, preferably at least 80%) identical, and more preferably at least 90% identical).
- the anti-smoothened polypeptide antibodies do not substantially cross react (i.e. does not react specifically) with a protein which is, for example, less than 85%>, 90% or 95% homologous with the selected smoothened.
- a protein which is, for example, less than 85%>, 90% or 95% homologous with the selected smoothened.
- not substantially cross react it is meant that the antibody has a binding affinity for a non-homologous protein which is at least one order of magnitude, more preferably at least 2 orders of magnitude, and even more preferably at least 3 orders of magnitude less than the binding affinity of the antibody for the intended target smoothened.
- anti-smoothened antisera can be obtained and, if desired, polyclonal anti- smoothened antibodies isolated from the serum.
- antibody-producing cells lymphocytes
- immortalizing cells such as myeloma cells to yield hybridoma cells.
- Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a smoothened polypeptide of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
- antibody as used herein is intended to include fragments thereof which are also specifically reactive with a smoothened polypeptide.
- Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab) 2 fragments can be generated by treating antibody with pepsin. The resulting F(ab) 2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.
- the antibody of the present invention is further intended to include bispecific and chimeric molecules having affinity for a smoothened protein conferred by at least one CDR region of the antibody.
- Both monoclonal and polyclonal antibodies (Ab) directed against authentic smoothened polypeptides, or smoothened variants, and antibody fragments such as Fab, F(ab)2, Fv and scFv can be used to block the action of a smoothened protein and allow the study of the role of these proteins in, for example, differentiation of tissue. Experiments of this nature can aid in deciphering the role of smoothened proteins that may be involved in control of proliferation versus differentiation, e.g., in patterning and tissue formation.
- Antibodies which specifically bind smoothened epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of each of the subject smoothened polypeptides.
- Anti-smoothened antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate smoothened protein levels in tissue as part of a clinical testing procedure. For instance, such measurements can be useful in predictive valuations of the onset or progression of proliferative or differentiative disorders.
- the ability to monitor smoothened protein levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder.
- the level of smoothened polypeptides may be measured from cells in bodily fluid, such as in samples of cerebral spinal fluid or amniotic fluid, or can be measured in tissue, such as produced by biopsy.
- Diagnostic assays using anti-smoothened antibodies can include, for example, immunoassays designed to aid in early diagnosis of a disorder, particularly ones which are manifest at birth. Diagnostic assays using anti-smoothened polypeptide antibodies can also include immunoassays designed to aid in early diagnosis and phenotyping neoplastic or hyperplastic disorders.
- anti-smoothened antibodies of the present invention is in the immunological screening of cDNA libraries constructed in expression vectors such as ⁇ gtl l, ⁇ gtl 8-23, ⁇ ZAP, and ⁇ ORF8.
- Messenger libraries of this type having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.
- ⁇ gtl 1 will produce fusion proteins whose amino termini consist of ⁇ - galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.
- Antigenic epitopes of a smoothened protein e.g. orthologs of the smoothened protein from other species, can then be detected with antibodies, as.
- nucleotide sequences determined from the cloning of smoothened genes from organisms will further allow for the generation of probes and primers designed for use in identifying and/or cloning smoothened homologs in other cell types, e.g. from other tissues, as well as smoothened homologs from other organisms.
- the present invention also provides a probe/primer comprising a substantially purified oligonucleotide, which oligonucleotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least 15, 20, 25 or 30 consecutive nucleotides of sense or anti-sense sequence selected from the group consisting of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 or SEQ ID No: 4 or naturally occurring mutants thereof.
- primers based on the nucleic acid represented in SEQ ID No: 1 , SEQ ID No: 2, SEQ ID No: 3 or SEQ ID No: 4 can be used in PCR reactions to clone smoothened homologs.
- probes based on the subject smoothened sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
- the probe further comprises a label group attached thereto and able to be detected, e.g. the label group is selected from amongst radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
- Such probes can also be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a smoothened protein, such as by measuring a level of a sm ⁇ t/ze/.et.-encoding nucleic acid in a sample of cells from a patient-animal; e.g. detecting smoothened mRNA levels or determining whether a genomic smoothened gene has been mutated or deleted.
- nucleotide probes can be generated from the subject smoothened genes which facilitate histological screening of intact tissue and tissue samples for the presence (or absence) of smoothened-encoding transcripts. Similar to the diagnostic uses of anti- smoothened antibodies, the use of probes directed to smoothened messages, or to genomic smoothened sequences, can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, degenerative disorders marked by loss of particular cell-types, apoptosis, neoplastic and/or hyperplastic disorders (e.g. unwanted cell growth) or abnormal differentiation of tissue.
- the oligonucleotide probes can help facilitate the determination of the molecular basis for a developmental disorder which may involve some abnormality associated with expression (or lack thereof) of a smoothened protein. For instance, variation in polypeptide synthesis can be differentiated from a mutation in a coding sequence.
- the present method provides a method for determining if a subject is at risk for a disorder characterized by aberrant apoptosis, cell proliferation and/or differentiation.
- method can be generally characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of (i) an alteration affecting the integrity of a gene encoding a smoothened-protein, or (ii) the mis-expression of the smoothened gene.
- such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from a smoothened gene, (ii) an addition of one or more nucleotides to a smoothened gene, (iii) a substitution of one or more nucleotides of a smoothened gene, (iv) a gross chromosomal rearrangement of a smoothened gene, (v) a gross alteration in the level of a messenger RNA transcript of a smoothened gene, (vii) aberrant modification of a smoothened gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a smoothened gene, (viii) a non-wild type level of a smoothened-protein, and (ix) inappropriate post-translational modification of a smoothened-protein.
- a nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of a smoothened gene, such as represented by any one of SEQ ID Nos: 1-4 and 9-14, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the subject smoothened genes or naturally occurring mutants thereof.
- the nucleic acid of a cell is rendered accessible for hybridization, the probe is exposed to nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected.
- Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.
- detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 :1077-1080; and Nakazawa et al. (1944) PNAS 91 :360-364), the later of which can be particularly useful for detecting point mutations in the smoothened gene.
- PCR polymerase chain reaction
- LCR ligation chain reaction
- the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to a smoothened gene under conditions such that hybridization and amplification of the smoothened gene (if present) occurs, and (iv) detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
- nucleic acid e.g., genomic, mRNA or both
- the level of a smoothened-protein can be detected by immunoassay.
- the cells of a biopsy sample can be dispersed, and the level of a smoothened-protein present on the surface of each cell can be quantitated by standard immunoassay techniques.
- aberrant methylation patterns of a smoothened gene can be detected by digesting genomic DNA from a patient sample with one or more restriction endonucleases that are sensitive to methylation and for which recognition sites exist in the smoothened gene (including in the flanking and intronic sequences). See, for example, Buiting et al. (1994) Human Mol Genet 3:893-895.
- Digested DNA is separated by gel electrophoresis, and hybridized with probes derived from, for example, genomic or cDNA sequences.
- the methylation status of the smoothened gene can be determined by comparison of the restriction pattern generated from the sample DNA with that for a standard of known methylation.
- a number of techniques exist in the art for identifying ligands to the smoothened receptor For instance, expression cloning can be carried out on a cDNA or genomic library by isolating cells which are decorated with a labeled form of the receptor, such as present in labeled liposomal preparations.
- the technique uses the smoothened receptor in an in situ assay for detecting smoothened ligands in cDNA cloned from tissue samples and whole organisms.
- the present invention makes use of the RAP- situ assay (for Receptor Affinity Probe) of Flanagan and Leder (see PCT publications WO 92/06220; and also Cheng et al. (1994) Cell 79:157-168).
- This system involves the use of an expression cloning system whereby a smoothened ligand can be cloned on the basis of a cDNA/alkaline phosphatase fusion protein binding to smoothened.
- the method comprises (i) providing a hybrid molecule (the affinity probe) including the cDNA-encoded protein covalently bonded to an enzymatically active tag, preferably for which chromogenic substrates exist, (ii) contacting a cell expressing smoothened to form complexes between the probe and smoothened, removing unbound probe, and (iii) detecting the affinity complex using a chromogenic substrate for the enzymatic activity associated with the affinity probe.
- the affinity probe including the cDNA-encoded protein covalently bonded to an enzymatically active tag, preferably for which chromogenic substrates exist
- the present invention facilitates the development of assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function of the subject smoothened proteins, or of their role in the pathogenesis of cellular maintenance, differentiation and/or proliferation and disorders related thereto.
- the assay evaluates the ability of a compound to modulate binding between a smoothened protein and a molecule, e.g., patched or a smoothened ligand, that interacts with the smoothened protein.
- Exemplary compounds which can be screened against such smoothened-mediated interactions include peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries, such as isolated from animals, plants, fungus and/or microbes.
- high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time.
- Assays which are performed in cell-free systems such as may be derived with purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound.
- a reaction mixture is generated to include a smoothened polypeptide, compound(s) of interest, and a "target molecule", e.g., a protein, which interacts with the smoothened polypeptide.
- target molecules include patched, extracellular ligands, as well as other protein and non-protein interacting molecules which interact with smoothened in the membrane or cytoplasm.
- Detection and quantification of interaction of the smoothened protein with the target molecule provides a means for determining a compound's efficacy at inhibiting (or potentiating) interaction between the smoothened and the target molecule.
- the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
- a control assay can also be performed to provide a baseline for comparison. In the control assay, interaction of the smoothened polypeptide and target molecule is quantitated in the absence of the test compound.
- Interaction between the smoothened polypeptide and the target molecule may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled smoothened polypeptides, by immunoassay, by chromatographic detection, or by detecting the intrinsic activity of the acetylase.
- detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled smoothened polypeptides
- immunoassay by immunoassay
- chromatographic detection or by detecting the intrinsic activity of the acetylase.
- the compound of interest is contacted with a mixture including a smoothened protein (e.g., a cell expressing smoothened) and a target molecule under conditions in which the two molecules are ordinarily capable of binding one another.
- a composition containing a test compound e.g., a cell expressing smoothened
- Detection and quantification of smoothened complexes provides a means for determining the test compound's efficacy at inhibiting (or potentiating) complex formation between the molecules.
- a control assay can also be performed to provide a baseline for comparison. In the control assay, formation of the complexes is quantitated in the absence of the test compound.
- the screening assay includes all or a suitable portion of the smoothened protein, which can be obtained from vertebrate or invertebrate sources.
- the smoothened protein can be provided in the screening assay as a whole protein (preferably expressed on the surface of a cell), or alternatively as a fragment of the full length protein which binds to a target molecule, e.g., an extracellular or intracellular domain(s) as appropriate.
- the protein can be provided as part of a liposomal preparation or expressed on the surface of a cell.
- the smoothened protein can derived from a recombinant gene, e.g., being ectopically expressed in a heterologous cell.
- the protein can be expressed on oocytes.
- mammalian cells e.g., COS, CHO, 3T3 or the like
- yeast cell by standard recombinant DNA techniques. These recombinant cells can be used for receptor binding, signal transduction or gene expression assays.
- Marigo et al. (1996) Development 122: 1225-1233 illustrates a binding assay of human hedgehog to chick patched protein ectopically expressed in Xenop us laevis oocytes.
- the assay system of Marigo et al. can be adapted to the present drug screening assays by ectopic or endogenous expression of a smoothened protein. In the illustrated assay, the amount of smoothened/patched complexes can be quantitated by immunoassay or the like.
- Complex formation between the smoothened polypeptide and a target molecule may be detected by a variety of techniques. For instance, modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabelled, fluorescently labeled, or enzymatically labeled hedgehog polypeptides, by immunoassay, or by chromatographic detection.
- detectably labeled proteins such as radiolabelled, fluorescently labeled, or enzymatically labeled hedgehog polypeptides
- a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
- glutathione-S-transferase/receptor (GST/receptor) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the target polypeptide, e.g.
- an labeled polypeptide and the test compound and incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired.
- the beads are washed to remove any unbound target polypeptide, and the matrix bead-bound radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are dissociated.
- the complexes can be dissociated from the bead, separated by SDS-PAGE gel, and the level of target polypeptide found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
- soluble portions of the smoothened protein can be immobilized utilizing conjugation of biotin and streptavidin.
- biotinylated receptor molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
- antibodies reactive with the smoothened but which do not interfere with, e.g., patched binding can be derivatized to the wells of the plate and the receptor trapped in the wells by antibody conjugation.
- preparations of a smoothened polypeptide and a test compound are incubated in the wells of the plate, and the amount of complex trapped in the well can be quantitated.
- Exemplary methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the smoothened or target polypeptide; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the smoothened or target polypeptide.
- the enzyme can be chemically conjugated or provided as a fusion protein, e.g., fused with alkaline phosphatase, and the amount of fusion protein in the complex can be assessed with a chromogenic substrate of the enzyme, e.g. paranitrophenylphosphate.
- a fusion protein including glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using l-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).
- the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in addition to the smoothened polypeptide, a second polypeptide for which antibodies are readily available (e.g. from commercial sources).
- the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety.
- Other useful epitope tags include myc-epitopes (e.g., see Ellison et al.
- liposomal vesicles can be used to provide manipulatable and isolatable sources of the protein.
- both authentic and recombinant forms of the smoothened protein can be reconstituted in artificial lipid vesicles (e.g. phosphatidylcholine liposomes) or in cell membrane-derived vesicles (see, for example, Bear et al. (1992) Cell 68:809-818; Newton et al. (1983) Biochemistry 22:6110-61 17; and Reber et al. (1987) J Biol Chem 262: 1 1369- 1 1374).
- artificial lipid vesicles e.g. phosphatidylcholine liposomes
- cell membrane-derived vesicles see, for example, Bear et al. (1992) Cell 68:809-818; Newton et al. (1983) Biochemistry 22:6110-61 17; and Reber et al. (1987) J Biol Chem 262
- the readily available source of smoothened genes provided by the subject invention also facilitates the generation of cell-based assays for identifying small molecule agonists and antagonists of smoothened activity, e.g., which can be used to mimic or inhibit the effect of hedgehog.
- the interaction of smoothened and patched proteins in a cell or liposome is assessed.
- cells which have been genetically engineered to ectopically express smoothened can be utilized for drug screening assays.
- cells which either express low levels or lack expression of the smoothened protein e.g. Xenopus laevis oocytes, COS cells or yeast cells
- the resulting recombinant cells e.g., which express a functional smoothened, can be utilized in receptor binding assays to identify agonist or antagonists its patched binding. Binding assays can be performed using whole cells.
- the recombinant cells of the present invention can be engineered to include other heterologous genes encoding proteins involved in smoot/.e..e_/-dependent signal pathways.
- the gene products of one or more of costal-2 and/or fused can be co-expressed with smoothened in the reagent cell, with assays being sensitive to the functional reconstitution of the smoothened signal transduction cascade.
- liposomal preparations using reconstituted smoothened protein can be utilized.
- Smoothened protein purified from detergent extracts from both authentic and recombinant origins can be reconstituted in artificial lipid vesicles (e.g. phosphatidylcholine liposomes) or in cell membrane-derived vesicles (see, for example, Bear et al. (1992) Cell 68:809-818; Newton et al. (1983) Biochemistry 22:61 10-61 17; and Reber et al. (1987) J Biol Chem 262: 1 1369-11374).
- the lamellar structure and size of the resulting liposomes can be characterized using electron microscopy. External orientation of the smoothened protein in the reconstituted membranes can be demonstrated, for example, by immunoelectron microscopy.
- modulators i.e., agonists of smoothened activities.
- candidate antagonists and antagonists of smoothened signaling can be identified (e.g., having a hedgehog-like or hedgehog- inhibitory activity).
- the GLI genes encode putative transcription factors having zinc finger DNA binding domains (Orenic et al. (1990) Genes & Dev 4:1053-1067; Kinzler et al. (1990) Mol Cell Biol 10:634-642). Transcription of the GLI gene has been reported to be upregulated in response to hedgehog in limb buds, while transcription of the GLI3 gene is downregulated in response to hedgehog induction (Marigo et al. (1996) Development 122:1225-1233). By selecting transcriptional regulatory sequences from such target genes, e.g.
- Reporter gene based assays of this invention measure the end stage of the above described cascade of events, e.g., transcriptional modulation.
- a reporter gene construct is inserted into the reagent cell in order to generate a detection signal dependent on smoothened signaling.
- nested deletions of genomic clones of the target gene can be constructed using standard techniques. See. for example, Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989); U.S. Patent 5,266,488; Sato et al.
- a nested set of DNA fragments from the gene's 5'-flanking region are placed upstream of a reporter gene, such as the luciferase gene, and assayed for their ability to direct reporter gene expression in smoothened expressing cells.
- a reporter gene such as the luciferase gene
- Host cells transiently transfected with reporter gene constructs can be scored for the induction of expression of the reporter gene in the presence and absence of hedgehog to determine regulatory sequences which are responsive to smoothened-dependent signaling.
- a reporter gene construct is inserted into the reagent cell in order to generate a detection signal dependent on second messengers generated by induction with hedgehog protein.
- the reporter gene construct will include a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the hedgehog activity, with the level of expression of the reporter gene providing the hedgehog-dependent and smoothened- dependent detection signal.
- the amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable. For example, mRNA expression from the reporter gene may be detected using RNAse protection or RNA-based PCR, or the protein product of the reporter gene may be identified by a characteristic stain or an intrinsic activity.
- the amount of expression from the reporter gene is then compared to the amount of expression in either the same cell in the absence of the test compound (or hedgehog) or it may be compared with the amount of transcription in a substantially identical cell that lacks the target receptor protein. Any statistically or otherwise significant difference in the amount of transcription indicates that the test compound has in some manner altered the signal transduction of the smoothened protein, e.g., the test compound is a potential ptc therapeutic.
- the gene product of the reporter is detected by an intrinsic activity associated with that product.
- the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence.
- the reporter or marker gene provides a selective growth advantage, e.g., the reporter gene may enhance cell viability, relieve a cell nutritional requirement, and/or provide resistance to a drug.
- reporter genes are those that are readily detectable.
- the reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties.
- reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell. Biol.
- Transcriptional control elements which may be included in a reporter gene construct include, but are not limited to, promoters, enhancers, and repressor and activator binding sites.
- Suitable transcriptional regulatory elements may be derived from the transcriptional regulatory regions of genes whose expression is induced after modulation of a smoothened signal transduction pathway.
- the characteristics of preferred genes from which the transcriptional control elements are derived include, but are not limited to, low or undetectable expression in quiescent cells, rapid induction at the transcriptional level within minutes of extracellular simulation, induction that is transient and independent of new protein synthesis, subsequent shut-off of transcription requires new protein synthesis, and mRNAs transcribed from these genes have a short half-life. It is not necessary for all of these properties to be present.
- second messenger generation can be measured directly in the detection step, such as mobilization of intracellular calcium, phospholipid metabolism or adenylate cyclase activity are quantitated, for instance, the products of phospholipid hydrolysis IP 3 , DAG or cAMP could be measured
- PKA protein kinase A
- High PKA activity has been shown to antagonize hedgehog signaling in these systems.
- inhibitors of PKA will mimic and/or potentiate the action of hedgehog.
- PKA acts directly downstream or in parallel with hedgehog signaling
- hedgehog signaling occurs via inhibition of PKA activity.
- detection of PKA activity provides a potential readout for the instant assays.
- Smoothened may, under certain circumstances, stimulate the activity of phospholipases.
- Inositol lipids can be extracted and analyzed using standard lipid extraction techniques. Water soluble derivatives of all three inositol lipids (IP ] , IP 2 , IP3) can also be quantitated using radiolabelling techniques or HPLC.
- the mobilization of intracellular calcium or the influx of calcium from outside the cell may also be a response to smoothe ed-dependent signaling, or lack there of.
- Calcium flux in the reagent cell can be measured using standard techniques.
- the choice of the appropriate calcium indicator, fluorescent, bioluminescent, metallochromic, or Ca ++ - sensitive microelectrodes depends on the cell type and the magnitude and time constant of the event under study (Borle (1990) Environ Health Perspect 84:45-56).
- Ca ++ detection cells could be loaded with the Ca ++ sensitive fluorescent dye fura-2 or indo-1, using standard methods, and any change in Ca ++ measured using a fluorometer.
- the gene fused (fu) which encodes a serine/threonine kinase has been identified as a potential downstream target in smoothened signaling. (Preat et al., 1990 Nature 347, 87-89; Therond et al. 1993, Mech. Dev. 44. 65- 80).
- the ability of compounds to modulate serine/threonine kinase activation could be screened using colony immunoblotting (Lyons and Nelson (1984) Proc. Natl. Acad. Sci. USA 81 :7426-7430) using antibodies against phosphorylated serine or threonine residues.
- Reagents for performing such assays are commercially available, for example, phosphoserine and phosphothreonine specific antibodies which measure increases in phosphorylation of those residues can be purchased from commercial sources.
- test compounds are identified as potential modulators of one or more bioactivities of a smoothened protein.
- the practitioner of the subject assay will continue to test the efficacy and specificity of the selected compounds both in vitro and in vivo.
- agents identified in the subject assay can be formulated in pharmaceutical preparations for in vivo administration to an animal, preferably a human.
- Another aspect of the present invention relates to a method of inducing and/or maintaining a differentiated state, enhancing survival, and/or inhibiting (or alternatively potentiating) proliferation of a cell, by contacting the cells with an agent which modulates _.m ⁇ t/.ette-/-dependent signal transduction pathways.
- the subject method could be used to generate and/or maintain an array of different tissue both in vitro and in vivo.
- a “smoothened therapeutic,” whether inhibitory or potentiating with respect to modulating the activity of a smoothened protein, can be, as appropriate, any of the preparations described above, including isolated smoothened polypeptides (including both agonist and antagonist forms), gene therapy constructs, antisense molecules, peptidomimetics, or agents identified in the drug assays provided herein, e.g., which inhibit or potentiate the interactions of smoothened and patched.
- the smoothened therapeutic compounds of the present invention are likely to play an important role in the modulation of cellular proliferation and maintenance of, for example, neuronal, testicular, osteogenic or chondrogenic tissues during disease states. It will also be apparent that, by transient use of modulators of smoothened activities, in vivo reformation of tissue can be accomplished, e.g. in the development and maintenance of organs such as ectodermal patterning, as well as certain mesodermal and endodermal differentiation processes. By controlling the prohferative and differentiative potential for different cells, the subject smoothened therapeutics can be used to reform injured tissue, or to improve grafting and morphology of transplanted tissue. For instance, smoothened antagonists and agonists can be employed in a differential manner to regulate different stages of organ repair after physical, chemical or pathological insult. The present method is also applicable to cell culture techniques.
- in vitro neuronal culture systems have proved to be fundamental and indispensable tools for the study of neural development, as well as the identification of neurotrophic factors such as nerve growth factor (NGF), ciliary trophic factors (CNTF), and brain derived neurotrophic factor (BDNF).
- NGF nerve growth factor
- CNTF ciliary trophic factors
- BDNF brain derived neurotrophic factor
- the present method provides a means for ensuring an adequately restrictive environment in order to maintain neuronal cells at various stages of differentiation, and can be employed, for instance, in cell cultures designed to test the specific activities of other trophic factors.
- the cultured cells can be contacted with a smoothened therapeutic, e.g., such as an agent identified in the assays described above which potentiate _vw ⁇ t ?e«e_ -dependent hedgehog bioactivities, in order to induce neuronal differentiation (e.g. of a stem cell), or to maintain the integrity of a culture of terminally-differentiated neuronal cells by preventing loss of differentiation.
- a smoothened therapeutic e.g., such as an agent identified in the assays described above which potentiate _vw ⁇ t ?e«e_ -dependent hedgehog bioactivities, in order to induce neuronal differentiation (e.g. of a stem cell), or to maintain the integrity of a culture of terminally-differentiated neuronal cells by preventing loss of
- intracerebral grafting has emerged as an additional approach to central nervous system therapies.
- one approach to repairing damaged brain tissues involves the transplantation of cells from fetal or neonatal animals into the adult brain (Dunnett et al. (1987) J Exp Biol 123:265-289; and Freund et al. (1985) J Neurosci 5:603-616).
- Fetal neurons from a variety of brain regions can be successfully incorporated into the adult brain, and such grafts can alleviate behavioral defects.
- grafts of embryonic dopaminergic neurons For example, movement disorder induced by lesions of dopaminergic projections to the basal ganglia can be prevented by grafts of embryonic dopaminergic neurons. Complex cognitive functions that are impaired after lesions of the neocortex can also be partially restored by grafts of embryonic cortical cells.
- the differential use of hedgehog agonists and antagonists in the culture can control the timing and type of differentiation accessible by the culture.
- yet another aspect of the present invention concerns the therapeutic application of a smoothened therapeutics to enhance survival of neurons and other neuronal cells in both the central nervous system and the peripheral nervous system.
- hedgehog protein to regulate neuronal differentiation during development of the nervous system and also presumably in the adult state indicates that certain of the hedgehog proteins, and accordingly smoothened therapeutic which modulate hedgehog bioactivities, can be reasonably expected to facilitate control of adult neurons with regard to maintenance, functional performance, and aging of normal cells; repair and regeneration processes in chemically or mechanically lesioned cells; and prevention of degeneration and premature death which result from loss of differentiation in certain pathological conditions.
- the present invention specifically contemplates applications of the subject smoothened therapeutics to the treatment of (prevention and/or reduction of the severity of) neurological conditions deriving from: (i) acute, subacute, or chronic injury to the nervous system, including traumatic injury, chemical injury, vasal injury and deficits (such as the ischemia resulting from stroke), together with infectious/inflammatory and tumor-induced injury; (ii) aging of the nervous system including Alzheimer's disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson's disease, Huntington's chorea, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations; and (iv) chronic immunological diseases of the nervous system or affecting the nervous system, including multiple sclerosis.
- Many neurological disorders are associated with degeneration of discrete populations of neuronal elements and may be treatable with a therapeutic regimen which includes a smoothened therapeutic that acts as a hedgehog agonist.
- Alzheimer's disease is associated with deficits in several neurotransmitter systems, both those that project to the neocortex and those that reside with the cortex.
- the nucleus basalis in patients with Alzheimer's disease have been observed to have a profound (75%>) loss of neurons compared to age-matched controls.
- Alzheimer's disease is by far the most common form of dementia, several other disorders can produce dementia.
- Several of these are degenerative diseases characterized by the death of neurons in various parts of the central nervous system, especially the cerebral cortex.
- Alzheimer's disease involves the degeneration of intrastraital and cortical cholinergic neurons and GABAergic neurons.
- Pick's disease is a severe neuronal degeneration in the neocortex of the frontal and anterior temporal lobes, sometimes accompanied by death of neurons in the striatum.
- Treatment of patients suffering from such degenerative conditions can include the application of smoothened therapeutics in order to control, for example, differentiation and apoptotic events which give rise to loss of neurons (e.g. to enhance survival of existing neurons) as well as promote differentiation and repopulation by progenitor cells in the area affected.
- a pharmaceutical preparation of one or more of the subject smoothened therapeutics can be applied opportunely in the treatment of neurodegenerative disorders which have manifestations of tremors and involuntary movements.
- Parkinson's disease for example, primarily affects subcortical structures and is characterized by degeneration of the nigrostriatal pathway, raphe nuclei, locus cereleus, and the motor nucleus of vagus. Ballism is typically associated with damage to the subthalmic nucleus, often due to acute vascular accident.
- neurogenic and myopathic diseases which ultimately affect the somatic division of the peripheral nervous system and are manifest as neuromuscular disorders.
- Examples include chronic atrophies such as amyotrophic lateral sclerosis, Guillain-Barre syndrome and chronic peripheral neuropathy, as well as other diseases which can be manifest as progressive bulbar palsies or spinal muscular atrophies.
- the present method is amenable to the treatment of disorders of the cerebellum which result in hypotonia or ataxia, such as those lesions in the cerebellum which produce disorders in the limbs ipsilateral to the lesion.
- a preparation of a smoothened therapeutic can used to treat a restricted form of cerebellar cortical degeneration involving the anterior lobes (vermis and leg areas) such as is common in alcoholic patients.
- the subject method is used to treat amyotrophic lateral sclerosis.
- ALS is a name given to a complex of disorders that comprise upper and lower motor neurons. Patients may present with progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, or a combination of these conditions.
- the major pathological abnormality is characterized by a selective and progressive degeneration of the lower motor neurons in the spinal cord and the upper motor neurons in the cerebral cortex.
- the therapeutic application of a smoothened therapeutic which is a hedgehog agonist can be used alone, or in conjunction with other neurotrophic factors such as CNTF, BDNF or NGF to prevent and/or reverse motor neuron degeneration in ALS patients.
- Smoothened therapeutics of the present invention can also be used in the treatment of autonomic disorders of the peripheral nervous system, which include disorders affecting the innervation of smooth muscle and endocrine tissue (such as glandular tissue).
- the subject method can be used to treat tachycardia or atrial cardiac arrythmias which may arise from a degenerative condition of the nerves innervating the striated muscle of the heart.
- compositions comprising smoothened therapeutics which agonize hedgehog activity, may be employed to support the survival and reprojection of several types of ganglionic neurons sympathetic and sensory neurons as well as motor neurons.
- Such therapeutic compositions may be useful in treatments designed to rescue, for example, various neurons from lesion-induced death as well as guiding reprojection of these neurons after such damage.
- diseases include, but are not limited to, CNS trauma infarction, infection (such as viral infection with varicella-zoster), metabolic disease, nutritional deficiency, toxic agents (such as cisplatin treatment).
- smoothened therapeutics e.g., which antagonize hedgehog induction
- smoothened therapeutics can be used in nerve prostheses for the repair of central and peripheral nerve damage.
- certain of smoothened therapeutics can be added to the prosthetic device to increase the rate of growth and regeneration of the dendridic processes.
- Exemplary nerve guidance channels are described in U.S. patents 5,092,871 and 4,955,892. Accordingly, a severed axonal process can be directed toward the nerve ending from which it was severed by a prosthesis nerve guide.
- the subject method can be used in the treatment of neoplastic or hyperplastic transformations such as may occur in the central nervous system.
- certain of the smoothened therapeutics which induce differentiation of neuronal cells can be utilized to cause such transformed cells to become either post-mitotic or apoptotic.
- Treatment with a smoothened therapeutic may facilitate disruption of autocrine loops, such as TGF- ⁇ or PDGF autostimulatory loops, which are believed to be involved in the neoplastic transformation of several neuronal tumors, smoothened therapeutics may, therefore, thus be of use in the treatment of, for example, malignant gliomas, medulloblastomas, neuroectodermal tumors, and ependymonas.
- hedgehog proteins are morphogenic signals involved in other vertebrate organogenic pathways in addition to neuronal differentiation as described above, having apparent roles in other endodermal patterning, as well as both mesodermal and endodermal differentiation processes.
- Shh plays a role in proper limb growth and patterning by initiating expression of signaling molecules, including Bmp-2 in the mesoderm and Fgf-4 in the ectoderm.
- compositions comprising certain of the smoothened therapeutics can also be utilized for both cell culture and therapeutic methods involving generation and maintenance of non- neuronal tissue.
- the present invention makes use of the discovery that hedgehog proteins, such as Shh, are apparently involved in controlling the development of stem cells responsible for formation of the digestive tract, liver, lungs, and other organs which derive from the primitive gut.
- Shh serves as an inductive signal from the endoderm to the mesoderm, which is critical to gut morphogenesis. Therefore, for example, hedgehog agonists can be employed in the development and maintenance of an artificial liver which can have multiple metabolic functions of a normal liver.
- a smoothened therapeutic which acts as a hedgehog agonist can be used to induce differentiation of digestive tube stem cells to form hepatocyte cultures which can be used to populate extracellular matrices, or which can be encapsulated in biocompatible polymers, to form both implantable and extracorporeal artificial livers.
- therapeutic compositions of hedgehog agonists can be utilized in conjunction with transplantation of such artificial livers, as well as embryonic liver structures, to promote intraperitoneal implantation, vascularization, and in vivo differentiation and maintenance of the engrafted liver tissue.
- smoothened therapeutics can be employed therapeutically to regulate such organs after physical, chemical or pathological insult.
- therapeutic compositions comprising hedgehog agonists can be utilized in liver repair subsequent to a partial hepatectomy.
- therapeutic compositions containing hedgehog agonists can be used to promote regeneration of lung tissue in the treatment of emphysema.
- compositions comprising smoothened therapeutics can be used in the in vitro generation of skeletal tissue, such as from skeletogenic stem cells, as well as the in vivo treatment of skeletal tissue deficiencies.
- the present invention particularly contemplates the use of smoothened therapeutics which agonize a hedgehog a skeletogenic activity, such as an ability to induce chondrogenesis and/or osteogenesis.
- skeletal tissue deficiency it is meant a deficiency in bone or other skeletal connective tissue at any site where it is desired to restore the bone or connective tissue, no matter how the deficiency originated, e.g. whether as a result of surgical intervention, removal of tumor, ulceration, implant, fracture, or other traumatic or degenerative conditions.
- the present invention makes available effective therapeutic methods and compositions for restoring cartilage function to a connective tissue.
- Such methods are useful in, for example, the repair of defects or lesions in cartilage tissue which is the result of degenerative wear such as that which results in arthritis, as well as other mechanical derangements which may be caused by trauma to the tissue, such as a displacement of torn meniscus tissue, meniscectomy, a taxation of a joint by a torn ligament, malignment of joints, bone fracture, or by hereditary disease.
- the present reparative method is also useful for remodeling cartilage matrix, such as in plastic or reconstructive surgery, as well as periodontal surgery.
- the present method may also be applied to improving a previous reparative procedure, for example, following surgical repair of a meniscus, ligament, or cartilage. Furthermore, it may prevent the onset or exacerbation of degenerative disease if applied early enough after trauma.
- the subject method comprises treating the afflicted connective tissue with a therapeutically sufficient amount of a hedgehog agonist, particularly smoothened therapeutic which agonizes Ihh activity, to generate a cartilage repair response in the connective tissue by stimulating the differentiation and/or proliferation of chondrocytes embedded in the tissue.
- a hedgehog agonist particularly smoothened therapeutic which agonizes Ihh activity
- Induction of chondrocytes by treatment with a hedgehog agonist can subsequently result in the synthesis of new cartilage matrix by the treated cells.
- Such connective tissues as articular cartilage, interarticular cartilage (menisci), costal cartilage (connecting the true ribs and the sternum), ligaments, and tendons are particularly amenable to treatment in reconstructive and/or regenerative therapies using the subject method.
- regenerative therapies include treatment of degenerative states which have progressed to the point of which impairment of the tissue is obviously manifest, as well as preventive treatments of tissue where degeneration is in its earliest stages or imminent.
- the subject method can further be used to prevent the spread of mineralisation into fibrotic tissue by maintaining a constant production of new cartilage.
- the subject method can be used to treat cartilage of a diarthroidal joint, such as a knee, an ankle, an elbow, a smoothened, a wrist, a knuckle of either a finger or toe, or a temperomandibular joint.
- the treatment can be directed to the meniscus of the joint, to the articular cartilage of the joint, or both.
- the subject method can be used to treat a degenerative disorder of a knee, such as which might be the result of traumatic injury (e.g., a sports injury or excessive wear) or osteoarthritis.
- an injection of a smoothened therapeutic into the joint with, for instance, an arthroscopic needle can be used to treat the afflicted cartilage.
- the injected agent can be in the form of a hydrogel or other slow release vehicle described above in order to permit a more extended and regular contact of the agent with the treated tissue.
- the present invention further contemplates the use of the subject method in the field of cartilage transplantation and prosthetic device therapies.
- the growth of new cartilage from either transplantation of autologous or allogenic cartilage has been largely unsuccessful.
- the zonal arrangement of these tissues may reflect a gradual change in mechanical properties, and failure occurs when implanted tissue, which has not differentiated under those conditions, lacks the ability to appropriately respond.
- the tissue undergoes a metaplasia to pure fibrous tissue.
- the subject method can be used to particularly addresses this problem, by causing the implanted cells to become more adaptive to the new environment and effectively resemble hypertrophic chondrocytes of an earlier developmental stage of the tissue.
- the action of chondrogenesis in the implanted tissue, as provided by the subject method, and the mechanical forces on the actively remodeling tissue can synergize to produce an improved implant more suitable for the new function to which it is to be put.
- the subject method can be applied to enhancing both the generation of prosthetic cartilage devices and to their implantation.
- the need for improved treatment has motivated research aimed at creating new cartilage that is based on collagen- glycosaminoglycan templates (Stone et al. (1990) Clin Orthop Relat Red 252:129), isolated chondrocytes (Grande et al. (1989) J Orthop Res 7:208; and Takigawa et al. (1987) Bone Miner 2:449), and chondrocytes attached to natural or synthetic polymers (Walitani et al. (1989) J Bone Jt Surg 71B:74; Vacanti et al.
- chondrocytes can be grown in culture on biodegradable, biocompatible highly porous scaffolds formed from polymers such as polyglycolic acid, polylactic acid, agarose gel, or other polymers which degrade over time as function of hydrolysis of the polymer backbone into innocuous monomers.
- the matrices are designed to allow adequate nutrient and gas exchange to the cells until engraftment occurs.
- the cells can be cultured in vitro until adequate cell volume and density has developed for the cells to be implanted.
- One advantage of the matrices is that they can be cast or molded into a desired shape on an individual basis, so that the final product closely resembles the patient's own ear or nose (by way of example), or flexible matrices can be used which allow for manipulation at the time of implantation, as in a joint.
- the implants are contacted with a smoothened therapeutic during the culturing process, such as an Ihh agonist, in order to induce and/or maintain differentiated chondrocytes in the culture in order as to further stimulate cartilage matrix production within the implant.
- the cultured cells can be caused to maintain a phenotype typical of a chondrogenic cell (i.e. hypertrophic), and hence continue the population of the matrix and production of cartilage tissue.
- the implanted device is treated with a smoothened therapeutic in order to actively remodel the implanted matrix and to make it more suitable for its intended function.
- a smoothened therapeutic in order to actively remodel the implanted matrix and to make it more suitable for its intended function.
- the artificial transplants suffer from the same deficiency of not being derived in a setting which is comparable to the actual mechanical environment in which the matrix is implanted.
- the activation of the chondrocytes in the matrix by the subject method can allow the implant to acquire characteristics similar to the tissue for which it is intended to replace.
- the subject method is used to enhance attachment of prosthetic devices.
- the subject method can be used in the implantation of a periodontal prosthesis, wherein the treatment of the surrounding connective tissue stimulates formation of periodontal ligament about the prosthesis, as well as inhibits formation of fibrotic tissue proximate the prosthetic device.
- the subject method can be employed for the generation of bone (osteogenesis) at a site in the animal where such skeletal tissue is deficient.
- Indian hedgehog is particularly associated with the hypertrophic chondrocytes that are ultimately replaced by osteoblasts.
- administration of a smoothened therapeutic of the present invention can be employed as part of a method for treating bone loss in a subject, e.g. to prevent and/or reverse osteoporosis and other osteopenic disorders, as well as to regulate bone growth and maturation.
- preparations comprising hedgehog agonists can be employed, for example, to induce endochondral ossification, at least so far as to facilitate the formation of cartilaginous tissue precursors to form the "model" for ossification.
- Therapeutic compositions of smoothened therapeutics can be supplemented, if required, with other osteoinductive factors, such as bone growth factors (e.g. TGF- ⁇ factors, such as the bone morphogenetic factors BMP-2 and BMP-4, as well as activin), and may also include, or be administered in combination with, an inhibitor of bone resorption such as estrogen, bisphosphonate, sodium fluoride, calcitonin, or tamoxifen, or related compounds.
- bone growth factors e.g. TGF- ⁇ factors, such as the bone morphogenetic factors BMP-2 and BMP-4, as well as activin
- an inhibitor of bone resorption such as estrogen, bisphosphonate, sodium fluoride, calcitonin, or tamoxifen, or related
- hedgehog proteins such as ⁇ hh and Shh are likely to be upstream of BMPs, e.g. treatment with a hedgehog agonist will have the advantage of initiating endogenous expression of BMPs along with other factors.
- the smoothened therapeutic of the present invention can be used in the treatment of testicular cells, so as to modulate spermatogenesis.
- hedgehog antagonist can be utilized to block the action of a naturally-occurring hedgehog protein.
- the smoothened therapeutic inhibits the biological activity of Dhh with respect to spermatogenesis, by competitively binding hedgehog in the testis. That is, the smoothened therapeutic can be administered as a contraceptive formulation.
- smoothened therapeutics which agonize the spermatogenic activity of Dhh can be used as fertility enhancers.
- hedgehog agonists and antagonists are potentially useful for modulating normal ovarian function.
- the source of the smoothened therapeutics to be formulated will depend on the particular form of the agent. Small organic molecules and peptidyl fragments can be chemically synthesized and provided in a pure form suitable for pharmaceutical/cosmetic usage. Products of natural extracts can be purified according to techniques known in the art.
- the smoothened therapeutic formulations used in the method of the invention are most preferably applied in the form of appropriate compositions.
- appropriate compositions there may be cited all compositions usually employed for systemically or locally (such as intrathecal) administering drugs.
- the pharmaceutically acceptable carrier should be substantially inert, so as not to act with the active component. Suitable inert carriers include water, alcohol polyethylene glycol, mineral oil or petroleum gel, propylene glycol and the like.
- compositions of this invention an effective amount of the particular smoothened therapeutic as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration.
- a pharmaceutically acceptable carrier which carrier may take a wide variety of forms depending on the form of preparation desired for administration.
- These pharmaceutical compositions are desirable in unitary dosage form suitable, particularly, for administration orally, rectally, percutaneously, or by parenteral injection.
- any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets. Because of their ease in administration, tablets and capsules represents the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed.
- the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included.
- Injectable solutions may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution.
- Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations.
- the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin.
- Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
- dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powders packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.
- the pharmaceutical preparations of the present invention can be used, as stated above, for the many applications which can be considered cosmetic uses.
- Cosmetic compositions known in the art are especially preferred, and include toilet waters, packs, lotions, skin milks or milky lotions.
- the preparations contain, besides the smoothened therapeutic, components usually employed in such preparations. Examples of such components are oils, fats, waxes, surfactants, humectants, thickening agents, antioxidants, viscosity stabilizers, chelating agents, buffers, preservatives, perfumes, dyestuffs, lower alkanols, and the like.
- further ingredients may be incorporated in the compositions, e.g. anti-inflammatory agents, antibacterials, antifungals, disinfectants, vitamins, sunscreens, antibiotics, or other anti-acne agents.
- oils comprise fats and oils such as olive oil and hydrogenated oils; waxes such as beeswax and lanolin; hydrocarbons such as liquid paraffin, ceresin, and squalane; fatty acids such as stearic acid and oleic acid; alcohols such as cetyl alcohol, stearyl alcohol, lanolin alcohol, and hexadecanol; and esters such as isopropyl myristate, isopropyl palmitate and butyl stearate.
- oils comprise fats and oils such as olive oil and hydrogenated oils; waxes such as beeswax and lanolin; hydrocarbons such as liquid paraffin, ceresin, and squalane; fatty acids such as stearic acid and oleic acid; alcohols such as cetyl alcohol, stearyl alcohol, lanolin alcohol, and hexadecanol; and esters such as isopropyl myristate, isopropyl palmitate and butyl
- anionic surfactants such as sodium stearate, sodium cetylsulfate, polyoxyethylene laurylether phosphate, sodium N-acyl glutamate; cationic surfactants such as stearyldimethylbenzylammonium chloride and stearyltrimethylammonium chloride; ampholytic surfactants such as alkylaminoethylglycine hydrochloride solutions and lecithin; and nonionic surfactants such as glycerin monostearate, sorbitan monostearate, sucrose fatty acid esters, propylene glycol monostearate, polyoxyethylene oleylether, polyethylene glycol monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene coconut fatty acid monoethanolamide, polyoxypropylene glycol (e.g.
- humectants include glycerin, 1 ,3-butylene glycol, and propylene glycol
- examples of lower alcohols include ethanol and isopropanol
- examples of thickening agents include xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyethylene glycol and sodium carboxymethyl cellulose
- examples of antioxidants comprise butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, citric acid and ethoxyquin
- examples of chelating agents include disodium edetate and ethanehydroxy diphosphate
- examples of buffers comprise citric acid, sodium citrate, boric acid, borax, and disodium hydrogen phosphate
- examples of preservatives are methyl parahydroxybenzoate, ethyl parahydroxybenzoate, dehydroacetic acid, salicylic acid and
- the carrier for example consists of 1 to 20%, in particular 5 to 15% of a humectant, 0.1 to 10%> in particular from 0.5 to 5% of a thickener and water; or said carrier may consist of 70 to 99%), in particular 20 to 95% of a surfactant, and 0 to 20%, in particular 2.5 to 15%.
- the carrier for example consists of 2 to 10%) of a lower alcohol, 0.1 to 10% or in particular 0.5 to 1% of a surfactant, 1 to 20%, in particular 3 to 7% of a humectant, 0 to 5%> of a buffer, water and small amounts ( ⁇ 2%>) of preservative, dyestuff and/or perfume.
- the carrier typically consists of 10-50%) of oil, 1 to 10%o of surfactant, 50-80% of water and 0 to 3% of preservative and/or perfume.
- all % symbols refer to weight by weight percentage.
- compositions for use in the method of the present invention are those wherein the smoothened therapeutic is formulated in liposome-containing compositions.
- Liposomes are artificial vesicles formed by amphiphatic molecules such as polar lipids, for example, phosphatidyl cholines, ethanolamines and serines, sphingomyelins, cardiolipins, plasmalogens, phosphatidic acids and cerebiosides. Liposomes are formed when suitable amphiphathic molecules are allowed to swell in water or aqueous solutions to form liquid crystals usually of multilayer structure comprised of many bilayers separated from each other by aqueous material (also referred to as coarse liposomes).
- lipid soluble active ingredient is encapsulated in the aqueous spaces between the molecular layers.
- the lipid soluble active ingredient of a smoothened therapeutic, such as an organic mimetic, is predominantly incorporated into the lipid iayers, although polar head groups may protrude from the layer into the aqueous space.
- the encapsulation of these compounds can be achieved by a number of methods.
- the method most commonly used involves casting a thin film of phospholipid onto the walls of a flask by evaporation from an organic solvent. When this film is dispersed in a suitable aqueous medium, multilamellar liposomes are formed. Upon suitable sonication, the coarse liposomes form smaller similarly closed vesicles.
- Water-soluble active ingredients are usually incorporated by dispersing the cast film with an aqueous solution of the compound. The unencapsulated compound is then removed by centrifugation, chromatography, dialysis or other art-known suitable procedures. The lipid-soluble active ingredient is usually incorporated by dissolving it in the organic solvent with the phospholipid prior to casting the film. If the solubility of the material in the lipid phase is not exceeded or the amount present is not in excess of that which can be bound to the lipid. liposomes prepared by the above method usually contain most of the material bound in the lipid bilayers; separation of the liposomes from unencapsulated material is not required.
- a particularly convenient method for preparing liposome formulated forms of smoothened therapeutics is the method described in EP-A-253,619, incorporated herein by reference.
- single bilayered liposomes containing encapsulated active ingredients are prepared by dissolving the lipid component in an organic medium, injecting the organic solution of the lipid component under pressure into an aqueous component while simultaneously mixing the organic and aqueous components with a high speed homogenizer or mixing means, whereupon the liposomes are formed spontaneously.
- the single bilayered liposomes containing the encapsulated smoothened therapeutic can be employed directly or they can be employed in a suitable pharmaceutically acceptable carrier for localized administration.
- the viscosity of the liposomes can be increased by the addition of one or more suitable thickening agents such as, for example xanthan gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose and mixtures thereof.
- the aqueous component may consist of water alone or it may contain electrolytes, buffered systems and other ingredients, such as, for example, preservatives.
- Suitable electrolytes which can be employed include metal salts such as alkali metal and alkaline earth metal salts.
- the preferred metal salts are calcium chloride, sodium chloride and potassium chloride.
- the concentration of the electrolyte may vary from zero to 260 mM, preferably from 5 mM to 160 mM.
- the aqueous component is placed in a suitable vessel which can be adapted to effect homogenization by effecting great turbulence during the injection of the organic component. Homogenization of the two components can be accomplished within the vessel, or, alternatively, the aqueous and organic components may be injected separately into a mixing means which is located outside the vessel. In the latter case, the liposomes are formed in the mixing means and then transferred to another vessel for collection purpose.
- the organic component consists of a suitable non-toxic, pharmaceutically acceptable solvent such as, for example ethanol, glycerol, propylene glycol and polyethylene glycol. and a suitable phospholipid which is soluble in the solvent.
- suitable phospholipids which can be employed include lecithin, phosphatidylcholine, phosphatydylserine, phosphatidylethanol-amine, phosphatidylinositol, lysophosphatidylcholine and phospha- tidyl glycerol, for example.
- Other lipophilic additives may be employed in order to selectively modify the characteristics of the liposomes. Examples of such other additives include stearylamine, phosphatidic acid, tocopherol, cholesterol and lanolin extracts.
- ingredients which can prevent oxidation of the phospholipids may be added to the organic component.
- examples of such other ingredients include tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, ascorbyl palmitate and ascorbyl oleate.
- Preservatives such a benzoic acid, methyl paraben and propyl paraben may also be added.
- Methods of introduction may also be provided by rechargeable or biodegradable devices.
- Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals.
- a variety of biocompatible polymers including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of an hh at a particular target site.
- An essential feature of certain embodiments of the implant can be the linear release of the therapeutic, which can be achieved through the manipulation of the polymer composition and form.
- the amount of water, porosity and consequent permeability characteristics can be controlled.
- the selection of the shape, size, polymer, and method for implantation can be determined on an individual basis according to the disorder to be treated and the individual patient response.
- the generation of such implants is generally known in the art. See, for example, Concise Encylopedia of Medical & Dental Materials, ed. by David Williams (MIT Press: Cambridge, MA, 1990); and the Sabel et al. U.S. Patent No. 4,883,666.
- a source of cells producing the therapeutic e.g., secreting a soluble form of a smoothened ligand
- Such fibers can be pre-spun and subsequently loaded with the cell source (Aebischer et al. U.S.
- Another aspect of the invention features transgenic non-human animals which express a heterologous smoothened gene of the present invention, and/or which have had one or more genomic smoothened genes disrupted in at least a tissue or cell-types of the animal.
- the invention features an animal model for developmental diseases, which animal has one or more smoothened allele which is mis-expressed. For example, an animal can be generated which has one or more smoothened alleles deleted or otherwise rendered inactive. Such a model can then be used to study disorders arising from mis- expressed smoothened genes, as well as for evaluating potential therapies for similar disorders.
- the transgenic animals of the present invention all include within a plurality of their cells a transgene of the present invention, which transgene alters the phenotype of the "host cell” with respect to regulation by the smoothened protein, e.g., of cell growth, death and/or differentiation. Since it is possible to produce transgenic organisms of the invention utilizing one or more of the transgene constructs described herein, a general description will be given of the production of transgenic organisms by referring generally to exogenous genetic material. This general description can be adapted by those skilled in the art in order to incorporate specific transgene sequences into organisms utilizing the methods and materials described herein and those generally known in the art. In one embodiment, the transgene construct is a knockout construct.
- transgene constructs usually are insertion-type or replacement-type constructs (Hasty et al. (1991) Mol Cell Biol 1 1 :4509).
- the transgene constructs for disruption of a smoothened gene are designed to facilitate homologous recombination with a portion of the genomic smoothened gene so as to prevent the functional expression of the endogenous smoothened gene.
- the nucleotide sequence used as the knockout construct can be comprised of (1) DNA from some portion of the endogenous smoothened gene (exon sequence, intron sequence, promoter sequences, etc.) which direct recombination and (2) a marker sequence which is used to detect the presence of the knockout construct in the cell.
- the knockout construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to prevent or interrupt transcription of the native smoothened gene. Such insertion can occur by homologous recombination, i.e., regions of the knockout construct that are homologous to the endogenous smoothened gene sequence hybridize to the genomic DNA and recombine with the genomic sequences so that the construct is incorporated into the corresponding position of the genomic DNA.
- the knockout construct can comprise (1) a full or partial sequence of one or more exons and/or introns of the smoothened gene to be disrupted, (2) sequences which flank the 5' and 3' ends of the coding sequence of the smoothened gene, or (3) a combination thereof.
- a preferred knockout construct will delete, by targeted homologous recombination, essential structural elements of an endogenous smoothened gene.
- the targeting construct can recombine with the genomic smoothened gene can delete a portion of the coding sequence, and/or essential transcriptional regulatory sequences of the gene.
- the knockout construct can be used to interrupt essential structural and/or regulatory elements of an endogenous smoothened gene by targeted insertion of a polynucleotide sequence.
- a knockout construct can recombine with a smoothened gene and insert a nonhomologous sequence, such as a neo expression cassette, into a structural element (e.g., an exon) and/or regulatory element (e.g., enhancer, promoter, intron splice site, polyadenylation site, etc.) to yield a targeted smoothened allele having an insertional disruption.
- the inserted nucleic acid can range in size from 1 nucleotide (e.g., to produce a frameshift) to several kilobases or more, and is limited only by the efficiency of the targeting technique.
- the transgene construct can be used to generate a transgenic animal in which substantially all expression of the targeted smoothened gene is inhibited in at least a portion of the animal's cells. If only regulatory elements are targeted, some low-level expression of the targeted gene may occur (i.e., the targeted allele is "leaky").
- the nucleotide sequence(s) comprising the knockout construct(s) can be obtained using methods well known in the art. Such methods include, for example, screening genomic libraries with smoothened cDNA probes in order to identify the corresponding genomic smoothened gene and regulatory sequences. Alternatively, where the cDNA sequence is to be used as part of the knockout construct, the cDNA may be obtained by screening a cDNA library as set out above.
- the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal.
- Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell.
- the specific line(s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness.
- the haplotype is a significant factor. For example, when transgenic mice are to be produced, strains such as C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, ME).
- Preferred strains are those with H-2 b , H-2 d or H-2 c l haplotypes such as C57BL/6 or DBA/1.
- the line(s) used to practice this invention may themselves be transgenics, and/or may be knockouts (i.e., obtained from animals which have one or more genes partially or completely suppressed) .
- the transgene construct is introduced into a single stage embryo.
- the zygote is the best target for micro-injection.
- the use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). As a consequence, all cells of the transgenic animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.
- transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
- the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention.
- the exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art. Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces. Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method.
- Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene.
- Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product.
- DNA is prepared from excised tissue and analyzed by Southern analysis or PCR for the transgene.
- the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
- Retroviral infection can also be used to introduce transgene into a non-human animal.
- the developing non-human embryo can be cultured in vitro to the blastocyst stage.
- the blastomeres can be targets for retroviral infection (Jaenich, R. (1 76) PNAS 73:1260-1264).
- Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).
- the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al. (1985) PNAS 82:6927-6931 ; Van der Putten et al.
- a third type of target cell for transgene introduction is the embryonal stem cell (ES).
- ES embryonal stem cell
- ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292: 154-156; Bradley et al. (1984) Nature 309:255-
- Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
- gene targeting which is a method of using homologous recombination to modify an animal's genome, can be used to introduce changes into cultured embryonic stem cells. By targeting the smoothened gene in ES cells, these changes can be introduced into the germlines of animals to generate chimeras.
- the gene targeting procedure is accomplished by introducing into tissue culture cells a DNA targeting construct that includes a segment homologous to a smoothened locus, and which also includes an intended sequence modification to the moothened genomic sequence (e.g., insertion, deletion, point mutation). The treated cells are then screened for accurate targeting to identify and isolate those which have been properly targeted.
- a DNA targeting construct that includes a segment homologous to a smoothened locus, and which also includes an intended sequence modification to the moothened genomic sequence (e.g., insertion, deletion, point mutation).
- Gene targeting in embryonic stem cells is in fact a scheme contemplated by the present invention as a means for disrupting a smoothened gene function through the use of a targeting transgene construct designed to undergo homologous recombination with smoothened genomic sequences.
- Targeting construct can be arranged so that, upon recombination with an element of a smoothened gene, a positive selection marker is inserted into (or replaces) coding sequences of the targeted smoothened gene.
- the inserted sequence functionally disrupts the smoothened gene, while also providing a positive selection trait.
- the embryonic stem cells (ES cells ) used to produce the knockout animals will be of the same species as the knockout animal to be generated.
- mouse embryonic stem cells will usually be used for generation of a smoothened- knockout mice.
- Embryonic stem cells are generated and maintained using methods well known to the skilled artisan such as those described by Doetschman et al. (1985) J. Embryol. Exp. Morphol. 87:27-45).
- Any line of ES cells can be used, however, the line chosen is typically selected for the ability of the cells to integrate into and become part of the germ line of a developing embryo so as to create germ line transmission of the knockout construct.
- any ES cell line that is believed to have this capability is suitable for use herein.
- the cells are cultured and prepared for knockout construct insertion using methods well known to the skilled artisan, such as those set forth by Robertson in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. IRL Press. Washington. D.C. [1987]); by Bradley et al. (1986) Current Topics in Devel. Biol. 20:357-371); and by Hogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1986]) .
- Insertion of the knockout construct into the ES cells can be accomplished using a variety of methods well known in the art including for example, electroporation, microinjection, and calcium phosphate treatment.
- a preferred method of insertion is electroporation .
- Each knockout construct to be inserted into the cell must first be in the linear form.
- linearization is accomplished by digesting the DNA with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the knockout construct sequence.
- the knockout construct is added to the ES cells under appropriate conditions for the insertion method chosen, as is known to the skilled artisan. Where more than one construct is to be introduced into the ES cell, each knockout construct can be introduced simultaneously or one at a time.
- the ES cells and knockout construct DNA are exposed to an electric pulse using an electroporation machine and following the manufacturer's guidelines for use. After electroporation, the ES cells are typically allowed to recover under suitable incubation conditions. The cells are then screened for the presence of the knockout construct .
- the ES cells may be cultured in the presence of an otherwise lethal concentration of antibiotic. Those ES cells that survive have presumably integrated the knockout construct. If the marker gene is other than an antibiotic resistance gene, a Southern blot of the ES cell genomic DNA can be probed with a sequence of DNA designed to hybridize only to the marker sequence Alternatively, PCR can be used. Finally, if the marker gene is a gene that encodes an enzyme whose activity can be detected (e.g., ⁇ -galactosidase), the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed.
- an enzyme whose activity can be detected (e.g., ⁇ -galactosidase)
- the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed.
- the knockout construct may integrate into several locations in the ES cell genome, and may integrate into a different location in each ES cell's genome due to the occurrence of random insertion events.
- the desired location of insertion is in a complementary position to the DNA sequence to be knocked out, e.g., the smoothened coding sequence, transcriptional regulatory sequence, etc.
- the smoothened coding sequence e.g., the smoothened coding sequence, transcriptional regulatory sequence, etc.
- less than about 1-5 percent of the ES cells that take up the knockout construct will actually integrate the knockout construct in the desired location.
- total DNA can be extracted from the ES cells using standard methods.
- the DNA can then be probed on a Southern blot with a probe or probes designed to hybridize in a specific pattern to genomic DNA digested with particular restriction enzyme(s).
- the genomic DNA can be amplified by PCR with probes specifically designed to amplify DNA fragments of a particular size and sequence (i.e., only those cells containing the knockout construct in the proper position will generate DNA fragments of the proper size).
- the cells can be inserted into an embryo. Insertion may be accomplished in a variety of ways known to the skilled artisan, however a preferred method is by microinjection. For microinjection, about 10-30 cells are collected into a micropipet and injected into embryos that are at the proper stage of development to permit integration of the foreign ES cell containing the knockout construct into the developing embryo. For instance, the transformed ES cells can be microinjected into blastocytes.
- the embryo may be implanted into the uterus of a pseudopregnant foster mother for gestation. While any foster mother may be used, the foster mother is typically selected for her ability to breed and reproduce well, and for her ability to care for the young. Such foster mothers are typically prepared by mating with vasectomized males of the same species. The stage of the pseudopregnant foster mother is important for successful implantation, and it is species dependent.
- Offspring that are born to the foster mother may be screened initially for smoothened disruptants, DNA from tissue of the offspring may be screened for the presence of the knockout construct using Southern blots and/or PCR as described above.
- Offspring that appear to be mosaics may then be crossed to each other, if they are believed to carry the knockout construct in their germ line, in order to generate homozygous knockout animals.
- Homozygotes may be identified by Southern blotting of equivalent amounts of genomic DNA from animals that are the product of this cross, as well as animals that are known heterozygotes and wild type animals.
- Northern blots can be used to probe the mRNA for the presence or absence of transcripts of either the smoothened gene, the marker gene, or both.
- Western blots can be used to assess the (loss of) level of expression of the smoothened gene knocked out in various tissues of the offspring by probing the Western blot with an antibody against the smoothened protein, or an antibody against the marker gene product, where this gene is expressed.
- in situ analysis such as fixing the cells and labeling with antibody
- FACS fluorescence activated cell sorting
- animals containing more than one knockout construct and/or more than one transgene expression construct are prepared in any of several ways. The preferred manner of preparation is to generate a series of animals, each containing a desired transgenic phenotypes.
- Such animals are bred together through a series of crosses, backcrosses and selections, to ultimately generate a single animal containing all desired knockout constructs and/or expression constructs, where the animal is otherwise congenic (genetically identical) to the wild type except for the presence of the knockout construct(s) and/or transgene(s).
- a transgenic avian species can be generated by breeding a first transgenic bird in which the wild-type smoothened gene is disrupted with a second transgenic bird which has been engineered to express a mutant smoothened which retains most other biological functions of the receptor.
- the transformed animals, their progeny, and cell lines of the present invention provide several important uses that will be readily apparent to one of ordinary skill in the art.
- the transgenic animals and cell lines are particularly useful in screening compounds that have potential as prophylactic or therapeutic treatments of diseases such as may involve aberrant expression, or loss, of a smoothened gene, or aberrant or unwanted activation of receptor signaling. Screening for a useful drug would involve administering the candidate drug over a range of doses to the transgenic animal, and assaying at various time points for the effect(s) of the drug on the disease or disorder being evaluated. Alternatively, or additionally, the drug could be administered prior to or simultaneously with exposure to induction of the disease, if applicable.
- candidate compounds are screened by being administered to the transgenic animal, over a range of doses, and evaluating the animal's physiological response to the compound(s) over time.
- Administration may be oral, or by suitable injection, depending on the chemical nature of the compound being evaluated. In some cases, it may be appropriate to administer the compound in conjunction with co-factors that would enhance the efficacy of the compound.
- test compound is added to the cell culture medium at the appropriate time, and the cellular response to the compound is evaluated over time using the appropriate biochemical and/or histological assays. In some cases, it may be appropriate to apply the compound of interest to the culture medium in conjunction with co- factors that would enhance the efficacy of the compound.
- hh is in turn maintained by wg activity, and these mutual regulatory interactions stabilize the expression domains of the two genes and hence the parasegment boundaries defined by their interfaces (reviewed in Ingham, P.W. & Martinas-Arias, A. (1992) Cell 68:221-235). In the absence of either gene activity, these boundaries disappear and all patterning and polarity of the segments is lost (Fig. 1 e, f). Embryos homozygous for mutations of the smoothened (smo) locus (formerly named smooth) (Nusslein-Volhard, C. et al. (1984) Roux 's Arch. Dev. Biol.
- smo- embryos lack wg transcription at their parasegment boundaries (Fig 2a, d). As wg transcription also disappears in wg mutant embryos (Bejsovec, A. & Martinez-Arias, A. (1991 ) Development 1 13:471-485; Ingham, P.W. & Hidalgo, A. (1993) Development 1 17:283-291 ; van den Huevel, M. et al.
- hh In the developing imaginal discs, expression of hh is restricted to cells of the posterior lineage compartment and, as in the embryo, its activity is required by neighboring anterior compartment cells for the transcription of other signal-encoding genes (reviewed in Blair, S.S. (1995) BioEssays 17:299-309).
- the principal target of hh activity is the decapentaplegic (dpp) gene, expression of which is restricted to a thin stripe of cells running along the anterior side of the compartment boundary (Raftery, L.A. et al. (1991) Development 1 13:27-33) (Fig 3a).
- dpp expression of dpp is lost, however, from clones of cells lacking smo activity when such clones are located within the normal dpp domain (Fig. 3b-e).
- smo is required in a cell-autonomous manner for dpp expression.
- Transcription of dpp can be activated in the wing disc independently of hh by removing the activity of cAMP-dependent protein kinase (PKA), suggesting that PKA acts downstream of hh to antagonize its activity (Kalderon, D. (1995) Curr. Biol. 5:580-582).
- PKA cAMP-dependent protein kinase
- Such clones express dpp at any position within the anterior compartment of the disc (Fig. 3f, g), indicating that smo is not absolutely required for dpp transcription; rather, it acts upstream of PKA to mediate activation of dpp by hh.
- Genomic PI clones (Smoller, D.A. et al. (1991) Chromasoma 100:487- 494) covering this region were obtained and used to screen genomic DNA from newly isolated -ray-induced alleles (Fig. 1) of smo by Southern blot hybridization.
- the complete nucleotide sequence of the smo transcription unit was determined by sequencing genomic and partial complementary DNA clones (Fig. 4d) to reveal the intron- exon structure shown in Fig. 4c.
- Conceptual translation of the sequence identified a single long open reading frame of 1 ,024 amino acids (Fig. 4d) preceded by a 5' untranslated leader of at least 296 nucleotides. Downstream of the translation termination codon is an untranslated region of 654 nucleotides.
- Hydropathy analysis of the amino acid sequence indicates a putative signal sequence followed by a further seven hydrophobic domains, each of which is long enough to span the membrane once.
- GPCR G-protein-coupled receptors
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Insects & Arthropods (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU83808/98A AU8380898A (en) | 1997-07-01 | 1998-07-01 | Vertebrate smoothened gene, gene products, and uses related thereto |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US5140797P | 1997-07-01 | 1997-07-01 | |
US60/051,407 | 1997-07-01 | ||
US89779897A | 1997-07-21 | 1997-07-21 | |
US08/897,798 | 1997-07-21 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999001468A2 true WO1999001468A2 (fr) | 1999-01-14 |
Family
ID=26729382
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1998/013793 WO1999001468A2 (fr) | 1997-07-01 | 1998-07-01 | Gene smoothened de vertebre, produits geniques et utilisations associees |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU8380898A (fr) |
WO (1) | WO1999001468A2 (fr) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001070980A2 (fr) * | 2000-03-23 | 2001-09-27 | Pe Corporation (Ny) | Recepteurs isoles couples a la proteine g, molecules d'acides nucleiques codant des proteines gpcr et leurs utilisations en tant que cibles insecticides |
US6492139B1 (en) | 1996-09-30 | 2002-12-10 | Genentech, Inc. | Vertebrate smoothened proteins |
US6639051B2 (en) | 1997-10-20 | 2003-10-28 | Curis, Inc. | Regulation of epithelial tissue by hedgehog-like polypeptides, and formulations and uses related thereto |
US6767888B1 (en) | 1997-06-27 | 2004-07-27 | Curis, Inc. | Neuroprotective methods and reagents |
US6884770B1 (en) | 1998-11-06 | 2005-04-26 | Curis, Inc. | Methods and compositions for treating or preventing peripheral neuropathies |
US7138492B2 (en) | 1997-07-24 | 2006-11-21 | Curis, Inc. | Method of treating dopaminergic and GABA-nergic disorders |
WO2008070357A2 (fr) * | 2006-10-31 | 2008-06-12 | Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Polypeptides lissés et procédés d'utilisation |
CN109071625A (zh) * | 2016-02-04 | 2018-12-21 | 柯瑞斯公司 | 平滑化突变体和其使用方法 |
-
1998
- 1998-07-01 AU AU83808/98A patent/AU8380898A/en not_active Abandoned
- 1998-07-01 WO PCT/US1998/013793 patent/WO1999001468A2/fr not_active Application Discontinuation
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6492139B1 (en) | 1996-09-30 | 2002-12-10 | Genentech, Inc. | Vertebrate smoothened proteins |
US6767888B1 (en) | 1997-06-27 | 2004-07-27 | Curis, Inc. | Neuroprotective methods and reagents |
US7138492B2 (en) | 1997-07-24 | 2006-11-21 | Curis, Inc. | Method of treating dopaminergic and GABA-nergic disorders |
US7144997B2 (en) | 1997-07-24 | 2006-12-05 | Curis, Inc. | Vertebrate embryonic patterning-inducing proteins, compositions and uses related therto |
US6639051B2 (en) | 1997-10-20 | 2003-10-28 | Curis, Inc. | Regulation of epithelial tissue by hedgehog-like polypeptides, and formulations and uses related thereto |
US6884770B1 (en) | 1998-11-06 | 2005-04-26 | Curis, Inc. | Methods and compositions for treating or preventing peripheral neuropathies |
WO2001070980A2 (fr) * | 2000-03-23 | 2001-09-27 | Pe Corporation (Ny) | Recepteurs isoles couples a la proteine g, molecules d'acides nucleiques codant des proteines gpcr et leurs utilisations en tant que cibles insecticides |
WO2001070980A3 (fr) * | 2000-03-23 | 2002-04-18 | Pe Corp Ny | Recepteurs isoles couples a la proteine g, molecules d'acides nucleiques codant des proteines gpcr et leurs utilisations en tant que cibles insecticides |
WO2008070357A2 (fr) * | 2006-10-31 | 2008-06-12 | Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Polypeptides lissés et procédés d'utilisation |
WO2008070357A3 (fr) * | 2006-10-31 | 2009-02-26 | Us Gov Health & Human Serv | Polypeptides lissés et procédés d'utilisation |
JP2010508305A (ja) * | 2006-10-31 | 2010-03-18 | アメリカ合衆国 | スムースンドポリペプチドおよび使用方法 |
EP2431388A1 (fr) * | 2006-10-31 | 2012-03-21 | Government of the United States of America, Represented by the Secretary, Department of Health and Human Services | Polypeptides lissés et procédés d'utilisation |
US8198402B2 (en) | 2006-10-31 | 2012-06-12 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Smoothened polypeptides and methods of use |
US8546524B2 (en) | 2006-10-31 | 2013-10-01 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Smoothened polypeptides and methods of use |
JP2013255500A (ja) * | 2006-10-31 | 2013-12-26 | Usa Government | スムースンドポリペプチドおよび使用方法 |
JP2015180630A (ja) * | 2006-10-31 | 2015-10-15 | アメリカ合衆国 | スムースンドポリペプチドおよび使用方法 |
CN109071625A (zh) * | 2016-02-04 | 2018-12-21 | 柯瑞斯公司 | 平滑化突变体和其使用方法 |
Also Published As
Publication number | Publication date |
---|---|
AU8380898A (en) | 1999-01-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7585671B2 (en) | Hedgehog interacting proteins and uses related thereto | |
US7060450B1 (en) | Screening assays for agonists and antagonists of the hedgehog signaling pathway | |
CA2179029C (fr) | Proteines de type herisson induisant une structure embryonnaire chez les vertebres | |
US6261786B1 (en) | Screening assays for hedgehog agonists and antagonists | |
US7781215B2 (en) | Vertebrate embryonic pattern-inducing proteins, and uses related thereto | |
US6165747A (en) | Nucleic acids encoding hedgehog proteins | |
US6379925B1 (en) | Angiogenic modulation by notch signal transduction | |
US6884775B1 (en) | Methods and compositions for regulating skeletogenic formation | |
JPH09507853A (ja) | 脊椎動物胎仔パターン誘導性ヘッジホッグ様蛋白質 | |
US6399326B1 (en) | Nucleic acids encoding neural/pancreatic receptor tyrosine phosphatase | |
WO1999001468A2 (fr) | Gene smoothened de vertebre, produits geniques et utilisations associees | |
AU726918B2 (en) | TGFbeta signal transduction proteins, genes, and uses related thereto | |
WO1997022697A9 (fr) | PROTEINES AGISSANT SUR LA TRANSDUCTION DE SIGNAL PAR TGFβ GENES ASSOCIES ET LEURS UTILISATIONS | |
US6309879B1 (en) | Human patched genes and proteins, and uses related thereto | |
WO1998022504A1 (fr) | Nouveaux genes de semaphorine (i) | |
WO1999007854A2 (fr) | Serine/threonine kinase et ses utilisations | |
WO1999029860A1 (fr) | Polypeptides de type cadherine, procedes et compositions associes | |
JP2002530104A (ja) | カリウムチャンネル相互作用物質及びその利用法 | |
AU8731901A (en) | Vertebrate embryonic pattern-inducing hedgehog-like proteins |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 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 GW 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 UZ VN YU ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 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 ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
NENP | Non-entry into the national phase in: |
Ref country code: CA |
|
NENP | Non-entry into the national phase in: |
Ref country code: JP Ref document number: 1999507379 Format of ref document f/p: F |
|
122 | Ep: pct application non-entry in european phase | ||
WA | Withdrawal of international application |