WO2017132420A1 - Compositions and methods for suppression and diagnosis of cancer metastasis - Google Patents
Compositions and methods for suppression and diagnosis of cancer metastasis Download PDFInfo
- Publication number
- WO2017132420A1 WO2017132420A1 PCT/US2017/015198 US2017015198W WO2017132420A1 WO 2017132420 A1 WO2017132420 A1 WO 2017132420A1 US 2017015198 W US2017015198 W US 2017015198W WO 2017132420 A1 WO2017132420 A1 WO 2017132420A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- regnase
- accordance
- cells
- composition
- cancer
- Prior art date
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 181
- 201000011510 cancer Diseases 0.000 title claims abstract description 117
- 206010027476 Metastases Diseases 0.000 title claims abstract description 82
- 230000009401 metastasis Effects 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 69
- 239000000203 mixture Substances 0.000 title claims description 138
- 238000003745 diagnosis Methods 0.000 title claims description 15
- 230000001629 suppression Effects 0.000 title description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 100
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 78
- 229920001184 polypeptide Polymers 0.000 claims abstract description 71
- 206010061289 metastatic neoplasm Diseases 0.000 claims abstract description 47
- 230000001394 metastastic effect Effects 0.000 claims abstract description 42
- 241000124008 Mammalia Species 0.000 claims abstract description 31
- 210000004027 cell Anatomy 0.000 claims description 347
- 102100023882 Endoribonuclease ZC3H12A Human genes 0.000 claims description 213
- 101710112715 Endoribonuclease ZC3H12A Proteins 0.000 claims description 209
- 239000013598 vector Substances 0.000 claims description 67
- 150000007523 nucleic acids Chemical group 0.000 claims description 66
- 241000282414 Homo sapiens Species 0.000 claims description 57
- 210000001519 tissue Anatomy 0.000 claims description 38
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 26
- 239000003814 drug Substances 0.000 claims description 26
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 25
- 208000024891 symptom Diseases 0.000 claims description 23
- 238000011282 treatment Methods 0.000 claims description 22
- 241001465754 Metazoa Species 0.000 claims description 20
- 230000001225 therapeutic effect Effects 0.000 claims description 19
- 201000010099 disease Diseases 0.000 claims description 17
- 229940124597 therapeutic agent Drugs 0.000 claims description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 14
- 208000037819 metastatic cancer Diseases 0.000 claims description 12
- 208000011575 metastatic malignant neoplasm Diseases 0.000 claims description 12
- 239000008194 pharmaceutical composition Substances 0.000 claims description 11
- 210000004962 mammalian cell Anatomy 0.000 claims description 10
- 238000002560 therapeutic procedure Methods 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 208000035475 disorder Diseases 0.000 claims description 8
- 238000001415 gene therapy Methods 0.000 claims description 8
- 239000002246 antineoplastic agent Substances 0.000 claims description 7
- 238000011321 prophylaxis Methods 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 229940127089 cytotoxic agent Drugs 0.000 claims description 6
- 239000003085 diluting agent Substances 0.000 claims description 6
- 210000000056 organ Anatomy 0.000 claims description 6
- 230000002265 prevention Effects 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 5
- 230000002159 abnormal effect Effects 0.000 claims description 4
- 230000007812 deficiency Effects 0.000 claims description 4
- 239000000032 diagnostic agent Substances 0.000 claims description 4
- 229940039227 diagnostic agent Drugs 0.000 claims description 4
- 230000004064 dysfunction Effects 0.000 claims description 4
- 208000014674 injury Diseases 0.000 claims description 4
- 238000001990 intravenous administration Methods 0.000 claims description 4
- 244000144972 livestock Species 0.000 claims description 4
- 239000003981 vehicle Substances 0.000 claims description 4
- 239000012190 activator Substances 0.000 claims description 3
- 239000012216 imaging agent Substances 0.000 claims description 3
- 239000002105 nanoparticle Substances 0.000 claims description 3
- 230000000069 prophylactic effect Effects 0.000 claims description 3
- 230000008733 trauma Effects 0.000 claims description 3
- 239000002257 antimetastatic agent Substances 0.000 claims description 2
- 230000000973 chemotherapeutic effect Effects 0.000 claims description 2
- 230000006378 damage Effects 0.000 claims description 2
- 239000011859 microparticle Substances 0.000 claims description 2
- 239000004094 surface-active agent Substances 0.000 claims description 2
- 238000007910 systemic administration Methods 0.000 claims description 2
- 208000027418 Wounds and injury Diseases 0.000 claims 1
- 230000007547 defect Effects 0.000 claims 1
- 239000002502 liposome Substances 0.000 claims 1
- 239000002353 niosome Substances 0.000 claims 1
- 150000003904 phospholipids Chemical class 0.000 claims 1
- 230000005855 radiation Effects 0.000 claims 1
- 208000016691 refractory malignant neoplasm Diseases 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 61
- 108020004999 messenger RNA Proteins 0.000 abstract description 42
- 230000000694 effects Effects 0.000 abstract description 28
- 230000002401 inhibitory effect Effects 0.000 abstract description 11
- 230000001965 increasing effect Effects 0.000 abstract description 8
- 239000000090 biomarker Substances 0.000 abstract description 7
- 239000012636 effector Substances 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 85
- 241000699670 Mus sp. Species 0.000 description 66
- 102000004169 proteins and genes Human genes 0.000 description 58
- 235000018102 proteins Nutrition 0.000 description 55
- 102000039446 nucleic acids Human genes 0.000 description 46
- 108020004707 nucleic acids Proteins 0.000 description 46
- 210000004072 lung Anatomy 0.000 description 40
- 102000005881 S100 Calcium Binding Protein A6 Human genes 0.000 description 39
- 108010005260 S100 Calcium Binding Protein A6 Proteins 0.000 description 39
- 206010006187 Breast cancer Diseases 0.000 description 36
- 208000026310 Breast neoplasm Diseases 0.000 description 36
- 150000001875 compounds Chemical class 0.000 description 35
- 108020004414 DNA Proteins 0.000 description 33
- 102000040430 polynucleotide Human genes 0.000 description 29
- 108091033319 polynucleotide Proteins 0.000 description 29
- 239000002157 polynucleotide Substances 0.000 description 29
- 230000000295 complement effect Effects 0.000 description 27
- 235000001014 amino acid Nutrition 0.000 description 26
- 229940024606 amino acid Drugs 0.000 description 25
- 150000001413 amino acids Chemical class 0.000 description 25
- 125000003729 nucleotide group Chemical group 0.000 description 22
- 108010085149 S100 Calcium-Binding Protein A4 Proteins 0.000 description 21
- 239000002773 nucleotide Substances 0.000 description 21
- 102000007460 S100 Calcium-Binding Protein A4 Human genes 0.000 description 20
- 239000000243 solution Substances 0.000 description 20
- 238000001727 in vivo Methods 0.000 description 19
- 230000010473 stable expression Effects 0.000 description 18
- 238000009472 formulation Methods 0.000 description 17
- 201000001441 melanoma Diseases 0.000 description 17
- 230000002018 overexpression Effects 0.000 description 17
- 210000004881 tumor cell Anatomy 0.000 description 17
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 16
- 230000006870 function Effects 0.000 description 16
- 230000000692 anti-sense effect Effects 0.000 description 15
- 239000002585 base Substances 0.000 description 14
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 13
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 13
- 108010083644 Ribonucleases Proteins 0.000 description 13
- 102000006382 Ribonucleases Human genes 0.000 description 13
- 239000005090 green fluorescent protein Substances 0.000 description 13
- 238000013518 transcription Methods 0.000 description 13
- 230000035897 transcription Effects 0.000 description 13
- 238000003556 assay Methods 0.000 description 12
- 238000000338 in vitro Methods 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 12
- 150000003839 salts Chemical class 0.000 description 12
- 239000000523 sample Substances 0.000 description 12
- 108020005345 3' Untranslated Regions Proteins 0.000 description 11
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 11
- 108091034117 Oligonucleotide Proteins 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
- 230000015556 catabolic process Effects 0.000 description 11
- 239000003623 enhancer Substances 0.000 description 11
- 238000006731 degradation reaction Methods 0.000 description 10
- 230000007246 mechanism Effects 0.000 description 10
- 230000001105 regulatory effect Effects 0.000 description 10
- 108091006146 Channels Proteins 0.000 description 9
- 230000027455 binding Effects 0.000 description 9
- -1 chemotherapeutics Substances 0.000 description 9
- 238000011161 development Methods 0.000 description 9
- 238000009396 hybridization Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 230000004048 modification Effects 0.000 description 9
- 238000012986 modification Methods 0.000 description 9
- 238000012755 real-time RT-PCR analysis Methods 0.000 description 9
- 210000003462 vein Anatomy 0.000 description 9
- 208000001382 Experimental Melanoma Diseases 0.000 description 8
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 8
- 239000004480 active ingredient Substances 0.000 description 8
- 230000008901 benefit Effects 0.000 description 8
- 238000003384 imaging method Methods 0.000 description 8
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 101000976212 Homo sapiens Endoribonuclease ZC3H12A Proteins 0.000 description 7
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 7
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 7
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 7
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 7
- 206010027458 Metastases to lung Diseases 0.000 description 7
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 7
- 241000700605 Viruses Species 0.000 description 7
- 125000000539 amino acid group Chemical group 0.000 description 7
- 230000004663 cell proliferation Effects 0.000 description 7
- 230000002255 enzymatic effect Effects 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 230000000683 nonmetastatic effect Effects 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 241000702421 Dependoparvovirus Species 0.000 description 6
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 150000001251 acridines Chemical class 0.000 description 6
- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 description 6
- 230000000875 corresponding effect Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 230000001323 posttranslational effect Effects 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 230000002103 transcriptional effect Effects 0.000 description 6
- 102000007469 Actins Human genes 0.000 description 5
- 108010085238 Actins Proteins 0.000 description 5
- 239000004475 Arginine Substances 0.000 description 5
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 5
- 108090000994 Catalytic RNA Proteins 0.000 description 5
- 102000053642 Catalytic RNA Human genes 0.000 description 5
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 5
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 5
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 5
- 239000004472 Lysine Substances 0.000 description 5
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 5
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 5
- 239000004473 Threonine Substances 0.000 description 5
- 239000013543 active substance Substances 0.000 description 5
- 235000004279 alanine Nutrition 0.000 description 5
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 5
- 235000009582 asparagine Nutrition 0.000 description 5
- 229960001230 asparagine Drugs 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 5
- 230000001276 controlling effect Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000004205 dimethyl polysiloxane Substances 0.000 description 5
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 5
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 5
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 5
- 239000003112 inhibitor Substances 0.000 description 5
- 229960000310 isoleucine Drugs 0.000 description 5
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 5
- 238000000386 microscopy Methods 0.000 description 5
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 5
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 5
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 5
- 108091092562 ribozyme Proteins 0.000 description 5
- 239000002356 single layer Substances 0.000 description 5
- 150000003384 small molecules Chemical class 0.000 description 5
- 239000004474 valine Substances 0.000 description 5
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 4
- 206010055113 Breast cancer metastatic Diseases 0.000 description 4
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 4
- 101100193633 Danio rerio rag2 gene Proteins 0.000 description 4
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 101000976218 Homo sapiens Probable ribonuclease ZC3H12B Proteins 0.000 description 4
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 4
- 101100193635 Mus musculus Rag2 gene Proteins 0.000 description 4
- 101710081124 NEDD4-binding protein 1 Proteins 0.000 description 4
- 102100038552 NEDD4-binding protein 1 Human genes 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- 102100023886 Probable ribonuclease ZC3H12C Human genes 0.000 description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 4
- 108010029485 Protein Isoforms Proteins 0.000 description 4
- 102000001708 Protein Isoforms Human genes 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 4
- 108091023040 Transcription factor Proteins 0.000 description 4
- 102000040945 Transcription factor Human genes 0.000 description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 4
- 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 Polymers 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 4
- 239000002253 acid Substances 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
- 238000013459 approach Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 238000011049 filling Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 210000005260 human cell Anatomy 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- 230000000977 initiatory effect Effects 0.000 description 4
- 239000002679 microRNA Substances 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 4
- 235000021317 phosphate Nutrition 0.000 description 4
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 4
- 229950010131 puromycin Drugs 0.000 description 4
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 238000011269 treatment regimen 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
- 230000003612 virological effect Effects 0.000 description 4
- OISVCGZHLKNMSJ-UHFFFAOYSA-N 2,6-dimethylpyridine Chemical compound CC1=CC=CC(C)=N1 OISVCGZHLKNMSJ-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000271566 Aves Species 0.000 description 3
- 108091035707 Consensus sequence Proteins 0.000 description 3
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 3
- 206010014759 Endometrial neoplasm Diseases 0.000 description 3
- 108010093099 Endoribonucleases Proteins 0.000 description 3
- 102000002494 Endoribonucleases Human genes 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101000921361 Homo sapiens Elongation of very long chain fatty acids protein 5 Proteins 0.000 description 3
- 101000974747 Homo sapiens Potassium channel subfamily K member 12 Proteins 0.000 description 3
- 101000976219 Homo sapiens Probable ribonuclease ZC3H12C Proteins 0.000 description 3
- 101000976215 Homo sapiens Probable ribonuclease ZC3H12D Proteins 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 3
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 206010033799 Paralysis Diseases 0.000 description 3
- 102100022801 Potassium channel subfamily K member 12 Human genes 0.000 description 3
- 102100023884 Probable ribonuclease ZC3H12D Human genes 0.000 description 3
- 108091081021 Sense strand Proteins 0.000 description 3
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 229940009098 aspartate Drugs 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- XDHNQDDQEHDUTM-UHFFFAOYSA-N bafliomycin A1 Natural products COC1C=CC=C(C)CC(C)C(O)C(C)C=C(C)C=C(OC)C(=O)OC1C(C)C(O)C(C)C1(O)OC(C(C)C)C(C)C(O)C1 XDHNQDDQEHDUTM-UHFFFAOYSA-N 0.000 description 3
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 description 3
- 230000012292 cell migration Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000004590 computer program Methods 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 231100000517 death Toxicity 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 229930195712 glutamate Natural products 0.000 description 3
- 238000003119 immunoblot Methods 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 239000002751 oligonucleotide probe Substances 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 229910052710 silicon Inorganic materials 0.000 description 3
- 239000010703 silicon Substances 0.000 description 3
- 239000004055 small Interfering RNA Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 239000005051 trimethylchlorosilane Substances 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 239000013603 viral vector 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
- NEQZWEXWOFPKOT-BYRRXHGESA-N 5Z-7-oxozeaenol Chemical compound O([C@@H](C)C\C=C/C(=O)[C@@H](O)[C@@H](O)C/C=C/1)C(=O)C=2C\1=CC(OC)=CC=2O NEQZWEXWOFPKOT-BYRRXHGESA-N 0.000 description 2
- 241000702423 Adeno-associated virus - 2 Species 0.000 description 2
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 2
- 108020005544 Antisense RNA Proteins 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000972773 Aulopiformes Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 102000012199 E3 ubiquitin-protein ligase Mdm2 Human genes 0.000 description 2
- 108050002772 E3 ubiquitin-protein ligase Mdm2 Proteins 0.000 description 2
- 102100032052 Elongation of very long chain fatty acids protein 5 Human genes 0.000 description 2
- 206010014733 Endometrial cancer Diseases 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- 101001050612 Homo sapiens Protein KHNYN Proteins 0.000 description 2
- 101001121714 Homo sapiens Protein NYNRIN Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 239000003458 I kappa b kinase inhibitor Substances 0.000 description 2
- 102100026888 Mitogen-activated protein kinase kinase kinase 7 Human genes 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 102100023883 Probable ribonuclease ZC3H12B Human genes 0.000 description 2
- 102000007066 Prostate-Specific Antigen Human genes 0.000 description 2
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 2
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 2
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 2
- 229940079156 Proteasome inhibitor Drugs 0.000 description 2
- 102100025467 Protein NYNRIN Human genes 0.000 description 2
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 2
- 230000006819 RNA synthesis Effects 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 102000039471 Small Nuclear RNA Human genes 0.000 description 2
- 108020003224 Small Nucleolar RNA Proteins 0.000 description 2
- 102000042773 Small Nucleolar RNA Human genes 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- 108091060271 Small temporal RNA Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 108700026226 TATA Box Proteins 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 108700029229 Transcriptional Regulatory Elements Proteins 0.000 description 2
- 108020004566 Transfer RNA Proteins 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 description 2
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine group Chemical group [C@@H]1([C@H](O)[C@H](O)[C@@H](CO)O1)N1C=NC=2C(N)=NC=NC12 OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 125000005600 alkyl phosphonate group Chemical group 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 239000000074 antisense oligonucleotide Substances 0.000 description 2
- 238000012230 antisense oligonucleotides Methods 0.000 description 2
- 229930192649 bafilomycin Natural products 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 229960001467 bortezomib Drugs 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 210000005056 cell body Anatomy 0.000 description 2
- 108091092328 cellular RNA Proteins 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 230000005757 colony formation Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 239000002254 cytotoxic agent Substances 0.000 description 2
- 231100000599 cytotoxic agent Toxicity 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 239000002612 dispersion medium Substances 0.000 description 2
- 201000003914 endometrial carcinoma Diseases 0.000 description 2
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 2
- NEQZWEXWOFPKOT-UHFFFAOYSA-N f152A1 Natural products C1=CCC(O)C(O)C(=O)C=CCC(C)OC(=O)C=2C1=CC(OC)=CC=2O NEQZWEXWOFPKOT-UHFFFAOYSA-N 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000004547 gene signature Effects 0.000 description 2
- 230000008303 genetic mechanism Effects 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 229940093915 gynecological organic acid Drugs 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 235000011167 hydrochloric acid Nutrition 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical class CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 2
- 108091070501 miRNA Proteins 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 150000007522 mineralic acids Chemical class 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 238000011275 oncology therapy Methods 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- CBHCDHNUZWWAPP-UHFFFAOYSA-N pecazine Chemical compound C1N(C)CCCC1CN1C2=CC=CC=C2SC2=CC=CC=C21 CBHCDHNUZWWAPP-UHFFFAOYSA-N 0.000 description 2
- 229950007538 pecazine Drugs 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 150000004713 phosphodiesters Chemical group 0.000 description 2
- 235000011007 phosphoric acid Nutrition 0.000 description 2
- 229920002120 photoresistant polymer Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 239000003207 proteasome inhibitor Substances 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 235000019515 salmon Nutrition 0.000 description 2
- 238000006748 scratching Methods 0.000 description 2
- 230000002393 scratching effect Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 108091029842 small nuclear ribonucleic acid Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 230000002459 sustained effect Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 108091008743 testicular receptors 4 Proteins 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 230000005740 tumor formation Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 1
- 125000000229 (C1-C4)alkoxy group Chemical group 0.000 description 1
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- LNETULKMXZVUST-UHFFFAOYSA-N 1-naphthoic acid Chemical compound C1=CC=C2C(C(=O)O)=CC=CC2=C1 LNETULKMXZVUST-UHFFFAOYSA-N 0.000 description 1
- TWJGQZBSEMDPQP-UHFFFAOYSA-N 2-chloro-n-[4-[5-(3,4-dichlorophenyl)-3-(2-methoxyethoxy)-1,2,4-triazol-1-yl]phenyl]acetamide Chemical compound C=1C=C(NC(=O)CCl)C=CC=1N1N=C(OCCOC)N=C1C1=CC=C(Cl)C(Cl)=C1 TWJGQZBSEMDPQP-UHFFFAOYSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- XWKFPIODWVPXLX-UHFFFAOYSA-N 2-methyl-5-methylpyridine Natural products CC1=CC=C(C)N=C1 XWKFPIODWVPXLX-UHFFFAOYSA-N 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- MXVJCCPMGYHBLH-UHFFFAOYSA-N 6-chloro-2-methoxy-n-pentylacridin-9-amine Chemical compound C1=C(OC)C=C2C(NCCCCC)=C(C=CC(Cl)=C3)C3=NC2=C1 MXVJCCPMGYHBLH-UHFFFAOYSA-N 0.000 description 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- 208000035657 Abasia Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 208000031277 Amaurotic familial idiocy Diseases 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 101001007348 Arachis hypogaea Galactose-binding lectin Proteins 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 108091033409 CRISPR Proteins 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000938605 Crocodylia Species 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 108010092160 Dactinomycin Proteins 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 208000001914 Fragile X syndrome Diseases 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 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 1
- 241000175212 Herpesvirales Species 0.000 description 1
- 241001272567 Hominoidea Species 0.000 description 1
- 101001030477 Homo sapiens NEDD4-binding protein 1 Proteins 0.000 description 1
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 1
- 241000484121 Human parvovirus Species 0.000 description 1
- 208000023105 Huntington disease Diseases 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 102000001284 I-kappa-B kinase Human genes 0.000 description 1
- 108060006678 I-kappa-B kinase Proteins 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
- 108700002232 Immediate-Early Genes Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical group 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-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
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 241000219745 Lupinus Species 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 208000025205 Mantle-Cell Lymphoma Diseases 0.000 description 1
- 102000029749 Microtubule Human genes 0.000 description 1
- 108091022875 Microtubule Proteins 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 102000013967 Monokines Human genes 0.000 description 1
- 108010050619 Monokines Proteins 0.000 description 1
- 102100034256 Mucin-1 Human genes 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 208000029578 Muscle disease Diseases 0.000 description 1
- 208000021642 Muscular disease Diseases 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 229930182474 N-glycoside Natural products 0.000 description 1
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 108700019961 Neoplasm Genes Proteins 0.000 description 1
- 102000048850 Neoplasm Genes Human genes 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229920002230 Pectic acid Polymers 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010038988 Peptide Hormones Proteins 0.000 description 1
- 102000015731 Peptide Hormones Human genes 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 208000002151 Pleural effusion Diseases 0.000 description 1
- 108010020346 Polyglutamic Acid Proteins 0.000 description 1
- 241000282405 Pongo abelii Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 101710131547 Potassium channel subfamily K member 12 Proteins 0.000 description 1
- 101710192813 Probable ribonuclease ZC3H12C Proteins 0.000 description 1
- 101710192814 Probable ribonuclease ZC3H12D Proteins 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100023409 Protein KHNYN Human genes 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 241000220010 Rhode Species 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 101150054830 S100A6 gene Proteins 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 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
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 1
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 1
- 102000039634 Untranslated RNA Human genes 0.000 description 1
- 108020004417 Untranslated RNA Proteins 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 1
- 229940122803 Vinca alkaloid Drugs 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N aldehydo-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 229940045799 anthracyclines and related substance Drugs 0.000 description 1
- 230000000181 anti-adherent effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000001028 anti-proliverative effect Effects 0.000 description 1
- 229940027998 antiseptic and disinfectant acridine derivative Drugs 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 208000005980 beta thalassemia Diseases 0.000 description 1
- 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 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000001574 biopsy Methods 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
- 229910052797 bismuth Inorganic materials 0.000 description 1
- JCXGWMGPZLAOME-UHFFFAOYSA-N bismuth atom Chemical compound [Bi] JCXGWMGPZLAOME-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000012152 bradford reagent Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 230000005773 cancer-related death Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000012829 chemotherapy agent Substances 0.000 description 1
- 238000009104 chemotherapy regimen Methods 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- WZHCOOQXZCIUNC-UHFFFAOYSA-N cyclandelate Chemical compound C1C(C)(C)CC(C)CC1OC(=O)C(O)C1=CC=CC=C1 WZHCOOQXZCIUNC-UHFFFAOYSA-N 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 230000009266 disease activity Effects 0.000 description 1
- 230000006334 disulfide bridging Effects 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 239000012776 electronic material Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 235000011087 fumaric acid Nutrition 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- LRBQNJMCXXYXIU-QWKBTXIPSA-N gallotannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@H]2[C@@H]([C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-QWKBTXIPSA-N 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 238000010842 high-capacity cDNA reverse transcription kit Methods 0.000 description 1
- 102000051702 human ELOVL5 Human genes 0.000 description 1
- 102000057605 human KHNYN Human genes 0.000 description 1
- 102000050964 human ZC3H12A Human genes 0.000 description 1
- 102000056250 human ZC3H12B Human genes 0.000 description 1
- 102000047985 human ZC3H12D Human genes 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- RLCFKYRNUBRPIK-UHFFFAOYSA-N hydron;10-[(1-methylpiperidin-3-yl)methyl]phenothiazine;chloride Chemical compound Cl.C1N(C)CCCC1CN1C2=CC=CC=C2SC2=CC=CC=C21 RLCFKYRNUBRPIK-UHFFFAOYSA-N 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 230000037417 hyperactivation Effects 0.000 description 1
- 230000003463 hyperproliferative effect Effects 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 229940124644 immune regulator Drugs 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 229940125721 immunosuppressive agent Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 238000013187 longer-term treatment Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000009607 mammography Methods 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000003358 metastasis assay Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 210000004688 microtubule Anatomy 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 230000001617 migratory effect Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 239000002077 nanosphere Substances 0.000 description 1
- YZMHQCWXYHARLS-UHFFFAOYSA-N naphthalene-1,2-disulfonic acid Chemical class C1=CC=CC2=C(S(O)(=O)=O)C(S(=O)(=O)O)=CC=C21 YZMHQCWXYHARLS-UHFFFAOYSA-N 0.000 description 1
- PSZYNBSKGUBXEH-UHFFFAOYSA-N naphthalene-1-sulfonic acid Chemical class C1=CC=C2C(S(=O)(=O)O)=CC=CC2=C1 PSZYNBSKGUBXEH-UHFFFAOYSA-N 0.000 description 1
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 230000001293 nucleolytic effect Effects 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 230000000771 oncological effect Effects 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 150000002892 organic cations Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 108700025694 p53 Genes Proteins 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 238000002559 palpation Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000000206 photolithography Methods 0.000 description 1
- 230000010399 physical interaction Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000010318 polygalacturonic acid Substances 0.000 description 1
- 229920002643 polyglutamic acid Polymers 0.000 description 1
- 210000004896 polypeptide structure Anatomy 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 208000037821 progressive disease Diseases 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- LLHKCFNBLRBOGN-UHFFFAOYSA-N propylene glycol methyl ether acetate Chemical compound COCC(C)OC(C)=O LLHKCFNBLRBOGN-UHFFFAOYSA-N 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- IGFXRKMLLMBKSA-UHFFFAOYSA-N purine Chemical compound N1=C[N]C2=NC=NC2=C1 IGFXRKMLLMBKSA-UHFFFAOYSA-N 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000004528 spin coating Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- WZMPOCLULGAHJR-UHFFFAOYSA-N thiophen-2-ol Chemical compound OC1=CC=CS1 WZMPOCLULGAHJR-UHFFFAOYSA-N 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000759 toxicological effect Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 238000011277 treatment modality Methods 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 239000000107 tumor biomarker Substances 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 229940099039 velcade Drugs 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 239000012130 whole-cell lysate Substances 0.000 description 1
- 238000011816 wild-type C57Bl6 mouse Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- 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
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
Definitions
- the present disclosure generally relates to the fields of medicine and cancer biology.
- the invention provides improved compositions for determining the metastatic potential of mammalian tumors and other cancers by monitoring the level of Regnase- 1 polypeptide in vivo.
- methods for altering the metastatic potential of certain types of mammalian cancers by directly or indirectly increasing the overall levels of regnase activity, by selectively increasing the enzymatic activity of (or by selectively inhibiting one or more negative effectors of) Regnase, or by selectively increasing Regnase- specific mRNA levels within cancer cells to alter, prevent, or reduce their ability to metastasize to other cells or tissues.
- a targeted gene signature for Regnase is also disclosed, which can be exploited as a molecular biomarker for predicting metastasis and/or determining patient outcomes.
- Regnase- 1 is an enzyme that breaks down mRNA, but itself is a protein controlled by post-translational mechanisms. For example, proteasomes that can degrade proteins can also degrade Regnase-1. Thus, pathways that result in hyper-activation of such proteasomes will also result in the inactivation of Regnase-1. Such pathways and specific proteasomes can therefore serve as a molecular signature for cancer metastasis in lieu of Regnase-1. These molecules and pathways are also potential targets to inhibit cancer metastasis. However, this remains in the context that low levels of Regnase- 1 protein itself indicate the high metastatic potential for cancer cells. Furthermore, as Regnase- 1 breaks down mRNAs or ncRNAs, the identity of those RNA, once revealed, can also serve as a molecular signature to predict cancer metastasis or serve as targets for therapeutic interventions.
- Loss of Regnase- 1 enzymatic activity in vivo may be associated with tumor transformation, or resistance to certain therapeutic agents. It is also possible that the loss of Regnase- 1 activity may identify tumor stem cells, which have high metastatic potential.
- cancers are extremely aggressive, showing propensity of spreading early, and even the some type of cancer (e.g., melanoma), not all cancer cells are metastatic, and cells that can spread and establish distal nodules are confined to a small subset with stem cell-like features, the presence or absence of Regnase may segregate tumor cells into such categories.
- the decrease or absence of Regnase activity in cancer biopsies may indicate poor prognosis for cancer patients, and/or may signal the need for more aggressive and/or more exhaustive chemotherapy regimens than would ordinarily be indicated.
- Over-expressed and/or secreted proteins from cancer cells are easy to detect in the sera of cancer patients, and as such, have been used clinically as diagnostic and prognostic markers for cancers for a number of years (Wu et al., 2005).
- Examples of secreted proteins used for cancer diagnosis include prostate specific antigen (PSA) for prostate cancer (Balk et al, 2003), carcinoembryonic antigen, CA125, for ovarian cancer (Raamanathan et al., 2012) and colony stimulating factor-1 (CSF-1) (Kacinski et al, 1990) for endometrial carcinoma.
- PSA prostate specific antigen
- CA125 carcinoembryonic antigen
- CA125 for ovarian cancer
- CSF-1 colony stimulating factor-1
- a few soluble biomarkers for breast cancer including carcinoembryonic antigens, CA 15.3 and CA 27.59 and vascular endothelial growth factor (VEGF) are of prognostic value (Harris
- CA 15-3 is the most widely used serum biomarker assayed in conjunction with diagnostic imaging for monitoring metastatic disease in breast cancer (Danova et al, 2011).
- this, and other existing breast cancer biomarkers e.g., TP A/TPS, etc.
- TP A/TPS TP A/TPS, etc.
- Duffy 2006
- identification of more specific markers to predict metastasis would be of clinical significance, and would provide a significant advance over the prior art.
- the present disclosure addresses unmet deficiencies inherent in the relevant oncological and pharmaceutical arts by providing Regnase-specific polypeptide and polynucleotide compositions, gene therapy systems for expressing Regnase proteins in specific mammalian cells, and methods for controlling or expressing biologically-active Regnase polypeptides, and exploiting Regnase compounds as biomarkers for predicting the metastatic potential of cancer cells.
- the present disclosure also provides compositions for determining the metastatic potential of mammalian tumors and other cancers, and methods for monitoring the level of Regnase- 1 protein both in vitro and in vivo.
- the disclosed Regnase compositions facilitate new methods in the area of cancer diagnostics, and particularly in cancer metastasis diagnosis, by serving as molecular markers (i.e. a molecular "signature") that correlate with the invasiveness of particular types of cancer cells.
- the Regnase compositions disclosed herein also represent important new tools in cancer treatment, through the induction or overexpression of nuclease- active Regnase- 1 polypeptides in mammalian cancer cells.
- Gene-therapy-based approaches for persistently expressing Regnase- 1 polypeptide in cancer cells including vector-based, RNA-based, and nanoparticle-based vectors and/or delivery systems also represent important aspects of the present disclosure.
- the use of viral- based gene-expression vectors which have already been approved for human use in cancer treatment, to persistently express Regnase- 1 in populations of mammalian cells (and particularly, human cancer cells), offers a ready platform for exploiting the disclosed Regnase compositions in a variety of cancer treatment modalities.
- the P53 gene construct rAd-p53, also known as Gendicine (Shenzhen SiBiono GeneTech, CHINA) is a recombinant, replication-incompetent, human serotype 5 adenovirus. Approved for use in China in 2003, it has been used to treat head and neck squamous cell carcinoma in a number of clinical trials. To construct this therapeutic adenovirus, the El region of the virus was replaced by a human, wild-type P53 expression cassette.
- Ad- Reg- 1 A similar strategy has been used to generate the Ad- Reg- 1 construct referred to herein as "rAd- Regnase- 1."
- the El region was replaced by a human wild-type or a truncated Regnase- 1 gene, and the resulting adenoviral vectors were prepared using 293T host cells for propagation.
- Regnase-1 By understanding what down-regulates or degrades Regnase- 1, various approaches and specific small molecules, can be devised to inhibit such mechanisms to achieve sustained Regnase expression in cancer cells. Once induced, Regnase-1 undergoes attrition in cells over time, which is particularly significant at the protein level, where Regnase-1 is inactivated via proteosomal- based degradation.
- Bortezomib (VELCADE®, Janssen-Cilag, Pty Ltd, Macquarie Park, NSW, AUSTRALIA) is an FDA-approved proteasome inhibitor for treatment of multiple myeloma and mantle-cell lymphoma. Drugs like bortezomib can also be used as non-specific therapeutics to rescue Regnase-1 from degradation thereby inhibiting metastatic cancer cells. Identifying specific Regnase-1 degradation proteasomes and signaling pathways have led to the creation of more specific inhibitors of Regnase-1 degradation, which can be used to stabilize (or even enhance) Regnase-1 levels, thereby inhibiting cancer metastasis.
- Another approach afforded by the present disclosure is the development of small molecules that can bind to Regnase-1 to stabilize the protein in a manner analogous to that of a chaperone.
- Such small molecules may also function by disrupting physical interactions between Regnase and proteases, and thereby reducing or preventing the degradation of Regnase polypeptide.
- the tumor suppressor, P53 is inactivated by the E3 ligase, MDM2.
- Small molecules, such as Nutlin or its analogs can mimic the p53 peptide thereby blocking the activity of MDM2.
- Such small molecules are currently under development in clinic to treat multiple cancers. Similar strategies can be exploited to block the activity of E3 ligases that degrade Regnase-1.
- Regnase-1 -specific gene signatures can also be used as predictors of cancer metastasis, and may serve as potential targets of cancer therapies.
- Regnase-1 functions as a ribonuclease to breakdown a set of RNAs to block cancer metastasis.
- RNA microarrays may be exploited to identify potential targets of Regnase-1. By identifying such gene transcripts, they can serve also as molecular signatures to predict cancer metastasis.
- the present disclosure provides a composition that comprises: a therapeutically-effective amount of a first isolated mammalian Regnase-1 peptide, a Regnase-1 polypeptide, a Regnase-1 activator, or a combination thereof, either alone, or in combination with one or more distinct therapeutic, diagnostic, prophylactic, or prognostic agents, and in particular, one or more agents useful in determining the metastatic potential of a mammalian cancer cell, and particularly a human cancer cell.
- compositions may also further optionally comprise an additional chemotherapeutic agent, one or more diagnostic agents, one or more imaging agents, or any combinations thereof.
- Such compositions may be formulated with one or more pharmaceutically- acceptable buffers, diluents, solvents, or solutions, and may be contained within a commercial kit that includes instructions for administration of the composition to a mammal in need thereof.
- composition disclosed herein find particular use in the diagnosis, the prophylaxis, the therapy, or the amelioration of one or more symptoms of a cancer or cancer metastasis in a mammal.
- Regnase-1 peptides or polypeptides preferably comprise an at least 50 amino acid sequence that is at least 95% identical to an at least 50 contiguous amino acid sequence from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: 11, more preferably, an at least 100 amino acid sequence that is at least 95% identical to an at least 100 contiguous amino acid sequence from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: l l, and more preferably still, an at least 250 amino acid sequence that is at least 95% identical to an at least 250 contiguous amino acid sequence from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
- the Regnase polypeptides in accordance with the present disclosure comprise, consist essentially or, or alternatively, consist of an amino acid sequence that is at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to any one of the polypeptide sequences set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: 11.
- Regnase-1 peptides or polypeptides may be produced in vitro or in vivo by a gene therapy vector system that comprises a nucleic acid sequence encoding a selected Regnase-1 peptide or polypeptide operably linked to a promoter that expresses the nucleic acid sequence to produce the encoded peptide or polypeptide in a mammalian cell that has been transformed with the vector system.
- a gene therapy vector system include, without limitation, an adenoviral, adeno-associated, lentiviral, or herpesviral vector system that operably expresses the peptide or the polypeptide in a population of mammalian cells transformed with the vector system.
- compositions disclosed herein may be used in diagnosis, prophylaxis, or therapy, or one or more combinations thereof.
- the compositions may be used in the manufacture of a medicament for diagnosing, treating, or ameliorating one or more symptoms of a mammalian cancer, and preferably, of a human cancer, and in particular, one or more human metastatic cancers.
- Regnase compositions may further optionally include one or more active agents, such as, for example, one or more prophylactic agents, one or more therapeutic agents, one or more diagnostic agents, one or more vaccines, one or more imaging agents, one or more radiolabels, one or more adjuvanting agents, one or more chemotherapeutic agents, one or more cytotoxic agents, or any combination thereof, and preferably in a pharmaceutical formulation suitable for administration to a patient.
- active agents such as, for example, one or more prophylactic agents, one or more therapeutic agents, one or more diagnostic agents, one or more vaccines, one or more imaging agents, one or more radiolabels, one or more adjuvanting agents, one or more chemotherapeutic agents, one or more cytotoxic agents, or any combination thereof, and preferably in a pharmaceutical formulation suitable for administration to a patient.
- the invention also provides therapeutic and/or diagnostic kits including one or more of the cancer metastasis prognostic Regnase compositions disclosed herein, typically in combination with one or more pharmaceutically acceptable carriers, one or more devices for administration of the compositions to a subject of interest, as well as one or more instruction sets for using the composition in the prevention, the diagnosis, or the treatment of a mammalian condition, disease, disorder, trauma, and/or dysfunction, including, without limitation, one or more mammalian cancers and such like.
- the Regnase compositions disclosed herein may also be used in the manufacture of a diagnostic reagent for determining the metastatic potential of one or more cancers in a mammalian subject.
- the invention also provides a method for diagnosing, treating, or ameliorating one or more symptoms of cancer in a mammal.
- a method includes at least the step of administering to a mammal in need thereof, an effective amount of a Regnase composition as disclosed herein, for a time sufficient to diagnose, treat, or ameliorate the one or more symptoms of cancer in the mammal, including, without limitation, a human that has, or is at risk for developing one or more cancer metastases.
- Such methods may further optionally include administering a therapeutically-effective amount of at least one anti-cancer agent or one anti- metastatic agent to the mammal.
- the invention also provides in an overall and general sense, methods for providing a Regnase composition to a mammalian cancer cell that include at least the step of administering to the subject, an effective amount of one or more of the Regnase compositions disclosed herein.
- the subject is at risk for, diagnosed with, or suspected of having one or more abnormal conditions, including, for example, one or more cancers or other hyperproliferative disorders, and in particular, one or more metastatic cancers.
- compositions of the present disclosure may be administered to the subject through any one or more conventional methods for administration, including, without limitation, orally, intranasally, intravenously, subcutaneously, or by direct injection to one or more cells or one or more tissues within or about the body of the subject.
- the present disclosure also provides a method for administering an active agent to one or more cells, tissues, organs, or systems of a mammalian subject in need thereof.
- the method generally involves providing to a mammalian subject in need thereof, one or more of the regnase- specific compositions disclosed herein in an amount and for a time effective to introduce the composition into one or more selected tissues, organs, systems, or cells within or about the body of the subject.
- the cells extracted for such ex vivo manipulation will be those of the actual patient undergoing treatment.
- the Regnase compositions of the present invention may be formulated for pharmaceutical administration, and preferably for administration to a human.
- Such compositions may further include one or more additional therapeutic agents, chemotherapeutics, adjuvants, or a second distinct population of Regnase-expressing vectors, or host cells comprising them.
- compositions of the present disclosure may be administered to a mammal in a single administration, or in a series of successive administrations over a selected time interval ranging, for example, from one or more days, to one or more weeks, or even to one or more months wherein longer-term treatment is indicated.
- the Regnase composition(s) may be administered substantially concurrently with the administration of one or more additional therapeutic and/or diagnostic agents.
- the Regnase compositions may be comprised within a population of adenoviral vectors that contain a gene expression cassette encoding the Regnase peptide or protein of interest, operably linked to a promoter suitable for expressing the Regnase composition in a mammal, and particularly, a human.
- FIG. 1A and FIG. IB show the inactivation of Regnase-1 in highly metastatic breast cancer cells.
- FIG. 1A shows the immunoblot analysis of Regnase-1 protein level in MCF7, MDA-MB-231, SUM159, and MDA-MB-468 cancer cell lines.
- the breast cancer cells MDA- MB-231 and SUM159 have high metastatic potential as compared to MCF7 and MDA-MB-468 cells.
- 293 T cells are non-cancer cells and used as controls.
- FIG. IB shows the real-time RT- PCR analysis of Regnase-1 mRNA levels in 293T, MCF7, MDA-MB-231, SUM159, and MDA- MB-468 cells;
- FIG. 2A and FIG. 2B demonstrate the forced expression of Regnase-1 in cancer cells does not affect their proliferation potential or morphology.
- FIG. 2A shows tMCF7, SUM-159 and B16 cell lines that with stable expression of wild type (WT), endoribonuclease dead (D225A) Regnase-1 or control vector were seeded into 6-well plates and cultured for 48 hours. Their morphology was examined by light microscopy. The micrographs revealed clusters of proliferating cells.
- the cancer cell lines were labeled with CFSE, a dye that tracks cell proliferation, at day 0, and levels of CFSE in labeled cells was examined by flow cytometer at day 1 and day 5. Dilution of CFSE indicates cell proliferation;
- FIG. 3A and FIG. 3B show the expression of Regnase-1 inhibits cell migration and invasiveness.
- SUM-159 and B 16 melanoma cells that stably express wild type (WT), endoribonuclease dead (D225A) Regnase-1 or control vector were seeded into trans- well plates and cultured for 24 hours. The cells were fixed with methanol and stained with 0.05% crystal violet dye for 10 min. The invasiveness of cancer cells was marked by enlarged dark blue cell bodies.
- FIG. 3B shows SUM-159 cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were seeded into 6-well plates.
- FIG. 4A, FIG. 4B, and FIG. 4C show the forced expression of Regnase-1, but not the D225A mutation (enzymatically inactive Regnase-1), completely inhibits melanoma metastasis in vivo.
- FIG. 4A B 16 melanoma cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were injected into B6 mice through the tail vein. After two weeks, the mice were sacrificed, and the tumor foci in the lungs were determined. Photographs shown are both lungs from mice, and the dark spots are melanoma tumor foci. In FIG.
- FIG. 4B paraffin embedded lung tissues from host B6 mice injected with indicated melanoma cells were stained by Hematoxylin and Eosin (H&E). Tissue histology showed the lung tissue and tumor nodules in the lungs.
- FIG. 4C shows the survival of host mice injected with B16 melanoma cells with forced expression of WT Regnase-1, D225A mutated Regnase-1 or control vector. The primary endpoint was defined as severe paralysis or moribund. There were 10 mice in each group and none of the mice with Regnase-1 -expressing melanoma cells died in the four week- study period;
- FIG. 5A, FIG. 5B, and FIG. 5C show the forced expression of WT but not D225A mutated Regnase-1 completely inhibits metastasis of breast cancer cells in vivo.
- FIG. 5A SUM- 159 cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were injected into Rag2 _/ ⁇ and yc ' ⁇ double knockout (DKO) mice through tail vein. After three weeks, the mice were sacrificed and the tumor foci in the lung were imaged. The red arrow depicts tumor nodules.
- FIG. 5A SUM- 159 cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were injected into Rag2 _/ ⁇ and yc ' ⁇ double knockout (DKO) mice through tail vein. After three weeks, the mice were sacrificed and the tumor foci in the lung were imaged. The red arrow depicts tumor nodules.
- FIG. 5B The histogram shows the number of visible tumor nodules in the lung from Rag2 _/ ⁇ and yc ' ⁇ DKO mice injected with indicated cells.
- FIG. 5C Paraffin embedded lung tissues from Rag2 _/ ⁇ and yc ' ⁇ DKO mice injected with indicated tumor cells were stained by Hematoxylin and Eosin (H&E). Pictures show the lung tissue and tumor nodules in the lung;
- FIG. 6A and FIG. 6B show the correlation of breast cancer metastasis with low expression of Regnase-1.
- FIG. 6A Representative image of breast cancer tissues stained by anti- Regnase-1 antibody in breast cancer with or without metastasis.
- FIG. 6B The number represents breast cancer tissues with different level of Regnase-1 in breast cancer with or without metastasis;
- FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E-1, FIG. 7E-2 FIG. 7E-3 FIG. 7E-4, and FIG. 7E-5 show a microfluidic chip-based cell clustering (MFCBCC) assay to monitor cell clustering.
- FIG. 7A shows a working scheme to briefly describe the MFCBCC assay.
- FIG. 7B shows MCF7, MDA-MB-468, MDA-MB-231 and SUM-159 cells seeded into microfluidic chips at the concentration of 3 to 4 cells per channel.
- the process of cell clustering is monitored by digital recording in 2-hour time period.
- the red circles indicate individual cells at the beginning of experiment or individual cells and cell clusters at the end of experiment.
- FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E-1, FIG. 7E-2 FIG. 7E-3 FIG. 7E-4, and FIG. 7E-5 show a microfluidic chip-based cell clustering
- FIG. 7C is a histogram that shows the percentage of observed clustering events in total monitored wells within 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields.
- FIG. 7D shows five channels containing 3 - 5 cells for each cell type were monitored and their dynamic clustering rates presented as the percentage of observed clustering events in total events.
- FIG. 7E-1, FIG. 7E-2 FIG. 7E-3 FIG. 7E-4, and FIG. 7E-5 illustrate two channels containing 4 cells for each cell type. The dynamic sum of cell number and cluster number is also shown;
- FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, and FIG. 8F show the inactivation of Regnase-1 in highly metastatic breast cancer cells and tissues.
- FIG. 8A is the real-time RT-PCR analysis of Regnase-1 mRNA level in 293T, MCF7, MDA-MB-231, sUM159 and MDA-MB-468 cells.
- FIG. 8B shows the immunoblot analysis of Regnase-1 protein level in MCF7, MDA-MB- 231, SUM159, and MDA-MB-468 cancer cell lines.
- the breast cancer cells MDA-MB-231 and SUM159 had a high metastatic potential as compared to MCF7 and MDA-MB-468 cells.
- FIG. 8C shows MCF7, MDA-MB-231 and SUM-159 cells treated with different doses of MG132, Baf (Bafilomycin Al), MI2, Mep (Mepazine), IKKII (IKK inhibitor II), 5Z7 ((5Z)-7- Oxozeaenol) for 4 hrs. After treatment, the level of Regnase-1 was examined by Western Blot. The expression of ⁇ -actin as the loading control.
- FIG. 8D MCF7 or MDA-MB-468 cells were co-cultured with different amount of MDA-MB-231 or SUM-159 cells for 2 days.
- FIG. 8E is a representative image of breast cancer tissues stained by anti-Regnase-1 antibody in breast cancer with or without metastasis.
- FIG. 8F shows the number of breast cancer tissues with differing levels of Regnase-1 in cancer cells with or without metastasis. Data from FIG. 8A to FIG. 8D were representative of at least three independent studies;
- FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G, FIG. 9H, FIG. 91, and FIG. 9 J show the forced expression of Regnase-1, but not the D225A mutation (enzymatically-inactive Regnase-1), completely inhibited metastasis in vivo, and cell clustering in vitro.
- FIG. 9A SUM-159 cells with stable expression of WT Regnase-1, the D225A mutant Regnase, or a control vector were seeded into array chips, and monitored as described above in the description of FIG. 7A-FIG. 7E. The histogram in FIG.
- FIG. 9B shows the percentage of observed clustering events in all monitored channels at 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields.
- FIG. 9C shows SUM-159 cells with stable expression of WT Regnase-1, Reg " mutant, D225A, or a control vector that were then injected into Rag2/rc double-knockout (DKO) mice via the tail vein. After three weeks, the mice were sacrificed and the tumor foci in the lungs were imaged. The red arrow depicts tumor nodules. Paraffin- embedded lung tissues from Rag2/INFr DKO mice injected with indicated tumor cells were stained by H&E. Photographs illustrate the lung tissue and tumor nodules therein. In FIG.
- the histogram shows the number of visible tumor nodules in the lung from Rag2/rc DKO mice injected with the indicated cell lines.
- B16-F10 cells with stable expression of WT Regnase-1, the Reg " mutant (D225A), or a control vector were seeded into array chips and monitored as described in the brief description of FIG. 7A-FIG. 7E.
- the histogram shows the percentage of observed clustering events in all monitored channels at 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields.
- FIG. 9G shows B 16 melanoma cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector injected into B6 mice via the tail vein. After two weeks, the mice were sacrificed and the tumor foci in the lungs were determined and imaged. Photographs illustrating both lungs from mice are shown; black spots are the melanoma tumor foci. In FIG. 9H, the histogram shows the number of visible tumor nodules in the lung from B6 mice injected with indicated cells.
- FIG. 91 shows paraffin embedded lung tissues from host B6 mice injected with indicated melanoma cells were stained by H&E. Tissue histology showed the lung tissue and tumor nodules in the lungs.
- FIG. 91 shows paraffin embedded lung tissues from host B6 mice injected with indicated melanoma cells were stained by H&E. Tissue histology showed the lung tissue and tumor nodules in the lungs.
- 9J describes survival of host mice injected with B 16 melanoma cells with forced expression of WT Regnase-1, the D225A mutant Regnase, or a control vector.
- the primary endpoint was defined as severe paralysis or moribund. There were 10 mice in each group, and none of the mice transformed with Regnase-1 -expressing melanoma cells died during the four-week study period;
- FIG. 10A, FIG. 10B, FIG. IOC, FIG. 10D, FIG. 10E, FIG. 10F, FIG. 10G, FIG. lOH-1, FIG. 10H-2, and FIG. 10H-3 show S100A6 is a downstream target gene of Regnase-1.
- FIG. 10A shows real-time RT-PCR analysis of S100A6 mRNA level in B 16-Vector, B 16-Regnase-1 WT and B 16-Regnase-1 D225A cells.
- FIG. 10B shows real-time RT-PCR analysis of S100A6 mRNA levels in MCF7, MDA-MB-231, SUM-159 and MDA-MB-468 cells.
- FIG. 10A shows real-time RT-PCR analysis of S100A6 mRNA levels in MCF7, MDA-MB-231, SUM-159 and MDA-MB-468 cells.
- IOC is a Western blot analysis demonstrating the levels of S100A4 and S100A6 protein in 293T, MCF7, MDA-MB-231, SUM-159 and MDA-MB-468 cells.
- FIG. 10D shows the real-time RT-PCR analysis of S100A6 mRNA levels in MDA-MB-231 cells transfected either with the vector alone, wild-type Regnase-1, or the Reg " mutant, D225A.
- FIG. 10E shows the real-time RT-PCR analysis of S100A6 mRNA levels in SUM-159 cells transfected either with control vector alone, wild- type Regnase-1, or the Reg " mutant, D225A.
- FIG. 10D shows the real-time RT-PCR analysis of S100A6 mRNA levels in MDA-MB-231 cells transfected either with the vector alone, wild-type Regnase-1, or the Reg " mutant, D225A.
- FIG. 10E shows the real-time RT-PCR analysis of S100
- FIG. 10F shows the conserved sequence region from the human and murine S100A6 3'UTR region, 5 '-AATCCAGTGGTGGGTA-3 ' (SEQ ID NO: 12).
- FIG. 10G shows a typical "stem-loop" structure of the resultant mRNA 5'-AAUCCAGUGGUGGGUA-3' (SEQ ID NO: 13) was identified using the online software, RNAstructure.
- FIG. lOH-1, FIG. 10H-2, and FIG. 10H-3 illustrate the overexpression of Regnase-1 wild-type (but not D225A mutant) protein inhibited the expression of GFP encoded by a GFP-CDS fused to the S100A6 3'UTR sequence;
- FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. HE, FIG. 1 IF, FIG. 11G, FIG. 11H, FIG. HI, and FIG. llj show S100A6 partially rescued the inhibitory function of Regnase-1 on cell clustering and cancer metastasis.
- FIG. HA shows the in vitro cell clustering analysis of SUM-159-Regnase-l WT-Vector, SUM-159-Regnase-l WT-S100A4, SUM-159- Regnase-1 WT-S100A6 cells.
- the histogram shows the ability of cell clustering at 2 hrs.
- FIG. llC shows SUM-159-Regnase-l WT cells with stable expression of S100A4, S100A6, or control vector that were injected into Rag2 _/ ⁇ rC _/ ⁇ mice via the tail vein. After four weeks, the mice were sacrificed, and the tumor foci at lung were determined and imaged. Photographs of the lungs are shown, and the metastatic foci are indicated by arrows.
- FIG. 11D is a histogram showing the number of visible tumor nodules in the lung from Nu(NCr)-Foxnl mice injected with indicated cells.
- HE shows the in vitro cell clustering analysis of B16-Regnase-1 WT- Vector, B16-Regnase-1 WT-S100A4, B16-Regnase-1 WT-S 100A6 cells.
- FIG. 1 IF the histogram shows ability of cell clustering within 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields.
- FIG. 11G B16-Regnase-1 WT melanoma cells with stable expression of S100A4, S100A6, or control vector were injected into B6 mice via the tail vein. After four weeks, the mice were sacrificed and the tumor foci at lung were determined. Photographs illustrate both lungs from the mice, and the black spots represent melanoma tumor foci.
- FIG. 11H the histogram shows the number of visible tumor nodules in the lung from B6 mice that were injected with the indicated cells.
- FIG. HI shows paraffin-embedded lung tissues from host B6 mice that were injected with the indicated melanoma cells and stained by H&E. Tissue histology showed the lung tissue and the tumor nodules present in the lungs.
- FIG. HJ shows the survival of host mice injected with B 16-Regnase-1-WT melanoma cells with forced expression of S100A4, S100A6, or the control vector. The primary endpoint was defined as severe paralysis or moribund. There were 5 mice in each group and mice with B16-Regnase-1 WT-expressing melanoma cells died after about six weeks. The mice with B16-Regnase-1 WT- S100A6 died after about 30 days;
- FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D show a microfluidic chip-based cell clustering (MFCBCC) assay to monitor cell clustering.
- FIG. 12A is a detailed procedure for preparation of the microfluidic chip.
- FIG. 12B B16-F10 cells were seeded into microfluidic chips at a concentration of 3 to 4 cells per channel. The process of cell clustering was monitored by a camera during the 2-hr experimental period. The red circles indicate individual cells at the beginning of the experiment, or individual cells and cell clusters at the end of the experiment.
- FIG. 12C the histogram shows the percentage of observed clustering events in total monitored wells within 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields.
- FIG. 12D shows five channels containing 3 - 5 cells for each cell type were monitored and their dynamic clustering rate presented as a percentage of observed clustering events in total events;
- FIG. 13A, FIG. 13B, and FIG. 13C show the forced expression of Regnase-1 in cancer cells does not affect their proliferation potential or their morphology.
- MCF7, SUM- 159 and B16 cell lines that with transformed with wild type Regnase-1, endoribonuclease- inactive Regnase-1 ((D225A mutant), or a control vector were each seeded into 6- well plates and cultured for 48 hrs. Morphology was examined by light microscopy, and the resulting micrographs showed clusters of proliferating cells.
- FIG. 13B MCF7, SUM-159 and B16 cells were seeded in six-well plates at ⁇ 10 2 /well.
- FIG. 13C illustrates cancer cell lines that were labeled with CSFE (a dye that tracks cell proliferation) at Day 0.
- the level of CSFE in labeled cells was determined by flow cytometry at Day 1 and Day 5. Dilution of CFSE indicated cell proliferation;
- FIG. 14A, FIG. 14B, and FIG. 14C show the expression of Regnase-1 inhibits cell migration and invasiveness.
- FIG. 14A show MCF7, SUM-159, B16, MDA-MB-231 cells containing the WT Regnase-1, the D225A mutant Regnase-1 or a control vector that were seeded in six-well plates containing 0.3% agar, and grown for 2 weeks, then stained with MTT for 4 hrs. Photomicrographs were recorded and colonies were counted.
- FIG. 14A show MCF7, SUM-159, B16, MDA-MB-231 cells containing the WT Regnase-1, the D225A mutant Regnase-1 or a control vector that were seeded in six-well plates containing 0.3% agar, and grown for 2 weeks, then stained with MTT for 4 hrs. Photomicrographs were recorded and colonies were counted.
- FIG. 14A show MCF7, SUM-159, B16, MDA-MB
- FIG. 14B shows SUM-159 cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were seeded into 6-well plates.
- FIG. 15A and FIG. 15B show the ectopic expression of Regnase-1 inhibits cancer cell clustering.
- FIG. 15A MDA-MB-231 cells with stable expression of WT Regnase-1, the D225A mutant Regnase-1 or control vector were seeded into array chips at the concentration of 3 to 4 cells per channel. The process of cells clustering was monitored by a camera over a 2-hr period. The red circle indicates each cell at the beginning of the experiment, or each cell and cell cluster at the end of the experiment.
- FIG. 15B Histogram showing the ability of cell clustering within 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields;
- FIG. 16A and FIG. 16B show the forced expression of Regnase-1, but not the D225A mutation completely inhibits melanoma metastasis Ragl KO mice in vivo.
- FIG. 16A B 16 melanoma cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were injected into Ragl KO mice via the tail vein. After two weeks, the mice were sacrificed and the tumor foci at lung were determined and imaged. Photographs illustrate both lungs from the mice; black spots are melanoma tumor foci.
- FIG. 16B the histogram shows the number of visible tumor nodules in the lung from Rag2/rc DKO mice injected with indicated cells;
- FIG. 17A and FIG. 17B show Regnase-1 slightly inhibits tumor growth but this inhibitory effect is independent its ribonuclease activity.
- FIG. 17A Representative image of B16 tumors. B16 melanoma cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were injected into each flank and the interscapular region of each B6 mouse. After two weeks, the mice were sacrificed and the subcutaneous tumor were determined and shown.
- FIG. 17B the histogram shows the weight of B 16 tumors from B6 mice subcutaneously injected with the indicated cells;
- FIG. 18A, FIG. 18B-1, FIG. 18B-2, FIG. 18B-3, FIG. 18C-1 and FIG. 18C-2 show the expression of S100A4 and S100A6 after overexpression of Regnase-1.
- FIG. 18A illustrates a real-time RT-PCR analysis of S100A4 mRNA level in MCF7, MDA-MB-231, SUM- 159 and MDA-MB-468 cells.
- FIG. 18B-1, FIG. 18B-2, and FIG. 18B-3 show real-time RT-PCR analysis of S100A4 mRNA level in B 16-F10, MDA-MB-231 and SUM- 159 cells transfected with vector, Regnase-1 WT or D225A mutant.
- FIG. 18C-1 and FIG. 18C-2 real-time RT-PCR analysis of S100A4 and S100A6 mRNA level in 293T cells transiently transfected with vector, Regnase-1 WT or D225A mutant is shown; and
- FIG. 19A-1, FIG. 19A-2, FIG. 19A-3, FIG. 19B-1, FIG. 19B-2, and FIG. 19B-3 show the overexpression of Regnase-1 has minimal effect on GFP CDS or GFP-S100A4-3'UTR.
- FIG. 19A-1, FIG. 19A-2, and FIG. 19A-3 show the overexpression of wild-type Regnase-1 had minimal effect on GFP expression encoding by the native GFP CDS.
- FIG. 19B-1, FIG. 19B- 2, and FIG. 19B-3 overexpression of WT Regnase-1 had minimal effect on GFP expression encoded by a construct in which GFP-CDS was fused with the S100A4 3'UTR.
- SEQ ID NO:l is an exemplary forward oligonucleotide primer that is specific for ZC3H12A, and used in accordance with one or more aspects of the present disclosure.
- SEQ ID NO:2 is an exemplary reverse oligonucleotide primer that is specific for
- SEQ ID NO:3 is an exemplary human Regnase-1 polypeptide (ZC3H12A,
- NP_079355.2 for use in accordance with one or more aspects of the present disclosure.
- SEQ ID NO:4 is an exemplary human ZC3H12B ribonuclease polypeptide
- SEQ ID NO:5 is an exemplary human ZC3H12C ribonuclease polypeptide
- SEQ ID NO:6 is an exemplary human ZC3H12D ribonuclease polypeptide (NP_997243.2) for use in accordance with one or more aspects of the present disclosure.
- SEQ ID NO:7 is an exemplary human KHNYN isoform 1 polypeptide (NP_056114.1) for use in accordance with one or more aspects of the present disclosure.
- SEQ ID NO:8 is an exemplary human NYNRIN protein (NP_079357.2) for use in accordance with one or more aspects of the present disclosure.
- SEQ ID NO:9 is an exemplary human ELOVL5 Elongation of VLC FA protein 5, isoform 2, polypeptide (NP_001229757.1) for use in accordance with one or more aspects of the present disclosure.
- SEQ ID NO: 10 is an exemplary human KCNK12 potassium channel subfamily K member 12 polypeptide (NP_071338.1) for use in accordance with one or more aspects of the present disclosure.
- SEQ ID NO:ll is an exemplary human N4BP1 NEDD4-binding protein 1 polypeptide (N4BP1 NP_694574.3) for use in accordance with one or more aspects of the present disclosure.
- SEQ ID NO: 12 is a nucleotide consensus sequence between human and murine S100A6 3'UTRs, as described further herein.
- SEQ ID NO: 13 is the resultant mRNA sequence transcribed from the mammalian consensus sequence shown in SEQ ID NO: 12, and as described further herein.
- Cancer is arguably one of the biggest global threats to public health. Cancer metastasis is a key feature of malignancy and contributes to more than 90% solid tumor-related deaths. Because of poorly understood mechanisms of cancer metastasis, there is no diagnosis/prognosis signature and specific treatment available to efficiently control tumor metastasis. In fact, metastasis is a complex process involving well-coordinated sequence of events where some tumor cells leave the primary lesion and take residence at distal sites. As not all cells in primary tumors have the capacity to metastasize, it is reasoned that metastatic cancer cells may possess special features that can provide valuable therapeutic targets.
- Breast cancer is the second leading cause of death among women in the United States. Approximately one woman in every ten will develop breast cancer in her lifetime. Recent statistics estimate that 44,000 women will die of breast cancer, while 150,000 new female cases of breast cancer will be diagnosed in the next year.
- Tumor metastasis is a key feature of malignancy and a leading cause of tumor-related death, but how tumor cell metastasis is regulated remains poorly understood.
- Regnase-1 a cytosolic ribonuclease which is post-translationally inactivated in metastatic cancer cells, has been shown to be a potent repressor of tumor metastasis.
- the metastatic activities of tumor cells were inversely related to Regnase-1 expression and that forced expression of Regnase-1 in highly metastatic tumor cells converted metastatic tumor cells to non-metastatic tumor cells.
- Regnase-1 regulates microcluster formation of tumor cells, a process that precedes metastasis, and that formation of microclusters requires the calcium binding protein S100A6. Importantly, Regnase-1 inhibited S100A6 expression through its ribonuclease domain, thus preventing tumor cells from forming microclusters. Results shown in the examples herein demonstrate that Regnase-1 is a master regulator in cell clustering and tumor metastasis, and these results indicate an important clinical role for Regnase compositions in cancer diagnosis, metastasis prediction, and cancer therapies.
- Regnase-1 acts as a key checkpoint in tumor metastasis. Namely, the loss of Regnase-1 is associated with high metastatic potential of tumor cells, and over expression of Regnase-1 in two aggressive tumor lines (melanoma and breast cancer) completely prevented tumor cells from spreading to other sites— including the lungs). Regnase-1 did not seem to affect tumor cell survival or proliferation, but significantly controlled the invasiveness of these cells.
- the present invention concerns chemotherapeutic compositions prepared in pharmaceutically-acceptable formulations for delivery to one or more cells or tissues of an animal, either alone, or in combination with one or more other modalities of diagnosis, prophylaxis and/or therapy.
- chemotherapeutic compositions prepared in pharmaceutically-acceptable formulations for delivery to one or more cells or tissues of an animal, either alone, or in combination with one or more other modalities of diagnosis, prophylaxis and/or therapy.
- the formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those of ordinary skill in the art, as is the development of suitable dosing and treatment regimens for using the particular Regnase-specific compositions described herein in a variety of diagnostic and cancer-prognostic regimens.
- compositions in suitably-formulated pharmaceutical vehicles by one or more standard delivery devices, including, without limitation, subcutaneously, parenterally, intravenously, intramuscularly, intrathecally, intratumorally, intraperitoneally, transdermally, topically, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs within or about the body of an animal.
- standard delivery devices including, without limitation, subcutaneously, parenterally, intravenously, intramuscularly, intrathecally, intratumorally, intraperitoneally, transdermally, topically, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs within or about the body of an animal.
- the methods of administration may also include those modalities as described in U.S. Patent Nos. 5,543,158; 5,641,515, and 5,399,363, each of which is specifically incorporated herein in its entirety by express reference thereto.
- Solutions of the active compounds as freebase or pharmacologically acceptable salts may be prepared in sterile water, and may be suitably mixed with one or more surfactants, such as hydroxypropylcellulose.
- Dispersions may also be prepared in glycerol, liquid polyethylene glycols, oils, or mixtures thereof. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
- aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, transdermal, subdermal, and/or intraperitoneal administration.
- the compositions of the present invention may be formulated in one or more pharmaceutically acceptable vehicles, including for example sterile aqueous media, buffers, diluents, etc.
- a given dosage of active ingredient(s) may be dissolved in a particular volume of an isotonic solution (e.g., an isotonic NaCl-based solution), and then injected at the proposed site of administration, or further diluted in a vehicle suitable for intravenous infusion (see, e.g., "REMINGTON'S PHARMACEUTICAL SCIENCES" 15th Edition, pp. 1035-1038 and 1570-1580). While some variation in dosage will necessarily occur depending on the condition of the subject being treated, the extent of the treatment, and the site of administration, the person responsible for administration will nevertheless be able to determine the correct dosing regimens appropriate for the individual subject using ordinary knowledge in the medical and pharmaceutical arts.
- an isotonic solution e.g., an isotonic NaCl-based solution
- Sterile injectable compositions may be prepared by incorporating the disclosed compositions in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions can be prepared by incorporating the selected sterilized active ingredient(s) into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the compositions disclosed herein may also be formulated in a neutral or salt form.
- Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein), and which are formed with inorganic acids such as, without limitation, hydrochloric or phosphoric acids, or organic acids such as, without limitation, acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, without limitation, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation, and in such amount as is effective for the intended application.
- inorganic acids such as, without limitation, hydrochloric or phosphoric acids
- organic acids such as, without limitation, acetic, oxalic, tartaric, mandelic, and the like.
- Salts formed with the free carboxyl groups can also be derived from inorganic bases
- Formulations of compounds of the present invention may be readily administered in a variety of dosage forms such as injectable solutions, topical preparations, oral formulations, including sustain-release capsules, hydrogels, colloids, viscous gels, transdermal reagents, intranasal and inhalation formulations, and the like.
- chemotherapeutics disclosed herein will be within the purview of the ordinary-skilled artisan having benefit of the present teaching. It is likely, however, that the administration of a diagnostically-effective (i.e. , a pharmaceutically-effective) amount of one or more of the disclosed compositions may be achieved by a single administration, such as, without limitation, a single injection of a sufficient quantity of the delivered agent to provide the desired benefit to the patient in need thereof.
- compositions may be desirable to provide multiple, or successive administrations of the disclosed compositions, either over a relatively short, or even a relatively prolonged period, as may be determined by the medical practitioner overseeing the administration of such compositions to the selected individual undergoing such procedure(s).
- formulations of one or more of the regnase- specific compositions described herein will contain at least an effective amount of a first active agent.
- the formulation may contain at least about 0.001% of each active ingredient, preferably at least about 0.01% of the active ingredient, although the percentage of the active ingredient(s) may, of course, be varied, and may conveniently be present in amounts from about 0.01 to about 90 weight % or volume %, or from about 0.1 to about 80 weight % or volume %, or more preferably, from about 0.2 to about 60 weight % or volume %, based upon the total formulation.
- the amount of active compound(s) in each composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound.
- Factors such as solubility, bioavailability, biological ti/2, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one of ordinary skill in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
- compositions disclosed herein be suitable for direct injection into one or more organs, tissues, or cell types in the body.
- compositions of the present invention may further comprise one or more excipients, buffers, or diluents that are particularly formulated for contact with mammalian cells, and in particular human cells, and/or for administration to a mammalian subject, such as a human patient.
- Compositions may further optionally comprise one or more diagnostic or prognostic agents, and/or may be formulated within a population of microspheres, microparticles, nanospheres, or nanoparticles, and may be formulated for administration to one or more cells, tissues, organs, or body of a human in particular.
- Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing, diagnostic, and/or treatment regimens for using the particular compositions described herein in a variety of modalities, including e.g., without limitation, oral, parenteral, intravenous, intranasal, intratumoral, and intramuscular routes of administration.
- compositions employed, and the particular time of administration, or dosage regimen for compositions employing the disclosed regnase-specific formulations will be within the purview of a person of ordinary skill in the art having benefit of the present teaching. It is likely, however, that the administration of the disclosed formulations may be achieved by administration of one or more doses of the formulation, during a time effective to provide the desired benefit to the patient undergoing such treatment. Such dosing regimens may be determined by the medical practitioner overseeing the administration of the compounds, depending upon the particular condition or the patient, the extent or duration of the therapy being administered, etc.
- compositions of the present invention are not in any way limited to use only in humans, or even to primates, or mammals.
- the methods and compositions disclosed herein may be employed using avian, amphibian, reptilian, or other animal species.
- the compositions of the present invention are preferably formulated for administration to a mammal, and in particular, to humans, in a variety of regimens for modulating the metastatic potential of cancer cells.
- compositions are not limited only to use in humans, but may also be formulated for veterinary administration, including, without limitation, to selected livestock, exotic or domesticated animals, companion animals (including pets and such like), non- human primates, as well as zoological or otherwise captive specimens, and such like.
- compositions for preventing, diagnosing, treating and/or ameliorating one or more symptoms of one or more diseases, dysfunctions, abnormal conditions, or disorders in an animal, including, for example, vertebrate mammals.
- Use of the disclosed compositions is particularly contemplated in the diagnosis and/or prognosis of cancer, in the detection or prediction of cancer metastasis or in monitoring the extent thereof, and/or for suppression of the metastatic potential of one or more cancer cell types.
- Such use generally involves administration to the mammal in need thereof one or more of the disclosed regnase-specific compositions, in an amount and for a time sufficient to diagnose, treat, lessen, or ameliorate one or more symptoms of cancer metastasis in an affected mammal.
- compositions including one or more of the disclosed regnase compositions also form part of the present invention, and particularly those compositions that further include at least a first pharmaceutically-acceptable excipient for use in the diagnosis, prophylaxis, therapy and/or amelioration of one or more symptoms of cancer in an affected mammal.
- GENE THERAPY VECTORS GENE THERAPY VECTORS
- Gene therapy has been exploited for the treatment of a wide variety of human diseases including those of the central nervous system such as Alzheimer's, Parkinson's, Batten's and Huntington's Diseases, ⁇ -thalassemia, oci-antitrypsin (AAT) deficiency, and Fragile-X Mental Retardation Syndrome. Many chronic and progressive diseases require sustained or regulatable administration of the therapeutic gene to achieve successful treatment.
- the inventors contemplate the use of such Regnase-expressing systems in the transfection of mammalian cancer cell for the persistent expression of Regnase protein in vivo.
- Exemplary gene therapy vector systems are known to those of ordinary skill in the art, and include, but are not limited to, Ad, AAV, HSV, lenti virus, and the like.
- Adeno-associated virus is a single- stranded DNA-containing, non-pathogenic human parvovirus that is being widely investigated as a therapeutic vector for a host of muscle disorders
- Recombinant adeno-associated virus (rAAV) vectors have been developed in which the rep and cap open reading frames of the wild-type virus have been completely replaced by a therapeutic or reporter gene, retaining only the characteristic inverted terminal repeats (ITRs), the sole ds-acting elements required for virus packaging.
- ITRs characteristic inverted terminal repeats
- helper plasmids expressing various combinations of the AAV2 rep and AAV-1, -2, and -5 cap genes, respectively, efficient cross packaging of AAV2 genomes into particles containing the AAV-1, -2, or -5 capsid protein has been demonstrated.
- the various serotype vectors have demonstrated distinct tropisms for different tissue types in vivo, due in part to their putative cell surface receptors.
- expression vector or construct means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In preferred embodiments, expression only includes transcription of the nucleic acid, for example, to generate a therapeutic agent from a transcribed gene that is comprised within a suitable gene expression cassette.
- vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter.
- a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
- phrases “operatively linked,” “operably linked,” “operatively positioned,” “under the control of or “under the transcriptional control of means that the promoter is in the correct location and orientation in relation to the nucleic acid segment that comprises the therapeutic gene to properly facilitate, control, or regulate RNA polymerase initiation and expression of the therapeutic gene to produce the therapeutic peptide, polypeptide, ribozyme, or antisense RNA molecule in the cells that comprise and express the genetic construct.
- a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with the particular therapeutic gene of interest in its natural environment.
- promoters may include promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell.
- promoter that effectively directs the expression of the Regnase-encoding nucleic acid segment in the cell type, organism, or even animal, chosen for expression.
- the use of promoter and cell type combinations for protein expression is generally known to those of ordinary skill in the molecular biology arts.
- the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high-level expression of the introduced Regnase- specific DNA segment.
- At least one module in a promoter functions to position the start site for RNA synthesis.
- TATA box in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
- Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
- the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
- the particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell.
- a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
- a promoter might include either a human or viral promoter, such as a ⁇ -actin, AAV, AV, CMV or HSV promoter.
- inducible promoters such as tetracycline-controlled promoters, are also contemplated to be useful in certain cell types.
- the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of transgenes.
- CMV cytomegalovirus
- the use of other viral or mammalian cellular or bacterial phage promoters that are well known in the art to achieve expression of a transgene is contemplated as well, provided that the levels of expression are sufficient for a given purpose. .
- Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
- enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
- engineered and recombinant cells are intended to refer to a cell into which an exogenous nucleic acid segment, such as DNA segments that lead to the transcription of a therapeutic agent, such as a therapeutic peptide, polypeptide, ribozyme, antisense, or catalytic mRNA molecule has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells, which do not contain a recombinantly introduced exogenous polynucleotide segment. Engineered cells are thus cells having nucleic acid segment introduced through the hand of man.
- a suitable gene expression vector that comprises at least a first sequence region that encodes a Regnase peptide, polypeptide, ribozyme, or antisense mRNA under the control of one or more promoters.
- a sequence "under the control of a promoter one positions the 5' end of the transcription initiation site of the transcriptional reading frame generally between about 1 and about 50 nucleotides "downstream" of (i.e. , 3' of) the chosen promoter.
- the "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded polypeptide. This is the meaning of "recombinant expression" in this context.
- Modification and changes may be made in the structure of the gene expression cassettes, or to the viral vectors comprising them, as well as modification to the the therapeutic agents encoded by them and still obtain functional vectors, viral particles, and virion that encode one or more therapeutic agents with desirable characteristics.
- the resulting encoded polypeptide sequence is altered by this mutation, or in other cases, the sequence of the polypeptide is unchanged by one or more mutations in the encoding polynucleotide.
- the amino acid changes may be achieved by changing one or more of the codons of the encoding DNA sequence, according to Table 1.
- certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
- the hydropathic index of amino acids may be considered.
- the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
- Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
- an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
- substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
- amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
- Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of ordinary skill in the art, and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
- the gene expression constructs of the invention, and the viral vectors comprising them will find utility in the delivery of one or more antisense oligonucleotides or polynucleotides for inhibiting the expression of a selected mammalian mRNA in a host cell that has been transformed with the construct.
- the letters, A, G, C, T, and U respectively indicate nucleotides in which the nucleoside is Adenosine (Ade), Guanosine (Gua), Cytidine (Cyt), Thymidine (Thy), and Uridine (Ura).
- compounds that are "antisense” to a particular PNA, DNA, or mRNA "sense" strand are nucleotide compounds that have a nucleoside sequence that is complementary to the sense strand. It will be understood by those skilled in the art that the present invention broadly includes oligonucleotide compounds that are capable of binding to the selected DNA or mRNA sense strand.
- mRNA includes not only the ribonucleotide sequences encoding a protein, but also regions including the 5 '-untranslated region, the 3 '-untranslated region, the 5 '-cap region and the intron/exon junction regions.
- the invention includes compounds that are not strictly antisense; the compounds of the invention also include those oligonucleotides that may have some bases that are not complementary to bases in the sense strand provided such compounds have sufficient binding affinity for the particular DNA or mRNA for which an inhibition of expression is desired.
- base modifications or the use of universal bases such as inosine in the oligonucleotides of the invention are contemplated within the scope of the subject invention.
- C1-4 alkyl means a branched or unbranched hydrocarbon having 1 to 4 carbon-atoms.
- the disclosed antisense compounds also may be substituted at the 3'-and/or 5'-ends by a substituted-acridine derivative.
- substituted-acridine means any acridine derivative capable of intercalating nucleotide strands such as DNA.
- Preferred substituted acridines are 2-methoxy-6-chloro-9-pentylaminoacridine, N-(6-chloro-2-methoxy acridinyl)-0-methoxydiisopropylamino-phosphinyl-3-aminopropanol, and N-(6-chloro- 2-methoxyacridinyl)-0-methoxydiisopropylamino-phosphinyl-5-aminopentanol.
- Other suitable acridine derivatives are readily apparent to persons skilled in the art.
- P(0)(0)-substituted acridine means a phosphate covalently linked to a substitute acridine.
- nucleotides includes nucleotides in which the phosphate moiety is replaced by phosphorothioate or alkylphosphonate and the nucleotides may be substituted by substituted acridines.
- the antisense compounds of the invention differ from native DNA by the modification of the phosphodiester backbone to extend the life of the antisense molecule.
- the phosphates can be replaced by phosphorothioates.
- the ends of the molecule may also be optimally substituted by an acridine derivative that intercalates nucleotide strands of DNA.
- the reaction scheme involves ⁇ -tetrazole-catalyzed coupling of phosphoramidites to give phosphate intermediates that are subsequently reacted with sulfur in 2,6-lutidine to generate phosphate compounds.
- Oligonucleotide compounds are prepared by treating the phosphate compounds with thiophenoxide (1:2:2 thiophenol/triethylamine/tetrahydrofuran, room temperature, 1 hr). The reaction sequence is repeated until an oligonucleotide compound of the desired length has been prepared. The compounds are cleaved from the support by treating with ammonium hydroxide at room temperature for 1 hr and then are further deprotected by heating at about 50°C overnight to yield preferred antisense compounds.
- T m secondary structure
- binding energy relative stability
- antisense compositions were selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell.
- Highly preferred target regions of the mRNA are those that are at or near the AUG translation initiation codon, and those sequences that were substantially complementary to 5'-regions of the mRNA.
- polynucleotides, nucleic acid segments, nucleic acid sequences, and the like include, but are not limited to, DNAs (including and not limited to genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.
- DNAs including and not limited to genomic or extragenomic DNAs
- genes include peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.
- PNAs peptide nucleic acids
- an "antigenic polypeptide” or an “immunogenic polypeptide” is a polypeptide which, when introduced into a vertebrate, reacts with the vertebrate's immune system molecules, i.e., is antigenic, and/or induces an immune response in the vertebrate, i.e., is immunogenic.
- biologically-functional equivalent is well understood in the art, and is further defined in detail herein. Accordingly, sequences that have about 85% to about 90%; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99%; of nucleotides that are identical or functionally-equivalent to one or more of the nucleotide sequences provided herein are particularly contemplated to be useful in the practice of the methods and compositions set forth in the instant application.
- buffer includes one or more compositions, or aqueous solutions thereof, that resist fluctuation in the pH when an acid or an alkali is added to the solution or composition that includes the buffer. This resistance to pH change is due to the buffering properties of such solutions, and may be a function of one or more specific compounds included in the composition. Thus, solutions or other compositions exhibiting buffering activity are referred to as buffers or buffer solutions. Buffers generally do not have an unlimited ability to maintain the pH of a solution or composition; rather, they are typically able to maintain the pH within certain ranges, for example from a pH of about 5 to 7.
- carrier is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert (s), or such like, or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal or acceptable for a therapeutic or diagnostic purpose, as applicable.
- DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments that have been isolated away from, or purified free from, total genomic DNA of the particular species from which they are obtained. Included within the term “DNA segment,” are DNA segments and smaller fragments of such segments, as well as recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
- the term "effective amount,” as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.
- heterologous is defined in relation to a predetermined referenced nucleic acid sequence.
- a heterologous promoter is defined as a promoter which does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation.
- a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements.
- homologous means, when referring to polynucleotides, sequences that have the same essential nucleotide sequence, despite arising from different origins. Typically, homologous nucleic acid sequences are derived from closely related genes or organisms possessing one or more substantially similar genomic sequences. By contrast, an "analogous" polynucleotide is one that shares the same function with a polynucleotide from a different species or organism, but may have a significantly different primary nucleotide sequence that encodes one or more proteins or polypeptides that accomplish similar functions or possess similar biological activity. Analogous polynucleotides may often be derived from two or more organisms that are not closely related (e.g., either genetically or phylogenetically).
- the term “homology” refers to a degree of complementarity between two or more polynucleotide or polypeptide sequences.
- the word “identity” may substitute for the word “homology” when a first nucleic acid or amino acid sequence has the exact same primary sequence as a second nucleic acid or amino acid sequence.
- Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods may be used to assess whether a given sequence is identical or homologous to another selected sequence.
- nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of ordinary skill) or by visual inspection.
- the phrase "in need of treatment” refers to a judgment made by a caregiver such as a physician or veterinarian that a patient requires (or will benefit in one or more ways) from treatment. Such judgment may made based on a variety of factors that are in the realm of a caregiver's expertise, and may include the knowledge that the patient is ill as the result of a disease state that is treatable by one or more compound or pharmaceutical compositions such as those set forth herein.
- isolated or “biologically pure” refer to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state.
- isolated polynucleotides or polypeptides in accordance with the present disclosure preferably do not contain materials normally associated with those polynucleotides or polypeptides in their natural, or in situ, environment.
- kit may be used to describe variations of the portable, self- contained enclosure that includes at least one set of reagents, components, or pharmaceutically- formulated compositions of the present invention.
- kit may include one or more sets of instructions for use of the enclosed compositions, such as, for example, in a laboratory or clinical application.
- Link refers to any method known in the art for functionally connecting one or more proteins, peptides, nucleic acids, or polynucleotides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, electrostatic bonding, and the like.
- naturally-occurring refers to the fact that an object can be found in nature.
- a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of man in a laboratory is naturally- occurring.
- laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally-occurring animals.
- nucleic acid includes one or more types of: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D- ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including abasic sites).
- nucleic acid also includes polymers of ribonucleosides or deoxyribonucleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like.
- Nucleic acids include single- and double- stranded DNA, as well as single- and double- stranded RNA.
- nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small temporal RNA (stRNA), and the like, and any combination thereof.
- operably linked refers to that the nucleic acid sequences being linked are typically contiguous, or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.
- patient also interchangeably referred to as “recipient” "host” or “subject” refers to any host that can serve as a recipient for one or more of the vascular access devices as discussed herein.
- the recipient will be a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being).
- a "patient” refers to any animal host, including but not limited to, human and non-human primates, avians, reptiles, amphibians, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, racines, vulpines, and the like, including, without limitation, domesticated livestock, herding or migratory animals or birds, exotics or zoological specimens, as well as companion animals, pets, and any animal under the care of a veterinary practitioner.
- compositions that do not produce an allergic or similar untoward reaction when administered to a human, and in particular, when administered to the human eye.
- aqueous composition that contains a protein as an active ingredient is well understood in the art.
- injectables either as liquid solutions or as suspensions.
- solid form suitable for solution or suspension in liquid prior to injection may be prepared.
- salts refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects.
- examples of such salts include, but are not limited to, acid-addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic (embonic) acid, alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; salts with polyvalent metal cations such as zinc,
- plasmid refers to a genetic construct that is composed of genetic material (i.e., nucleic acids).
- a plasmid or a vector contains an origin of replication that is functional in bacterial host cells, e.g., Escherichia coli, and selectable markers for detecting bacterial host cells including the plasmid.
- Plasmids and vectors of the present invention may include one or more genetic elements as described herein arranged such that an inserted coding sequence can be transcribed and translated in a suitable expression cells.
- the plasmid or vector may include one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments that are obtained from or derived from one or more natural and/or artificial sources.
- polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and includes any chain or chains of two or more amino acids.
- terms including, but not limited to “peptide,” “dipeptide,” “tripeptide,” “protein,” “enzyme,” “amino acid chain,” and “contiguous amino acid sequence” are all encompassed within the definition of a “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with, any of these terms.
- polypeptides that have undergone one or more post-translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids.
- post-translational modification(s) including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids.
- Conventional nomenclature exists in the art for polynucleotide and polypeptide structures.
- amino acids Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; He), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys).
- Amino acid residues described herein are preferred to be in the "L” isomeric form. However, residues in the "D" isomeric form may be substituted for L-amino acid residue
- the terms "prevent,” “preventing,” “prevention,” “suppress,” “suppressing,” and “suppression” as used herein refer to administering a compound either alone or as contained in a pharmaceutical composition prior to the onset of clinical symptoms of a disease state so as to prevent any symptom, aspect or characteristic of the disease state. Such preventing and suppressing need not be absolute to be deemed medically useful.
- Protein is used herein interchangeably with “peptide” and “polypeptide,” and includes both peptides and polypeptides produced synthetically, recombinantly, or in vitro and peptides and polypeptides expressed in vivo after nucleic acid sequences are administered into a host animal or human subject.
- polypeptide is preferably intended to refer to any amino acid chain length, including those of short peptides from about two to about 20 amino acid residues in length, oligopeptides from about 10 to about 100 amino acid residues in length, and longer polypeptides including from about 100 amino acid residues or more in length.
- polypeptides and proteins of the present invention also include polypeptides and proteins that are or have been post-translationally-modified, and include any sugar or other derivative(s) or conjugate(s) added to the backbone amino acid chain.
- “Purified,” as used herein, means separated from many other compounds or entities.
- a compound or entity may be partially purified, substantially purified, or pure.
- a compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e. , is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure.
- a partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the material with which it is naturally found, e.g., cellular material such as cellular proteins and/or nucleic acids.
- the term "recombinant” indicates that the material (e.g., a polynucleotide or a polypeptide) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within or removed from, its natural environment, or native state. Specifically, e.g., a promoter sequence is "recombinant” when it is produced by the expression of a nucleic acid segment engineered by the hand of man.
- a "recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis
- a "recombinant polypeptide” or “recombinant protein” is a polypeptide or protein which is produced by expression of a recombinant nucleic acid
- a "recombinant virus,” e.g., a recombinant AAV virus is produced by the expression of a recombinant nucleic acid.
- regulatory element refers to a region or regions of a nucleic acid sequence that regulates transcription.
- exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and such like.
- RNA segment refers to an RNA molecule that has been isolated free of total cellular RNA of a particular species. Therefore, RNA segments can refer to one or more RNA segments (either of native or synthetic origin) that have been isolated away from, or purified free from, other RNAs. Included within the term “RNA segment,” are RNA segments and smaller fragments of such segments.
- sequence essentially as set forth in SEQ ID NO:X means that the sequence substantially corresponds to a portion of SEQ ID NO:X and has relatively few nucleotides (or amino acids in the case of polypeptide sequences) that are not identical to, or a biologically functional equivalent of, the nucleotides (or amino acids) of SEQ ID NO:X.
- biologically functional equivalent is well understood in the art, and is further defined in detail herein.
- sequences that have about 85% to about 90%; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99%; of nucleotides that are identical or functionally equivalent to one or more of the nucleotide sequences provided herein are particularly contemplated to be useful in the practice of the invention.
- Suitable standard hybridization conditions for nucleic acids for use in the present invention include, for example, hybridization in 50% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 ⁇ g/mL of denatured salmon sperm DNA at 42°C for 16 hr followed by 1 hr sequential washes with O.lx SSC, 0.1% SDS solution at 60°C to remove the desired amount of background signal.
- Lower stringency hybridization conditions for the present invention include, for example, hybridization in 35% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 ⁇ g/mL denatured salmon sperm DNA or E.
- structural gene is intended to generally describe a polynucleotide, such as a gene, that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.
- subject describes an organism, including mammals such as primates, to which treatment with the compositions according to the present invention can be provided.
- Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, apes; chimpanzees; orangutans; humans; monkeys; domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.
- substantially complementary when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to all or a portion of the selected sequence, and thus will specifically bind to a portion of an mRNA encoding the selected sequence.
- sequences will be highly complementary to the mRNA "target" sequence, and will have no more than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 or so base mismatches throughout the complementary portion of the sequence.
- sequences may be exact matches, i.e., be completely complementary to the sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch.
- highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product.
- Substantially complementary nucleic acid sequences will be greater than about 80 percent complementary (or "% exact-match") to a corresponding nucleic acid target sequence to which the nucleic acid specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds.
- nucleic acid sequences will be greater than about 90 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and even up to and including about 96%, about 97%, about 98%, about 99%, and even about 100% exact match complementary to all or a portion of the target sequence to which the designed nucleic acid specifically binds.
- Percent similarity or percent complementary of any of the disclosed nucleic acid sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
- the GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences.
- the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
- Nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules.
- complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to one or more of the specific nucleic acid segments disclosed herein under relatively stringent conditions such as those described immediately above.
- the term “substantially free” or “essentially free” in connection with the amount of a component preferably refers to a composition that contains less than about 10 weight percent, preferably less than about 5 weight percent, and more preferably less than about 1 weight percent of a compound. In preferred embodiments, these terms refer to less than about 0.5 weight percent, less than about 0.1 weight percent, or less than about 0.01 weight percent.
- Probes and primers for use in the present invention may be of any suitable length. By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, etc. , an algorithm defining all probes or primers contained within a given sequence can be proposed:
- n to n + y where n is an integer from 1 to the last number of the sequence and y is the length of the probe or primer minus one, where n + y does not exceed the last number of the sequence.
- the collection of probes or primers correspond to bases 1 to 25, bases 2 to 26, bases 3 to 27, bases 4 to 28, and so on over the entire length of the sequence.
- exemplary primer or probe sequence include, without limitation, sequences corresponding to bases 1 to 35, bases 2 to 36, bases 3 to 37, bases 4 to 38, and so on over the entire length of the sequence.
- probes or primers may correspond to the nucleotides from the first basepair to bp 40, from the second bp of the sequence to bp 41, from the third bp to bp 42, and so forth
- probes or primers may correspond to a nucleotide sequence extending from bp 1 to bp 50, from bp 2 to bp 51, from bp 3 to bp 52, from bp 4 to bp 53, and so forth.
- the term "therapeutically-practical period” means the period of time that is necessary for one or more active agents to be therapeutically effective.
- the term “therapeutically-effective” refers to reduction in severity and/or frequency of one or more symptoms, elimination of one or more symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and the improvement or a remediation of damage.
- a “therapeutic agent” may be any physiologically or pharmacologically active substance that may produce a desired biological effect in a targeted site in a subject.
- the therapeutic agent may be a chemotherapeutic agent, an immunosuppressive agent, a cytokine, a cytotoxic agent, a nucleolytic compound, a radioactive isotope, a receptor, and a pro-drug activating enzyme, which may be naturally occurring, produced by synthetic or recombinant methods, or a combination thereof.
- Drugs that are affected by classical multidrug resistance such as vinca alkaloids (e.g., vinblastine and vincristine), the anthracyclines (e.g., doxorubicin and daunorubicin), RNA transcription inhibitors (e.g., actinomycin-D) and microtubule stabilizing drugs (e.g., paclitaxel) may have particular utility as the therapeutic agent.
- Cytokines may be also used as the therapeutic agent. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.
- a cancer chemotherapy agent may be a preferred therapeutic agent.
- anticancer agents and other therapeutic agents those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician's Desk Reference and Hardman and Limbird (2001).
- transcription factor recognition site and a “transcription factor binding site” refer to a polynucleotide sequence(s) or sequence motif(s), which are identified as being sites for the sequence-specific interaction of one or more transcription factors, frequently taking the form of direct protein-DNA binding.
- transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, and the like, and/or can be predicted based on known consensus sequence motifs, or by other methods known to those of ordinary skill in the art.
- Transcriptional regulatory element refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences.
- a transcriptional regulatory element can, for example, comprise one or more promoters, one or more response elements, one or more negative regulatory elements, and/or one or more enhancers.
- Transcriptional unit refers to a polynucleotide sequence that comprises at least a first structural gene operably linked to at least a first ds-acting promoter sequence and optionally linked operably to one or more other cw-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequences, and at least a first distal regulatory element as may be required for the appropriate tissue-specific and developmental transcription of the structural gene sequence operably positioned under the control of the promoter and/or enhancer elements, as well as any additional cis- sequences that are necessary for efficient transcription and translation (e.g., polyadenylation site(s), mRNA stability controlling sequence(s), etc.
- transformation is intended to generally describe a process of introducing an exogenous polynucleotide sequence (e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule) into a host cell or protoplast in which the exogenous polynucleotide is incorporated into at least a first chromosome or is capable of autonomous replication within the transformed host cell.
- an exogenous polynucleotide sequence e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule
- Transfection, electroporation, and "naked" nucleic acid uptake all represent examples of techniques used to transform a host cell with one or more polynucleotides.
- transformed cell is intended to mean a host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into that cell.
- the terms “treat,” “treating,” and “treatment” refer to the administration of one or more compounds (either alone or as contained in one or more pharmaceutical compositions) after the onset of clinical symptoms of a disease state so as to reduce, or eliminate any symptom, aspect or characteristic of the disease state. Such treating need not be absolute to be deemed medically useful.
- the terms “treatment,” “treat,” “treated,” or “treating” may refer to therapy, or to the amelioration or the reduction, in the extent or severity of disease, of one or more symptom thereof, whether before or after its development afflicts a patient.
- Treating may include any administration or application of a compound or composition of the invention to a subject for purposes such as curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of a disease, disorder, or condition or one or more symptoms or manifestations of a disease, disorder, or condition.
- the compositions of the present invention may also be administered prophylactically, i.e. , before development of any symptom or manifestation of the condition, where such prophylaxis is warranted.
- the subject will be one that has been diagnosed for being "at risk” of developing such a disease or disorder, either as a result of familial history, medical record, or the completion of one or more diagnostic or prognostic tests indicative of a propensity for subsequently developing such a disease or disorder.
- the tern “vector,” as used herein, refers to a nucleic acid molecule (typically comprised of DNA) capable of replication in a host cell and/or to which another nucleic acid segment can be operatively linked so as to bring about replication of the attached segment.
- a plasmid, cosmid, or a virus is an exemplary vector.
- indicator compounds and compositions are known in the art for labeling oligonucleotide probes, including, without limitation, fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, etc., which are capable of being detected in a suitable assay.
- fluorescent labels or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally less- desirable reagents.
- colorimetric, chromogenic, or fluorogenic indicator substrates are known that can be employed to provide a method for detecting the sample that is visible to the human eye, or by analytical methods such as scintigraphy, fluorimetry, spectrophotometry, and the like, to identify specific hybridization with samples containing one or more complementary or substantially complementary nucleic acid sequences.
- multiplexing assays where two or more labeled probes are detected either simultaneously or sequentially, it may be desirable to label a first oligonucleotide probe with a first label having a first detection property or parameter (for example, an emission and/or excitation spectral maximum), which also labeled a second oligonucleotide probe with a second label having a second detection property or parameter that is different (i.e., discreet or discernible from the first label.
- first detection property or parameter for example, an emission and/or excitation spectral maximum
- a microfluidic chip approach has been utilized to examine behaviors of metastatic (MDA-MB-231, SUM-159) vs. non-metastatic (MDA-MB-468, MCF7) breast cancer cell lines (FIG. 7 A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E-1, FIG. 7E-2, FIG. 7E-3, FIG. 7E-4, and FIG. 7E- 5).
- MDA-MB-231 and SUM-159 cells belong to basal B type and have higher metastatic ability as compared to MCF7 and MDA-MB-468 cells.
- the microfluidic chips were fabricated in a way that cancer cells were precisely seeded in the microchips in desired numbers (2-5 cells per chamber) (FIG.
- FIG. 7A and FIG. 16A cells could be monitored in real time.
- FIG. 7B-FIG. 7E-5 striking differences in the clustering ability were observed between metastatic and non-metastatic cancer cells.
- the highly metastatic breast cancer cells MDA-MB-231 and SUM-159 readily clustered together (FIG. 7B), and within 2 hrs, more than 80% individual MDA-MB-231 and SUM-159 cells form cell clusters.
- less than 30% single MCF7 and MDA-MB-468 cells formed clusters (FIG. 7C).
- FIG. 7D More than 50% metastatic cells formed clusters within 1 hr (FIG. 7D).
- Two representative wells containing 4 cells for each cell type further indicated the apparent clustering difference between metastatic and non-metastatic cells (FIG. 7E-1-FIG. 7E-5).
- B 16 melanoma cells, which are highly metastatic, were also included as a control; these cells exhibited strong tendency in forming cell clusters (FIG. 12B, FIG. 12C, and FIG. 12D).
- Regnase-1 is a cytosolic ribonuclease that degrades a set of mRNAs, and is initially found in innate and adaptive immune cells. Regnase-1, however, is also expressed in other tissues and cell types with unknown function.
- Regnase-1 protein was readily detected in the non-metastatic cell lines, MCF7 and MDA-MB-468. In these studies, 293T cells were included as controls, wherein Regnase-1 was expressed at high levels (FIG. 8B).
- MCF7 cells were co-cultured with differing numbers of MDA-MB-231 or SUM-159 cells. Co-cultures with MDA-MB-231 or SUM-159 cells could not trigger the degradation of Regnase-1 in MCF7 cells (FIG. 8D). Similar results were also observed in MDA-MB-468 cells (FIG. 8D), which suggested that the post-translational inactivation of Regnase-1 was intrinsic in highly-metastatic cancer cells, but not due to cytokine(s) or the microenvironment. To further confirm that Regnase-1 was also inactivated in breast tissue, the protein level of Regnase-1 was measured in breast cancer samples.
- Regnase-1 was involved in cell clustering and cancer metastasis.
- a series of Regnase-1 mutants were constructed, including a mutant that contained an Aspartic Acid to Alanine substitution (Asp— >Ala) at amino acid residue 225 (D225A), which lacked Regnase enzymatic activity.
- Wild-type Regnase-1 or the D225A mutant was stably introduced into MCF7, MDA-MB-231, SUM-159, and B 16 cells. Re- introduction of Regnase-1 into these cells, however, did not change cell morphology, cell proliferation, or affect attachment-dependent colony formation in vitro (FIG. 13A, FIG. 13B, and FIG.
- Regnase-1 could inhibit cell clustering in an established microchip-based assay. Surprisingly, Regnase-1 completely inhibited metastatic cell clustering (FIG. 9A and FIG. 9B). Since cell clustering ability is strongly associated with metastatic potential, the function of Regnase-1 in cancer metastasis in vivo was also examined. SUM- 159 cells that expressed either wild-type Regnase-1, or the enzymatically-inactive mutant, D225A, were injected into Rag2 _/ ⁇ rC _/ ⁇ knockout mice via the tail vein, and the resulting tumor nodules in the lungs were examined.
- mice injected with B 16 cells transduced with either the vector alone or the enzymatically-inactive mutant, D225A showed extensive metastatic nodules in the lungs (-150 metastases).
- Overexpression of wild-type Regnase-1 in B16 cells however, completely prevented their in vivo metastasis in lung (FIG. 9G and FIG. 9H), which was confirmed by H&E staining (FIG. 91).
- B6 mice transferred with B 16-Vector or B16-D225A mutant cells all died within three weeks (FIG. 9J).
- B6 mice transformedwith B16 cells transduced with wild-type Regnase-1 were all alive 4 weeks later (FIG. 9J).
- Regnase-1 could recognize the "stem-loop" structure at 3'UTR to degrade targeting mRNAs.
- a conserved RNA sequence was found (FIG. 10F). This conserved RNA sequence could form a typical "stem- loop" structure (FIG. 10G), but no similar structure was found in the 3'UTR of S100A4 mRNA.
- the 3'UTR sequence was cloned into the end of GFP coding sequencing (CDS).
- mice transferred with B 16-Regl-WT-S100A6 cells The number of metastatic foci in mice transferred with B 16-Regl-WT-S100A6 cells is significantly higher than it in mice transferred with B 16-Regl-WT- vector or B 16-Regl-WT-S100A4 cells (FIG. 11G).
- B6 mice injected with B16 cells will normally die within 3 weeks (FIG. 11J).
- mice injected with B 16 cells overexpressing wild- type Regnase-1 survived for more than 7 weeks.
- Additional expression of S100A6 in B 16-Regl-WT cells rendered the cells aggressive again, and upon in vivo injection, all recipient mice died within 5 weeks (FIG. 11 J).
- Regnase-1 was purchased from GE Healthcare Dharmacon, Inc. (Lafayette, CO, USA).
- a Regnase-1 -specific antibody (MAB7875) used in Western hybridization analyses was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).
- the S100A4- and SI 00 A6- specific antibodies (ab27957 and abl81975, respectively) were purchased from Abeam, PLC (Cambridge, MA, USA).
- the anti-FLAG antibody, F1804, and the ⁇ -actin-specific antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA).
- the anti-C-Myc antibody, SC-40 was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
- Anti-mouse (7076), and anti-rabbit (7074) secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).
- the breast cancer tissue array (BC081120a) was purchased from US BioMax, Inc. (Rockville, MD, USA). MG132 was purchased from EMD Millipore (474780) (Darmstadt, GERMANY). Bafilomycin Al (tlrl-bafl) was purchased from Invivogen (San Diego, CA, USA). MI2 (4848) was purchased from Tocris Bioscience (Bristol, UNITED KINGDOM). The TAK1 inhibitor (5Z)-7-Oxozeaenol (499610) was purchased from EMD Millipore. Mepazine hydrochloride 500500 was purchased from Calbiochem (San Diego, CA, USA).
- Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA).
- SU-8 2100 photoresist was purchased from Rohm and Haas Electronic Materials, LLC (Marlborough, MA, USA).
- PDMS GE 615 RTV
- Trimethylchlorosilane was purchased from Sigma-Aldrich (St. Louis, MO, USA).
- the full-length Regnase-1 gene was amplified by PCR and ligated into pMYs-Puro vectors using the restriction enzymes Bglll and EcoRI.
- the expression vectors encoding S100A4 and S100A6 were purchased from OriGene Technologies (Rockville, MD, USA).
- the coding sequence (CDS) of green-fluorescent protein (GFP) was amplified by PCR and ligated into pMYs-Puro vector using the restriction enzymes BamHI and EcoRI.
- the 3'UTRs of the S100A6 and S100A4 genes were amplified by PCR and ligated to the end of the GFP CDS by EcoRI and Xbal digestion.
- plasmids encoding either wild- type (WT) or the D225A Reg " mutant were transfected into MCF7, MDA-MB-231, B 16-F10 and SUM- 159 cells. The transfected cells were selected by puromycin for two weeks. After puromycin selection, the Regnase-1 WT cells were further transfected with plasmids encoding S100A4 or S100A6. These transfected cells were further selected under G418 for two weeks.
- Real-Time PCR Real-time PCR.
- GPDH glyceraldehyde-3 -phosphate dehydrogenase
- Trans-Well Migration Assay The migration and invasion ability of interested cells was measured by using a 24-well Transwell system (Corning, Inc., Corning, NY, USA). Briefly, selected cells were harvested and counted using a hemocytometer. Then, cells were suspended in culture medium and 2 x 10 5 cells were added into the upper chamber of the trans- well insert. The same culture medium was added into the lower chamber. Cells were incubated at 37°C for 24 hrs. The non-migrated cells in the upper chamber were removed by wiping with a cotton swab. Migrated cells were then fixed with 4% formaldehyde in PBS, and stained with 2% crystal violet in 2% ethanol. The migrated cells, which were on the lower surface of membrane, were counted using light microscopy.
- CFSE Proliferation Assays Established stable cell lines were seeded into 12-well plates and further incubate at 37°C overnight. Cells were washed twice with pre-warmed PBS and incubated with CFSE dye at a final concentration of 5 ⁇ . After 20 min, the remaining culture medium was washed twice to remove excess CFSE dye. Labeled cells were allowed to grow for several other days. At different days, cells were harvested and analyzed by flow cytometry.
- Microstructure patterns were first designed in AutoCAD software and fabricated using standard photolithography and molding processes.
- the masters were prepared by spin coating SU-8 2100 negative photoresist onto a silicon wafer and UV crosslinking for 30 min. Subsequently, the designed pattern was developed using SU-8 developer (MicroChem Corp., Westborough, MA, USA) and cleaned with isopropyl alcohol under nitrogen gas.
- the silicon masters were baked at 150°C for 30 min and treated with the anti-adhesive agent, TMCS, via vapor reaction for 4 hr. Next, the structure on the silicon wafer was used to fabricate the PDMS layer.
- the mold and PDMS layers were then baked at 80°C for 4 hr, and the cured PDMS was cut and peeled off. After treatment with oxygen plasma, the PDMS layer was reacted immediately with PEG5000 for 30 min at 150°C. Finally, the device was clean with PBS. Suspended cells in the concentration of 1 x 10 6 /mL were dropped into a microfluidic chip. Chip channels containing 2-5 cells were monitored by camera to determine their clustering ability.
- mice For SUM-159 cells, 1 x 10 6 cells in 200 ⁇ L ⁇ cell solution were injected into the Rag2 _/ ⁇ rc _/ ⁇ double-knockout or Nu(NCr)-Foxnl mice. Typically after two weeks, the mice that received B 16-F10 cells were sacrificed and dissected. Mice that received SUM-159 B 16-Regnase-1-WT-S100A6 were sacrificed and dissected at 4 weeks. Mice that received SUM-159-Regnase-l WT-S100A6 were sacrificed and dissected at 8 weeks. Images of lungs with metastasis were recorded, and the extent of lung metastasis was observed under a dissecting microscope. The number of detectable metastases on both sides were counted, with the number of lung metastases on all the four lobes added together for the total number of lung metastases.
- Subcutaneous Tumor Formation Cell preparation was performed as the same as performed at the experimental metastasis. Subcutaneous injections of 1 x 10 6 cells were performed into each flank and the interscapular region of each mouse. Tumor formation was evaluated regularly by palpation of injection sites. When tumor reached the designated diameters, mice were sacrificed and subcutaneous tumors were dissected for imaging.
- N4BP1 NP_694574.3 NEDD4-binding protein 1 SEQ ID NO:ll:
- Cancer metastasis is a leading cause of cancer-related death due to poorly defined molecular mechanisms, and our treatment strategies are also limited.
- the immune regulator Regnase-1 which is regulated by post-translational mechanisms, is critically involved in cancer metastasis. Stable expression of Regnase-1 in cancer cells could dramatically inhibit metastasis through its ribonuclease activity, and tumors with lower levels of Regnase- 1 are much more aggressive in metastasis than those with higher levels of Regnase-1.
- the antiproliferative and anti-inflammatory activities of Regnase- 1 were shown not to be responsible for its effects in suppressing tumor metastasis.
- Regnase-1 clearly employs new mechanisms in the control of cancer metastasis and acts as a critical checkpoint in cancer treatment. Given extensive efforts in identifying genetic mechanisms in cancer metastasis, these results provide new evidence on post-translational mechanisms, especially at the level of protein stability, in the control of cancer metastasis.
- the components targeting the signaling networks that control Regnase-1 stability as well as molecular targets that are controlled by Regnase-1 are novel and ideal therapeutic targets for prevention of cancer metastasis.
- ACETO N et al, "Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis," Cell, 158(5): 1110- 1122 (Aug. 2014).
- ADAMS J. and M. KAUFFMAN, "Development of the proteasome inhibitor Velcade (Bortezomib), “ Cancer Invest, 22(2):304-311 (2004).
- ALTSCHUL, SF et al "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucl. Acids &&,25( ⁇ 7):338 -3 02 (1997).
- FERNANDEZ Y et al, "Inhibition of apoptosis in human breast cancer cells: role in tumor progression to the metastatic state," Int. J. Cancer, 101(4):317-326 (Oct. 2002).
- FIDLER, IJ "The relationship of embolic homogeneity, number, size and viability to the incidence of experimental metastasis," Eur. J. Cancer, 9(3):223-227 (Mar. 1973).
- GRIBSKOV, M, and BURGESS, RR "Sigma factors from E. coli, B. subtilis, phage SPOl, and phage T4 are homologous proteins," Nucleic Acids Res., 14(16):6745-6763 (Aug. 1986).
- HALE, WG, and MARGHAM, JP "HARPER COLLINS DICTIONARY OF BIOLOGY,” HarperPerennial, New York (1991).
- HARRIS MP et al, "Adenovirus -mediated p53 gene transfer inhibits growth of human tumor cells expressing mutant p53 protein, " Cancer Gene Ther., 3(2): 121-130 (Mar. 1996).
- IWASAKI H et al, "The IkappaB kinase complex regulates the stability of cytokine- encoding mRNA induced by TLR-IL-1R by controlling degradation of Regnase-1, " Nat. Immunol, 12(12): 1167-1175 (Oct. 2011).
- KACINSKI BM et al, "The cytokine CSF-1 (M-CSF) expressed by endometrial carcinomas in vivo and in vitro, may also be a circulating tumor marker of neoplastic disease activity in endometrial carcinoma patients," Int. J. Radiat. Oncol. Biol. Phys., 19(3):619-626 (1990).
- M-CSF cytokine CSF-1
- MATSUSHITA, K et al, "Zc3hl2a is an RNase essential for controlling immune responses by regulating mRNA decay, " Nature, 458(7242): 1185-90 (Mar. 2009).
- NEEDLEMAN, SB and WUNSCH, CD "A general method applicable to the search for similarities in the amino acid sequence of two proteins," /. Mol. Biol , 48(3):443-453 (Mar. 1970).
- VASSILEV LT et al., "In vivo activation of the p53 pathway by small-molecule antagonists of MDM2, " Science, 303(5659):844-848 (Feb. 2004).
- compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the invention have been described herein in terms of illustrative embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods, and/or the steps or the sequence of steps of the methods without departing from the spirit, scope, and concept of the invention. More specifically, it will be apparent that certain compounds, which are chemically- and/or physiologically-related, may be substituted for one or more of the compounds described herein, while still achieving the same or similar results. All such substitutions and/or modifications, as apparent to one or more of ordinary skill in the relevant arts, are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Disclosed are methods for determining the metastatic potential of mammalian tumors and other cancers. Also disclosed are methods for altering the metastatic potential of mammalian cancers, by a) directly or indirectly increasing the level of Regnase peptide or polypeptide; b) selectively increasing the activity or specificity of Regnase peptide or polypeptide; c) selectively increasing the amount or stability of Regnase-specific mRNA; or d) selectively inhibiting one or more negative effectors of Regnase within a cancer cell in an amount and for a time effective to reduce, delay, retard, or prevent subsequent metastasis of the cancer within the body of a mammal. Also disclosed are methods for predicting cancer cell metastasis, and methods for determining the likelihood of patient outcomes by quantitating Regnase-specific expression or activity thereby providing a biomarker for determining the metastatic potential of a given cancer.
Description
DESCRIPTION
COMPOSITIONS AND METHODS FOR SUPPRESSION AND DIAGNOSIS OF CANCER METASTASIS BACKGROUND OF THE INVENTION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to United States Provisional Patent Application No. 62/287,188, filed January 26, 2016 (pending; Atty. Dkt. No. 37182.199PV01); the contents of which is specifically incorporated herein in its entirety by express reference thereto.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant No. R56-AI070315, awarded by the National Institutes of Health. The government has certain rights in the invention. NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT
[0003] Not Applicable.
FIELD OF THE INVENTION
[0004] The present disclosure generally relates to the fields of medicine and cancer biology. In particular, the invention provides improved compositions for determining the metastatic potential of mammalian tumors and other cancers by monitoring the level of Regnase- 1 polypeptide in vivo. Also disclosed are methods for altering the metastatic potential of certain types of mammalian cancers by directly or indirectly increasing the overall levels of regnase activity, by selectively increasing the enzymatic activity of (or by selectively inhibiting one or more negative effectors of) Regnase, or by selectively increasing Regnase- specific mRNA levels within cancer cells to alter, prevent, or reduce their ability to metastasize to other cells or tissues. A targeted gene signature for Regnase is also disclosed, which can be exploited as a molecular biomarker for predicting metastasis and/or determining patient outcomes. REGNASE
[0005] Regnase- 1 is an enzyme that breaks down mRNA, but itself is a protein controlled by post-translational mechanisms. For example, proteasomes that can degrade proteins can also degrade Regnase-1. Thus, pathways that result in hyper-activation of such proteasomes will also result in the inactivation of Regnase-1. Such pathways and specific proteasomes can therefore serve as a molecular signature for cancer metastasis in lieu of Regnase-1. These molecules and
pathways are also potential targets to inhibit cancer metastasis. However, this remains in the context that low levels of Regnase- 1 protein itself indicate the high metastatic potential for cancer cells. Furthermore, as Regnase- 1 breaks down mRNAs or ncRNAs, the identity of those RNA, once revealed, can also serve as a molecular signature to predict cancer metastasis or serve as targets for therapeutic interventions.
[0006] Loss of Regnase- 1 enzymatic activity in vivo may be associated with tumor transformation, or resistance to certain therapeutic agents. It is also possible that the loss of Regnase- 1 activity may identify tumor stem cells, which have high metastatic potential.
[0007] It is known that some cancers are extremely aggressive, showing propensity of spreading early, and even the some type of cancer (e.g., melanoma), not all cancer cells are metastatic, and cells that can spread and establish distal nodules are confined to a small subset with stem cell-like features, the presence or absence of Regnase may segregate tumor cells into such categories. As an early screening marker, the decrease or absence of Regnase activity in cancer biopsies may indicate poor prognosis for cancer patients, and/or may signal the need for more aggressive and/or more exhaustive chemotherapy regimens than would ordinarily be indicated.
BIOMARKERS FOR PREDICTING BREAST CANCER METASTASIS
[0008] Over-expressed and/or secreted proteins from cancer cells are easy to detect in the sera of cancer patients, and as such, have been used clinically as diagnostic and prognostic markers for cancers for a number of years (Wu et al., 2005). Examples of secreted proteins used for cancer diagnosis include prostate specific antigen (PSA) for prostate cancer (Balk et al, 2003), carcinoembryonic antigen, CA125, for ovarian cancer (Raamanathan et al., 2012) and colony stimulating factor-1 (CSF-1) (Kacinski et al, 1990) for endometrial carcinoma. A few soluble biomarkers for breast cancer including carcinoembryonic antigens, CA 15.3 and CA 27.59 and vascular endothelial growth factor (VEGF) are of prognostic value (Harris et al, 2007).
DEFICIENCIES IN THE PRIOR ART
[0009] Currently, CA 15-3 is the most widely used serum biomarker assayed in conjunction with diagnostic imaging for monitoring metastatic disease in breast cancer (Danova et al, 2011). However, this, and other existing breast cancer biomarkers (e.g., TP A/TPS, etc.) lack sensitivity for early disease, and are not very specific (Duffy, 2006). Thus, identification of more specific markers to predict metastasis would be of clinical significance, and would provide a significant advance over the prior art.
BRIEF SUMMARY OF THE INVENTION
[0010] The present disclosure addresses unmet deficiencies inherent in the relevant oncological and pharmaceutical arts by providing Regnase-specific polypeptide and polynucleotide compositions, gene therapy systems for expressing Regnase proteins in specific mammalian cells, and methods for controlling or expressing biologically-active Regnase polypeptides, and exploiting Regnase compounds as biomarkers for predicting the metastatic potential of cancer cells.
[0011] The present disclosure also provides compositions for determining the metastatic potential of mammalian tumors and other cancers, and methods for monitoring the level of Regnase- 1 protein both in vitro and in vivo.
[0012] The disclosed Regnase compositions facilitate new methods in the area of cancer diagnostics, and particularly in cancer metastasis diagnosis, by serving as molecular markers (i.e. a molecular "signature") that correlate with the invasiveness of particular types of cancer cells. The Regnase compositions disclosed herein also represent important new tools in cancer treatment, through the induction or overexpression of nuclease- active Regnase- 1 polypeptides in mammalian cancer cells.
[0013] Gene-therapy-based approaches for persistently expressing Regnase- 1 polypeptide in cancer cells, including vector-based, RNA-based, and nanoparticle-based vectors and/or delivery systems also represent important aspects of the present disclosure. In particular, the use of viral- based gene-expression vectors, which have already been approved for human use in cancer treatment, to persistently express Regnase- 1 in populations of mammalian cells (and particularly, human cancer cells), offers a ready platform for exploiting the disclosed Regnase compositions in a variety of cancer treatment modalities.
[0014] For example, the P53 gene construct, rAd-p53, also known as Gendicine (Shenzhen SiBiono GeneTech, CHINA) is a recombinant, replication-incompetent, human serotype 5 adenovirus. Approved for use in China in 2003, it has been used to treat head and neck squamous cell carcinoma in a number of clinical trials. To construct this therapeutic adenovirus, the El region of the virus was replaced by a human, wild-type P53 expression cassette. A similar strategy has been used to generate the Ad- Reg- 1 construct referred to herein as "rAd- Regnase- 1." In this gene cassette, the El region was replaced by a human wild-type or a truncated Regnase- 1 gene, and the resulting adenoviral vectors were prepared using 293T host cells for propagation.
[0015] By understanding what down-regulates or degrades Regnase- 1, various approaches and specific small molecules, can be devised to inhibit such mechanisms to achieve sustained Regnase
expression in cancer cells. Once induced, Regnase-1 undergoes attrition in cells over time, which is particularly significant at the protein level, where Regnase-1 is inactivated via proteosomal- based degradation.
[0016] Bortezomib (VELCADE®, Janssen-Cilag, Pty Ltd, Macquarie Park, NSW, AUSTRALIA) is an FDA-approved proteasome inhibitor for treatment of multiple myeloma and mantle-cell lymphoma. Drugs like bortezomib can also be used as non-specific therapeutics to rescue Regnase-1 from degradation thereby inhibiting metastatic cancer cells. Identifying specific Regnase-1 degradation proteasomes and signaling pathways have led to the creation of more specific inhibitors of Regnase-1 degradation, which can be used to stabilize (or even enhance) Regnase-1 levels, thereby inhibiting cancer metastasis.
[0017] Another approach afforded by the present disclosure is the development of small molecules that can bind to Regnase-1 to stabilize the protein in a manner analogous to that of a chaperone. Such small molecules may also function by disrupting physical interactions between Regnase and proteases, and thereby reducing or preventing the degradation of Regnase polypeptide. For example, the tumor suppressor, P53, is inactivated by the E3 ligase, MDM2. Small molecules, such as Nutlin or its analogs, however, can mimic the p53 peptide thereby blocking the activity of MDM2. Such small molecules are currently under development in clinic to treat multiple cancers. Similar strategies can be exploited to block the activity of E3 ligases that degrade Regnase-1.
[0018] In another aspect of the disclosure, Regnase-1 -specific gene signatures can also be used as predictors of cancer metastasis, and may serve as potential targets of cancer therapies. Regnase-1 functions as a ribonuclease to breakdown a set of RNAs to block cancer metastasis. RNA microarrays may be exploited to identify potential targets of Regnase-1. By identifying such gene transcripts, they can serve also as molecular signatures to predict cancer metastasis.
[0019] In an overall and general sense, the present disclosure provides a composition that comprises: a therapeutically-effective amount of a first isolated mammalian Regnase-1 peptide, a Regnase-1 polypeptide, a Regnase-1 activator, or a combination thereof, either alone, or in combination with one or more distinct therapeutic, diagnostic, prophylactic, or prognostic agents, and in particular, one or more agents useful in determining the metastatic potential of a mammalian cancer cell, and particularly a human cancer cell.
[0020] Such compositions may also further optionally comprise an additional chemotherapeutic agent, one or more diagnostic agents, one or more imaging agents, or any combinations thereof. Such compositions may be formulated with one or more pharmaceutically- acceptable buffers, diluents, solvents, or solutions, and may be contained within a commercial kit
that includes instructions for administration of the composition to a mammal in need thereof.
[0021] The pharmaceutical composition disclosed herein find particular use in the diagnosis, the prophylaxis, the therapy, or the amelioration of one or more symptoms of a cancer or cancer metastasis in a mammal.
[0022] In particular embodiments, Regnase-1 peptides or polypeptides preferably comprise an at least 50 amino acid sequence that is at least 95% identical to an at least 50 contiguous amino acid sequence from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: 11, more preferably, an at least 100 amino acid sequence that is at least 95% identical to an at least 100 contiguous amino acid sequence from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: l l, and more preferably still, an at least 250 amino acid sequence that is at least 95% identical to an at least 250 contiguous amino acid sequence from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: 11.
[0023] In certain aspects, the Regnase polypeptides in accordance with the present disclosure with comprise, consist essentially or, or alternatively, consist of an amino acid sequence that is at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to any one of the polypeptide sequences set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: 11.
[0024] In certain aspects, Regnase-1 peptides or polypeptides may be produced in vitro or in vivo by a gene therapy vector system that comprises a nucleic acid sequence encoding a selected Regnase-1 peptide or polypeptide operably linked to a promoter that expresses the nucleic acid sequence to produce the encoded peptide or polypeptide in a mammalian cell that has been transformed with the vector system. Exemplary gene therapy systems include, without limitation, an adenoviral, adeno-associated, lentiviral, or herpesviral vector system that operably expresses the peptide or the polypeptide in a population of mammalian cells transformed with the vector system.
[0025] Compositions disclosed herein may be used in diagnosis, prophylaxis, or therapy, or one or more combinations thereof. In certain embodiments, the compositions may be used in the manufacture of a medicament for diagnosing, treating, or ameliorating one or more symptoms of a mammalian cancer, and preferably, of a human cancer, and in particular, one or more human metastatic cancers.
[0026] As described herein, Regnase compositions may further optionally include one or more
active agents, such as, for example, one or more prophylactic agents, one or more therapeutic agents, one or more diagnostic agents, one or more vaccines, one or more imaging agents, one or more radiolabels, one or more adjuvanting agents, one or more chemotherapeutic agents, one or more cytotoxic agents, or any combination thereof, and preferably in a pharmaceutical formulation suitable for administration to a patient.
[0027] In related embodiments, the invention also provides therapeutic and/or diagnostic kits including one or more of the cancer metastasis prognostic Regnase compositions disclosed herein, typically in combination with one or more pharmaceutically acceptable carriers, one or more devices for administration of the compositions to a subject of interest, as well as one or more instruction sets for using the composition in the prevention, the diagnosis, or the treatment of a mammalian condition, disease, disorder, trauma, and/or dysfunction, including, without limitation, one or more mammalian cancers and such like.
[0028] The Regnase compositions disclosed herein may also be used in the manufacture of a diagnostic reagent for determining the metastatic potential of one or more cancers in a mammalian subject.
[0029] The invention also provides a method for diagnosing, treating, or ameliorating one or more symptoms of cancer in a mammal. In an overall and general sense, such a method includes at least the step of administering to a mammal in need thereof, an effective amount of a Regnase composition as disclosed herein, for a time sufficient to diagnose, treat, or ameliorate the one or more symptoms of cancer in the mammal, including, without limitation, a human that has, or is at risk for developing one or more cancer metastases. Such methods may further optionally include administering a therapeutically-effective amount of at least one anti-cancer agent or one anti- metastatic agent to the mammal.
[0030] The invention also provides in an overall and general sense, methods for providing a Regnase composition to a mammalian cancer cell that include at least the step of administering to the subject, an effective amount of one or more of the Regnase compositions disclosed herein. In certain embodiments, the subject is at risk for, diagnosed with, or suspected of having one or more abnormal conditions, including, for example, one or more cancers or other hyperproliferative disorders, and in particular, one or more metastatic cancers.
[0031] As noted herein, the compositions of the present disclosure may be administered to the subject through any one or more conventional methods for administration, including, without limitation, orally, intranasally, intravenously, subcutaneously, or by direct injection to one or more cells or one or more tissues within or about the body of the subject.
[0032] In another embodiment, the present disclosure also provides a method for administering
an active agent to one or more cells, tissues, organs, or systems of a mammalian subject in need thereof. The method generally involves providing to a mammalian subject in need thereof, one or more of the regnase- specific compositions disclosed herein in an amount and for a time effective to introduce the composition into one or more selected tissues, organs, systems, or cells within or about the body of the subject.
[0033] As further described herein, in certain applications, it may be desirable to contact a population of cells obtained from a subject ex vivo with one or more regnase compositions, and then, subsequently, to reintroduce the resulting contacted cells into the body of the subject. Such ex vivo therapy is particularly contemplated to be useful in introducing the disclosed Regnase compositions to populations of human cells, allowing the active ingredients to be contacted with the cells, and then to re-introduce the resulting cells back into the body of the animal. Preferably, the cells extracted for such ex vivo manipulation will be those of the actual patient undergoing treatment.
[0034] In particular embodiments, the Regnase compositions of the present invention may be formulated for pharmaceutical administration, and preferably for administration to a human. Such compositions may further include one or more additional therapeutic agents, chemotherapeutics, adjuvants, or a second distinct population of Regnase-expressing vectors, or host cells comprising them.
[0035] The compositions of the present disclosure may be administered to a mammal in a single administration, or in a series of successive administrations over a selected time interval ranging, for example, from one or more days, to one or more weeks, or even to one or more months wherein longer-term treatment is indicated. In certain applications, the Regnase composition(s) may be administered substantially concurrently with the administration of one or more additional therapeutic and/or diagnostic agents.
[0036] In particular embodiments, the Regnase compositions may be comprised within a population of adenoviral vectors that contain a gene expression cassette encoding the Regnase peptide or protein of interest, operably linked to a promoter suitable for expressing the Regnase composition in a mammal, and particularly, a human. BRIEF DESCRIPTION OF THE DRAWINGS :
[0037] The following drawings form part of the present specification and are included to demonstrate certain aspects of the present invention. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0038] For promoting an understanding of the principles of the invention, reference will now be made to the embodiments, or examples, illustrated in the drawings and specific language will be used to describe the same. It will, nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one of ordinary skill in the art to which the invention relates.
[0039] The invention may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:
[0040] FIG. 1A and FIG. IB show the inactivation of Regnase-1 in highly metastatic breast cancer cells. FIG. 1A shows the immunoblot analysis of Regnase-1 protein level in MCF7, MDA-MB-231, SUM159, and MDA-MB-468 cancer cell lines. The breast cancer cells MDA- MB-231 and SUM159 have high metastatic potential as compared to MCF7 and MDA-MB-468 cells. 293 T cells are non-cancer cells and used as controls. FIG. IB shows the real-time RT- PCR analysis of Regnase-1 mRNA levels in 293T, MCF7, MDA-MB-231, SUM159, and MDA- MB-468 cells;
[0041] FIG. 2A and FIG. 2B demonstrate the forced expression of Regnase-1 in cancer cells does not affect their proliferation potential or morphology. FIG. 2A shows tMCF7, SUM-159 and B16 cell lines that with stable expression of wild type (WT), endoribonuclease dead (D225A) Regnase-1 or control vector were seeded into 6-well plates and cultured for 48 hours. Their morphology was examined by light microscopy. The micrographs revealed clusters of proliferating cells. In FIG. 2B, the cancer cell lines were labeled with CFSE, a dye that tracks cell proliferation, at day 0, and levels of CFSE in labeled cells was examined by flow cytometer at day 1 and day 5. Dilution of CFSE indicates cell proliferation;
[0042] FIG. 3A and FIG. 3B show the expression of Regnase-1 inhibits cell migration and invasiveness. In FIG. 3A, SUM-159 and B 16 melanoma cells that stably express wild type (WT), endoribonuclease dead (D225A) Regnase-1 or control vector were seeded into trans- well plates and cultured for 24 hours. The cells were fixed with methanol and stained with 0.05% crystal violet dye for 10 min. The invasiveness of cancer cells was marked by enlarged dark blue cell bodies. FIG. 3B shows SUM-159 cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were seeded into 6-well plates. When the cells forming about 90% confluent monolayer, a gap in the confluent monolayer was made by scratching the cells with sterilized tip. 24 hours later, cell proliferation in filling the gaps was determined by light
microscopy. The higher the migration potential of cells, the faster in filling the gaps;
[0043] FIG. 4A, FIG. 4B, and FIG. 4C show the forced expression of Regnase-1, but not the D225A mutation (enzymatically inactive Regnase-1), completely inhibits melanoma metastasis in vivo. FIG. 4A: B 16 melanoma cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were injected into B6 mice through the tail vein. After two weeks, the mice were sacrificed, and the tumor foci in the lungs were determined. Photographs shown are both lungs from mice, and the dark spots are melanoma tumor foci. In FIG. 4B paraffin embedded lung tissues from host B6 mice injected with indicated melanoma cells were stained by Hematoxylin and Eosin (H&E). Tissue histology showed the lung tissue and tumor nodules in the lungs. FIG. 4C shows the survival of host mice injected with B16 melanoma cells with forced expression of WT Regnase-1, D225A mutated Regnase-1 or control vector. The primary endpoint was defined as severe paralysis or moribund. There were 10 mice in each group and none of the mice with Regnase-1 -expressing melanoma cells died in the four week- study period;
[0044] FIG. 5A, FIG. 5B, and FIG. 5C show the forced expression of WT but not D225A mutated Regnase-1 completely inhibits metastasis of breast cancer cells in vivo. FIG. 5A: SUM- 159 cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were injected into Rag2_/~ and yc '~ double knockout (DKO) mice through tail vein. After three weeks, the mice were sacrificed and the tumor foci in the lung were imaged. The red arrow depicts tumor nodules. FIG. 5B: The histogram shows the number of visible tumor nodules in the lung from Rag2_/~ and yc '~ DKO mice injected with indicated cells. FIG. 5C: Paraffin embedded lung tissues from Rag2_/~ and yc '~ DKO mice injected with indicated tumor cells were stained by Hematoxylin and Eosin (H&E). Pictures show the lung tissue and tumor nodules in the lung;
[0045] FIG. 6A and FIG. 6B show the correlation of breast cancer metastasis with low expression of Regnase-1. FIG. 6A: Representative image of breast cancer tissues stained by anti- Regnase-1 antibody in breast cancer with or without metastasis. FIG. 6B: The number represents breast cancer tissues with different level of Regnase-1 in breast cancer with or without metastasis;
[0046] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E-1, FIG. 7E-2 FIG. 7E-3 FIG. 7E-4, and FIG. 7E-5 show a microfluidic chip-based cell clustering (MFCBCC) assay to monitor cell clustering. FIG. 7A shows a working scheme to briefly describe the MFCBCC assay. FIG. 7B shows MCF7, MDA-MB-468, MDA-MB-231 and SUM-159 cells seeded into microfluidic chips at the concentration of 3 to 4 cells per channel. The process of cell clustering is monitored by digital recording in 2-hour time period. The red circles indicate individual cells at the beginning of experiment or individual cells and cell clusters at the end of experiment.
FIG. 7C is a histogram that shows the percentage of observed clustering events in total monitored wells within 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields. FIG. 7D shows five channels containing 3 - 5 cells for each cell type were monitored and their dynamic clustering rates presented as the percentage of observed clustering events in total events. FIG. 7E-1, FIG. 7E-2 FIG. 7E-3 FIG. 7E-4, and FIG. 7E-5 illustrate two channels containing 4 cells for each cell type. The dynamic sum of cell number and cluster number is also shown;
[0047] FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, and FIG. 8F show the inactivation of Regnase-1 in highly metastatic breast cancer cells and tissues. FIG. 8A is the real-time RT-PCR analysis of Regnase-1 mRNA level in 293T, MCF7, MDA-MB-231, sUM159 and MDA-MB-468 cells. FIG. 8B shows the immunoblot analysis of Regnase-1 protein level in MCF7, MDA-MB- 231, SUM159, and MDA-MB-468 cancer cell lines. The breast cancer cells MDA-MB-231 and SUM159 had a high metastatic potential as compared to MCF7 and MDA-MB-468 cells. 293T cells are non-cancer cells that were used as controls. Beta-actin was included as a loading control. FIG. 8C shows MCF7, MDA-MB-231 and SUM-159 cells treated with different doses of MG132, Baf (Bafilomycin Al), MI2, Mep (Mepazine), IKKII (IKK inhibitor II), 5Z7 ((5Z)-7- Oxozeaenol) for 4 hrs. After treatment, the level of Regnase-1 was examined by Western Blot. The expression of β-actin as the loading control. In FIG. 8D, MCF7 or MDA-MB-468 cells were co-cultured with different amount of MDA-MB-231 or SUM-159 cells for 2 days. Then, the expression level of Regnase-1 in MCF7 and MDA-MB-468 cells was examined by WB. The expression of β-actin was used to indicate the number of cells. FIG. 8E is a representative image of breast cancer tissues stained by anti-Regnase-1 antibody in breast cancer with or without metastasis. FIG. 8F shows the number of breast cancer tissues with differing levels of Regnase-1 in cancer cells with or without metastasis. Data from FIG. 8A to FIG. 8D were representative of at least three independent studies;
[0048] FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G, FIG. 9H, FIG. 91, and FIG. 9 J show the forced expression of Regnase-1, but not the D225A mutation (enzymatically-inactive Regnase-1), completely inhibited metastasis in vivo, and cell clustering in vitro. In FIG. 9A, SUM-159 cells with stable expression of WT Regnase-1, the D225A mutant Regnase, or a control vector were seeded into array chips, and monitored as described above in the description of FIG. 7A-FIG. 7E. The histogram in FIG. 9B shows the percentage of observed clustering events in all monitored channels at 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields. FIG. 9C shows SUM-159 cells with stable expression of WT Regnase-1, Reg" mutant, D225A, or a control vector that were then injected into Rag2/rc
double-knockout (DKO) mice via the tail vein. After three weeks, the mice were sacrificed and the tumor foci in the lungs were imaged. The red arrow depicts tumor nodules. Paraffin- embedded lung tissues from Rag2/INFr DKO mice injected with indicated tumor cells were stained by H&E. Photographs illustrate the lung tissue and tumor nodules therein. In FIG. 9D, the histogram shows the number of visible tumor nodules in the lung from Rag2/rc DKO mice injected with the indicated cell lines. In FIG. 9E, B16-F10 cells with stable expression of WT Regnase-1, the Reg" mutant (D225A), or a control vector were seeded into array chips and monitored as described in the brief description of FIG. 7A-FIG. 7E. In FIG. 9F, the histogram shows the percentage of observed clustering events in all monitored channels at 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields. FIG. 9G shows B 16 melanoma cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector injected into B6 mice via the tail vein. After two weeks, the mice were sacrificed and the tumor foci in the lungs were determined and imaged. Photographs illustrating both lungs from mice are shown; black spots are the melanoma tumor foci. In FIG. 9H, the histogram shows the number of visible tumor nodules in the lung from B6 mice injected with indicated cells. FIG. 91 shows paraffin embedded lung tissues from host B6 mice injected with indicated melanoma cells were stained by H&E. Tissue histology showed the lung tissue and tumor nodules in the lungs. FIG. 9J describes survival of host mice injected with B 16 melanoma cells with forced expression of WT Regnase-1, the D225A mutant Regnase, or a control vector. The primary endpoint was defined as severe paralysis or moribund. There were 10 mice in each group, and none of the mice transformed with Regnase-1 -expressing melanoma cells died during the four-week study period;
[0049] FIG. 10A, FIG. 10B, FIG. IOC, FIG. 10D, FIG. 10E, FIG. 10F, FIG. 10G, FIG. lOH-1, FIG. 10H-2, and FIG. 10H-3 show S100A6 is a downstream target gene of Regnase-1. FIG. 10A shows real-time RT-PCR analysis of S100A6 mRNA level in B 16-Vector, B 16-Regnase-1 WT and B 16-Regnase-1 D225A cells. FIG. 10B shows real-time RT-PCR analysis of S100A6 mRNA levels in MCF7, MDA-MB-231, SUM-159 and MDA-MB-468 cells. FIG. IOC is a Western blot analysis demonstrating the levels of S100A4 and S100A6 protein in 293T, MCF7, MDA-MB-231, SUM-159 and MDA-MB-468 cells. FIG. 10D shows the real-time RT-PCR analysis of S100A6 mRNA levels in MDA-MB-231 cells transfected either with the vector alone, wild-type Regnase-1, or the Reg" mutant, D225A. FIG. 10E shows the real-time RT-PCR analysis of S100A6 mRNA levels in SUM-159 cells transfected either with control vector alone, wild- type Regnase-1, or the Reg" mutant, D225A. FIG. 10F shows the conserved sequence region from the human and murine S100A6 3'UTR region, 5 '-AATCCAGTGGTGGGTA-3 ' (SEQ ID NO: 12). FIG. 10G shows a typical "stem-loop"
structure of the resultant mRNA 5'-AAUCCAGUGGUGGGUA-3' (SEQ ID NO: 13) was identified using the online software, RNAstructure. FIG. lOH-1, FIG. 10H-2, and FIG. 10H-3 illustrate the overexpression of Regnase-1 wild-type (but not D225A mutant) protein inhibited the expression of GFP encoded by a GFP-CDS fused to the S100A6 3'UTR sequence;
[0050] FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. HE, FIG. 1 IF, FIG. 11G, FIG. 11H, FIG. HI, and FIG. llj show S100A6 partially rescued the inhibitory function of Regnase-1 on cell clustering and cancer metastasis. FIG. HA shows the in vitro cell clustering analysis of SUM-159-Regnase-l WT-Vector, SUM-159-Regnase-l WT-S100A4, SUM-159- Regnase-1 WT-S100A6 cells. In FIG. 11B, the histogram shows the ability of cell clustering at 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields. FIG. llC shows SUM-159-Regnase-l WT cells with stable expression of S100A4, S100A6, or control vector that were injected into Rag2_/~ rC_/~ mice via the tail vein. After four weeks, the mice were sacrificed, and the tumor foci at lung were determined and imaged. Photographs of the lungs are shown, and the metastatic foci are indicated by arrows. FIG. 11D is a histogram showing the number of visible tumor nodules in the lung from Nu(NCr)-Foxnl mice injected with indicated cells. FIG. HE shows the in vitro cell clustering analysis of B16-Regnase-1 WT- Vector, B16-Regnase-1 WT-S100A4, B16-Regnase-1 WT-S 100A6 cells. In FIG. 1 IF, the histogram shows ability of cell clustering within 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields. In FIG. 11G, B16-Regnase-1 WT melanoma cells with stable expression of S100A4, S100A6, or control vector were injected into B6 mice via the tail vein. After four weeks, the mice were sacrificed and the tumor foci at lung were determined. Photographs illustrate both lungs from the mice, and the black spots represent melanoma tumor foci. In FIG. 11H, the histogram shows the number of visible tumor nodules in the lung from B6 mice that were injected with the indicated cells. FIG. HI shows paraffin-embedded lung tissues from host B6 mice that were injected with the indicated melanoma cells and stained by H&E. Tissue histology showed the lung tissue and the tumor nodules present in the lungs. FIG. HJ shows the survival of host mice injected with B 16-Regnase-1-WT melanoma cells with forced expression of S100A4, S100A6, or the control vector. The primary endpoint was defined as severe paralysis or moribund. There were 5 mice in each group and mice with B16-Regnase-1 WT-expressing melanoma cells died after about six weeks. The mice with B16-Regnase-1 WT- S100A6 died after about 30 days;
[0051] FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D show a microfluidic chip-based cell clustering (MFCBCC) assay to monitor cell clustering. FIG. 12A is a detailed procedure for preparation of the microfluidic chip. In FIG. 12B, B16-F10 cells were seeded into microfluidic
chips at a concentration of 3 to 4 cells per channel. The process of cell clustering was monitored by a camera during the 2-hr experimental period. The red circles indicate individual cells at the beginning of the experiment, or individual cells and cell clusters at the end of the experiment. In FIG. 12C, the histogram shows the percentage of observed clustering events in total monitored wells within 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields. FIG. 12D shows five channels containing 3 - 5 cells for each cell type were monitored and their dynamic clustering rate presented as a percentage of observed clustering events in total events;
[0052] FIG. 13A, FIG. 13B, and FIG. 13C show the forced expression of Regnase-1 in cancer cells does not affect their proliferation potential or their morphology. In FIG. 13A, MCF7, SUM- 159 and B16 cell lines that with transformed with wild type Regnase-1, endoribonuclease- inactive Regnase-1 ((D225A mutant), or a control vector were each seeded into 6- well plates and cultured for 48 hrs. Morphology was examined by light microscopy, and the resulting micrographs showed clusters of proliferating cells. In FIG. 13B, MCF7, SUM-159 and B16 cells were seeded in six-well plates at ~102/well. Cells were then incubated in DMEM medium for 2 weeks. The resultant colonies were fixed, stained with crystal violet dye, and then photographed. FIG. 13C illustrates cancer cell lines that were labeled with CSFE (a dye that tracks cell proliferation) at Day 0. The level of CSFE in labeled cells was determined by flow cytometry at Day 1 and Day 5. Dilution of CFSE indicated cell proliferation;
[0053] FIG. 14A, FIG. 14B, and FIG. 14C show the expression of Regnase-1 inhibits cell migration and invasiveness. FIG. 14A show MCF7, SUM-159, B16, MDA-MB-231 cells containing the WT Regnase-1, the D225A mutant Regnase-1 or a control vector that were seeded in six-well plates containing 0.3% agar, and grown for 2 weeks, then stained with MTT for 4 hrs. Photomicrographs were recorded and colonies were counted. In FIG. 14B, MCF7, SUM-159, B16 and MDA-MB-231 melanoma cells that stably express wild type (WT), endoribonuclease dead (D225A) Regnase-1 or control vector were seeded into transwell plates and cultured for 24 hrs. The cells were fixed with methanol and stained with 0.05% crystal violet dye for 10 min. The invasiveness of cancer cells was marked by enlarged dark blue cell bodies. FIG. 14C shows SUM-159 cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were seeded into 6-well plates. When the cells formed a -90% confluent monolayer, a gap in the confluent monolayer was made by scratching the cells with a sterilized tip. 24-hrs later, cell proliferation in filling the gaps was determined by light microscopy. The higher the migration potential of cells, the faster in filling the gaps;
[0054] FIG. 15A and FIG. 15B show the ectopic expression of Regnase-1 inhibits cancer cell
clustering. FIG. 15A: MDA-MB-231 cells with stable expression of WT Regnase-1, the D225A mutant Regnase-1 or control vector were seeded into array chips at the concentration of 3 to 4 cells per channel. The process of cells clustering was monitored by a camera over a 2-hr period. The red circle indicates each cell at the beginning of the experiment, or each cell and cell cluster at the end of the experiment. FIG. 15B: Histogram showing the ability of cell clustering within 2 hrs. Cell clusters containing 2 to 10 cells were counted from at least 3 imaging fields;
[0055] FIG. 16A and FIG. 16B show the forced expression of Regnase-1, but not the D225A mutation completely inhibits melanoma metastasis Ragl KO mice in vivo. FIG. 16A: B 16 melanoma cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were injected into Ragl KO mice via the tail vein. After two weeks, the mice were sacrificed and the tumor foci at lung were determined and imaged. Photographs illustrate both lungs from the mice; black spots are melanoma tumor foci. In FIG. 16B, the histogram shows the number of visible tumor nodules in the lung from Rag2/rc DKO mice injected with indicated cells;
[0056] FIG. 17A and FIG. 17B show Regnase-1 slightly inhibits tumor growth but this inhibitory effect is independent its ribonuclease activity. FIG. 17A: Representative image of B16 tumors. B16 melanoma cells with stable expression of WT Regnase-1, D225A mutated Regnase-1 or control vector were injected into each flank and the interscapular region of each B6 mouse. After two weeks, the mice were sacrificed and the subcutaneous tumor were determined and shown. In FIG. 17B, the histogram shows the weight of B 16 tumors from B6 mice subcutaneously injected with the indicated cells;
[0057] FIG. 18A, FIG. 18B-1, FIG. 18B-2, FIG. 18B-3, FIG. 18C-1 and FIG. 18C-2 show the expression of S100A4 and S100A6 after overexpression of Regnase-1. FIG. 18A illustrates a real-time RT-PCR analysis of S100A4 mRNA level in MCF7, MDA-MB-231, SUM- 159 and MDA-MB-468 cells. FIG. 18B-1, FIG. 18B-2, and FIG. 18B-3 show real-time RT-PCR analysis of S100A4 mRNA level in B 16-F10, MDA-MB-231 and SUM- 159 cells transfected with vector, Regnase-1 WT or D225A mutant. In FIG. 18C-1 and FIG. 18C-2, real-time RT-PCR analysis of S100A4 and S100A6 mRNA level in 293T cells transiently transfected with vector, Regnase-1 WT or D225A mutant is shown; and
[0058] FIG. 19A-1, FIG. 19A-2, FIG. 19A-3, FIG. 19B-1, FIG. 19B-2, and FIG. 19B-3 show the overexpression of Regnase-1 has minimal effect on GFP CDS or GFP-S100A4-3'UTR. FIG. 19A-1, FIG. 19A-2, and FIG. 19A-3 show the overexpression of wild-type Regnase-1 had minimal effect on GFP expression encoding by the native GFP CDS. In FIG. 19B-1, FIG. 19B- 2, and FIG. 19B-3, overexpression of WT Regnase-1 had minimal effect on GFP expression
encoded by a construct in which GFP-CDS was fused with the S100A4 3'UTR. BRIEF DESCRIPTION OF THE SEQUENCES:
[0059] SEQ ID NO:l is an exemplary forward oligonucleotide primer that is specific for ZC3H12A, and used in accordance with one or more aspects of the present disclosure.
[0060] SEQ ID NO:2 is an exemplary reverse oligonucleotide primer that is specific for
ZC3H12A, and used in accordance with one or more aspects of the present disclosure.
[0061] SEQ ID NO:3 is an exemplary human Regnase-1 polypeptide (ZC3H12A,
NP_079355.2) for use in accordance with one or more aspects of the present disclosure.
[0062] SEQ ID NO:4 is an exemplary human ZC3H12B ribonuclease polypeptide
(NP_001010888.3) for use in accordance with one or more aspects of the present disclosure.
[0063] SEQ ID NO:5 is an exemplary human ZC3H12C ribonuclease polypeptide
(NP_203748) for use in accordance with one or more aspects of the present disclosure.
[0064] SEQ ID NO:6 is an exemplary human ZC3H12D ribonuclease polypeptide (NP_997243.2) for use in accordance with one or more aspects of the present disclosure.
[0065] SEQ ID NO:7 is an exemplary human KHNYN isoform 1 polypeptide (NP_056114.1) for use in accordance with one or more aspects of the present disclosure.
[0066] SEQ ID NO:8 is an exemplary human NYNRIN protein (NP_079357.2) for use in accordance with one or more aspects of the present disclosure.
[0067] SEQ ID NO:9 is an exemplary human ELOVL5 Elongation of VLC FA protein 5, isoform 2, polypeptide (NP_001229757.1) for use in accordance with one or more aspects of the present disclosure.
[0068] SEQ ID NO: 10 is an exemplary human KCNK12 potassium channel subfamily K member 12 polypeptide (NP_071338.1) for use in accordance with one or more aspects of the present disclosure.
[0069] SEQ ID NO:ll is an exemplary human N4BP1 NEDD4-binding protein 1 polypeptide (N4BP1 NP_694574.3) for use in accordance with one or more aspects of the present disclosure.
[0070] SEQ ID NO: 12 is a nucleotide consensus sequence between human and murine S100A6 3'UTRs, as described further herein.
[0071] SEQ ID NO: 13 is the resultant mRNA sequence transcribed from the mammalian consensus sequence shown in SEQ ID NO: 12, and as described further herein.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0072] Illustrative embodiments of the invention are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be
appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.
CANCER
[0073] Cancer is arguably one of the biggest global threats to public health. Cancer metastasis is a key feature of malignancy and contributes to more than 90% solid tumor-related deaths. Because of poorly understood mechanisms of cancer metastasis, there is no diagnosis/prognosis signature and specific treatment available to efficiently control tumor metastasis. In fact, metastasis is a complex process involving well-coordinated sequence of events where some tumor cells leave the primary lesion and take residence at distal sites. As not all cells in primary tumors have the capacity to metastasize, it is reasoned that metastatic cancer cells may possess special features that can provide valuable therapeutic targets.
BREAST CANCER
[0074] Breast cancer is the second leading cause of death among women in the United States. Approximately one woman in every ten will develop breast cancer in her lifetime. Recent statistics estimate that 44,000 women will die of breast cancer, while 150,000 new female cases of breast cancer will be diagnosed in the next year.
[0075] It has been shown that screening for breast cancer can reduce breast cancer mortality. Among women aged 50 and older, studies have demonstrated a 20% to 40% reduction in breast cancer mortality for women screened by mammography and clinical breast examination. However, among women between 40 to 49 years of age, the mortality rate is reduced only 13% to 23%. These results suggest that further methods of screening could potentially reduce the mortality in the younger age group of women. TUMOR METASTASIS
[0076] Tumor metastasis is a key feature of malignancy and a leading cause of tumor-related death, but how tumor cell metastasis is regulated remains poorly understood. Regnase-1, a cytosolic ribonuclease which is post-translationally inactivated in metastatic cancer cells, has been shown to be a potent repressor of tumor metastasis. The metastatic activities of tumor cells were
inversely related to Regnase-1 expression and that forced expression of Regnase-1 in highly metastatic tumor cells converted metastatic tumor cells to non-metastatic tumor cells. Mechanistically, Regnase-1 regulates microcluster formation of tumor cells, a process that precedes metastasis, and that formation of microclusters requires the calcium binding protein S100A6. Importantly, Regnase-1 inhibited S100A6 expression through its ribonuclease domain, thus preventing tumor cells from forming microclusters. Results shown in the examples herein demonstrate that Regnase-1 is a master regulator in cell clustering and tumor metastasis, and these results indicate an important clinical role for Regnase compositions in cancer diagnosis, metastasis prediction, and cancer therapies.
[0077] Regnase-1 acts as a key checkpoint in tumor metastasis. Namely, the loss of Regnase-1 is associated with high metastatic potential of tumor cells, and over expression of Regnase-1 in two aggressive tumor lines (melanoma and breast cancer) completely prevented tumor cells from spreading to other sites— including the lungs). Regnase-1 did not seem to affect tumor cell survival or proliferation, but significantly controlled the invasiveness of these cells.
PHARMACEUTICAL FORMULATIONS
[0078] In certain embodiments, the present invention concerns chemotherapeutic compositions prepared in pharmaceutically-acceptable formulations for delivery to one or more cells or tissues of an animal, either alone, or in combination with one or more other modalities of diagnosis, prophylaxis and/or therapy. The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those of ordinary skill in the art, as is the development of suitable dosing and treatment regimens for using the particular Regnase-specific compositions described herein in a variety of diagnostic and cancer-prognostic regimens.
[0079] In certain circumstances it will be desirable to deliver the disclosed regnase-specific compositions in suitably-formulated pharmaceutical vehicles by one or more standard delivery devices, including, without limitation, subcutaneously, parenterally, intravenously, intramuscularly, intrathecally, intratumorally, intraperitoneally, transdermally, topically, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs within or about the body of an animal.
[0080] The methods of administration may also include those modalities as described in U.S. Patent Nos. 5,543,158; 5,641,515, and 5,399,363, each of which is specifically incorporated herein in its entirety by express reference thereto. Solutions of the active compounds as freebase or pharmacologically acceptable salts may be prepared in sterile water, and may be suitably mixed with one or more surfactants, such as hydroxypropylcellulose. Dispersions may also be prepared
in glycerol, liquid polyethylene glycols, oils, or mixtures thereof. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0081] For administration of an injectable aqueous solution, without limitation, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, transdermal, subdermal, and/or intraperitoneal administration. In this regard, the compositions of the present invention may be formulated in one or more pharmaceutically acceptable vehicles, including for example sterile aqueous media, buffers, diluents, etc. For example, a given dosage of active ingredient(s) may be dissolved in a particular volume of an isotonic solution (e.g., an isotonic NaCl-based solution), and then injected at the proposed site of administration, or further diluted in a vehicle suitable for intravenous infusion (see, e.g., "REMINGTON'S PHARMACEUTICAL SCIENCES" 15th Edition, pp. 1035-1038 and 1570-1580). While some variation in dosage will necessarily occur depending on the condition of the subject being treated, the extent of the treatment, and the site of administration, the person responsible for administration will nevertheless be able to determine the correct dosing regimens appropriate for the individual subject using ordinary knowledge in the medical and pharmaceutical arts.
[0082] Sterile injectable compositions may be prepared by incorporating the disclosed compositions in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the selected sterilized active ingredient(s) into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above. The compositions disclosed herein may also be formulated in a neutral or salt form.
[0083] Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein), and which are formed with inorganic acids such as, without limitation, hydrochloric or phosphoric acids, or organic acids such as, without limitation, acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, without limitation, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation, and in such amount as is effective for the intended application. Formulations of compounds of the present invention may be readily administered in
a variety of dosage forms such as injectable solutions, topical preparations, oral formulations, including sustain-release capsules, hydrogels, colloids, viscous gels, transdermal reagents, intranasal and inhalation formulations, and the like.
[0084] The amount, dosage regimen, formulation, and administration of chemotherapeutics disclosed herein will be within the purview of the ordinary-skilled artisan having benefit of the present teaching. It is likely, however, that the administration of a diagnostically-effective (i.e. , a pharmaceutically-effective) amount of one or more of the disclosed compositions may be achieved by a single administration, such as, without limitation, a single injection of a sufficient quantity of the delivered agent to provide the desired benefit to the patient in need thereof. Alternatively, in some circumstances, it may be desirable to provide multiple, or successive administrations of the disclosed compositions, either over a relatively short, or even a relatively prolonged period, as may be determined by the medical practitioner overseeing the administration of such compositions to the selected individual undergoing such procedure(s).
[0085] Typically, formulations of one or more of the regnase- specific compositions described herein will contain at least an effective amount of a first active agent. Preferably, the formulation may contain at least about 0.001% of each active ingredient, preferably at least about 0.01% of the active ingredient, although the percentage of the active ingredient(s) may, of course, be varied, and may conveniently be present in amounts from about 0.01 to about 90 weight % or volume %, or from about 0.1 to about 80 weight % or volume %, or more preferably, from about 0.2 to about 60 weight % or volume %, based upon the total formulation. Naturally, the amount of active compound(s) in each composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological ti/2, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one of ordinary skill in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
[0086] While systemic administration is contemplated to be effective in many embodiments of the invention, it is also contemplated that formulations disclosed herein be suitable for direct injection into one or more organs, tissues, or cell types in the body. Direct administration of the disclosed compositions to particular discreet locations within the body, or directly to tumor or cancerous tissues, e.g., may be conducted using suitable means as known to those of ordinary skill in the relevant medical oncology arts.
[0087] The pharmaceutical formulations of the present invention may further comprise one or more excipients, buffers, or diluents that are particularly formulated for contact with mammalian
cells, and in particular human cells, and/or for administration to a mammalian subject, such as a human patient. Compositions may further optionally comprise one or more diagnostic or prognostic agents, and/or may be formulated within a population of microspheres, microparticles, nanospheres, or nanoparticles, and may be formulated for administration to one or more cells, tissues, organs, or body of a human in particular.
[0088] Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing, diagnostic, and/or treatment regimens for using the particular compositions described herein in a variety of modalities, including e.g., without limitation, oral, parenteral, intravenous, intranasal, intratumoral, and intramuscular routes of administration.
[0089] The particular amount of compositions employed, and the particular time of administration, or dosage regimen for compositions employing the disclosed regnase-specific formulations will be within the purview of a person of ordinary skill in the art having benefit of the present teaching. It is likely, however, that the administration of the disclosed formulations may be achieved by administration of one or more doses of the formulation, during a time effective to provide the desired benefit to the patient undergoing such treatment. Such dosing regimens may be determined by the medical practitioner overseeing the administration of the compounds, depending upon the particular condition or the patient, the extent or duration of the therapy being administered, etc.
[0090] Pharmaceutical formulations comprising one or more active ingredients as disclosed herein are not in any way limited to use only in humans, or even to primates, or mammals. In certain embodiments, the methods and compositions disclosed herein may be employed using avian, amphibian, reptilian, or other animal species. In preferred embodiments, however, the compositions of the present invention are preferably formulated for administration to a mammal, and in particular, to humans, in a variety of regimens for modulating the metastatic potential of cancer cells. As noted above, such compositions are not limited only to use in humans, but may also be formulated for veterinary administration, including, without limitation, to selected livestock, exotic or domesticated animals, companion animals (including pets and such like), non- human primates, as well as zoological or otherwise captive specimens, and such like.
COMPOSITIONS FOR THE PREPARATION OF MEDICAMENTS
[0091] Another important aspect of the present invention concerns methods for using the disclosed compositions (as well as formulations including them) in the preparation of medicaments for preventing, diagnosing, treating and/or ameliorating one or more symptoms of
one or more diseases, dysfunctions, abnormal conditions, or disorders in an animal, including, for example, vertebrate mammals. Use of the disclosed compositions is particularly contemplated in the diagnosis and/or prognosis of cancer, in the detection or prediction of cancer metastasis or in monitoring the extent thereof, and/or for suppression of the metastatic potential of one or more cancer cell types.
[0092] Such use generally involves administration to the mammal in need thereof one or more of the disclosed regnase-specific compositions, in an amount and for a time sufficient to diagnose, treat, lessen, or ameliorate one or more symptoms of cancer metastasis in an affected mammal.
[0093] Pharmaceutical formulations including one or more of the disclosed regnase compositions also form part of the present invention, and particularly those compositions that further include at least a first pharmaceutically-acceptable excipient for use in the diagnosis, prophylaxis, therapy and/or amelioration of one or more symptoms of cancer in an affected mammal. GENE THERAPY VECTORS
[0094] Gene therapy has been exploited for the treatment of a wide variety of human diseases including those of the central nervous system such as Alzheimer's, Parkinson's, Batten's and Huntington's Diseases, β-thalassemia, oci-antitrypsin (AAT) deficiency, and Fragile-X Mental Retardation Syndrome. Many chronic and progressive diseases require sustained or regulatable administration of the therapeutic gene to achieve successful treatment. The inventors contemplate the use of such Regnase-expressing systems in the transfection of mammalian cancer cell for the persistent expression of Regnase protein in vivo. Exemplary gene therapy vector systems are known to those of ordinary skill in the art, and include, but are not limited to, Ad, AAV, HSV, lenti virus, and the like.
ADENO-ASSOCIATED VIRUS (AAV)
[0095] Adeno-associated virus is a single- stranded DNA-containing, non-pathogenic human parvovirus that is being widely investigated as a therapeutic vector for a host of muscle disorders Recombinant adeno-associated virus (rAAV) vectors have been developed in which the rep and cap open reading frames of the wild-type virus have been completely replaced by a therapeutic or reporter gene, retaining only the characteristic inverted terminal repeats (ITRs), the sole ds-acting elements required for virus packaging. Using helper plasmids expressing various combinations of the AAV2 rep and AAV-1, -2, and -5 cap genes, respectively, efficient cross packaging of AAV2 genomes into particles containing the AAV-1, -2, or -5 capsid protein has been demonstrated.
The various serotype vectors have demonstrated distinct tropisms for different tissue types in vivo, due in part to their putative cell surface receptors.
PROMOTERS AND ENHANCERS
[0096] Recombinant vectors capable of expressing Regnase polypeptides form important aspects of the present invention. The term "expression vector or construct" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In preferred embodiments, expression only includes transcription of the nucleic acid, for example, to generate a therapeutic agent from a transcribed gene that is comprised within a suitable gene expression cassette.
[0097] Particularly useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operatively linked," "operably linked," "operatively positioned," "under the control of or "under the transcriptional control of means that the promoter is in the correct location and orientation in relation to the nucleic acid segment that comprises the therapeutic gene to properly facilitate, control, or regulate RNA polymerase initiation and expression of the therapeutic gene to produce the therapeutic peptide, polypeptide, ribozyme, or antisense RNA molecule in the cells that comprise and express the genetic construct.
[0098] In preferred embodiments, it is contemplated that certain advantages will be gained by positioning the Regnase-encoding polynucleotide segment under the control of one or more recombinant, or heterologous, promoter(s). As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with the particular therapeutic gene of interest in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell.
[0099] Naturally, it will be important to employ a promoter that effectively directs the expression of the Regnase-encoding nucleic acid segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of ordinary skill in the molecular biology arts. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high-level expression of the introduced Regnase- specific DNA segment.
[0100] At least one module in a promoter functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
[0101] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
[0102] The particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter, such as a β-actin, AAV, AV, CMV or HSV promoter. In certain aspects of the invention, inducible promoters, such as tetracycline-controlled promoters, are also contemplated to be useful in certain cell types.
[0103] In various other embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of transgenes. The use of other viral or mammalian cellular or bacterial phage promoters that are well known in the art to achieve expression of a transgene is contemplated as well, provided that the levels of expression are sufficient for a given purpose. .
[0104] Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
[0105] The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a
promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
[0106] As used herein, the terms "engineered" and "recombinant" cells are intended to refer to a cell into which an exogenous nucleic acid segment, such as DNA segments that lead to the transcription of a therapeutic agent, such as a therapeutic peptide, polypeptide, ribozyme, antisense, or catalytic mRNA molecule has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells, which do not contain a recombinantly introduced exogenous polynucleotide segment. Engineered cells are thus cells having nucleic acid segment introduced through the hand of man.
[0107] To express a Regnase-encoding nucleic acid sequence in accordance with the present invention one would prepare a suitable gene expression vector that comprises at least a first sequence region that encodes a Regnase peptide, polypeptide, ribozyme, or antisense mRNA under the control of one or more promoters. To bring a sequence "under the control of a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame generally between about 1 and about 50 nucleotides "downstream" of (i.e. , 3' of) the chosen promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded polypeptide. This is the meaning of "recombinant expression" in this context.
BIOLOGICAL FUNCTIONAL EQUIVALENTS
[0108] Modification and changes may be made in the structure of the gene expression cassettes, or to the viral vectors comprising them, as well as modification to the the therapeutic agents encoded by them and still obtain functional vectors, viral particles, and virion that encode one or more therapeutic agents with desirable characteristics. As mentioned above, it is often desirable to introduce one or more mutations into a specific polynucleotide sequence. In certain circumstances, the resulting encoded polypeptide sequence is altered by this mutation, or in other cases, the sequence of the polypeptide is unchanged by one or more mutations in the encoding polynucleotide.
[0109] When it is desirable to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, second-generation molecule, the amino acid changes may be achieved by changing one or more of the codons of the encoding DNA sequence, according to Table 1.
[0110] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
TABLE 1
AMINO ACIDS CODONS
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG
Phenylalanine Phe F UUC UUU
Glycine Gly GGA GGC GGG GGU
G
Histidine His H CAC CAU
Isoleucine lie I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA cue CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA ccc CCG ecu
Glutamine Gin Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Trp w UGG
Tyrosine Tyr Y UAC UAU
[0111] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by
reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
[0112] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively based on hydrophilicity. U.S. Patent No. 4,554,101 (specifically incorporated herein in its entirety by express reference thereto), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
[0113] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.
[0114] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of ordinary skill in the art,
and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
ANTISENSE OLIGONUCLEOTIDES
[0115] In certain embodiments, the gene expression constructs of the invention, and the viral vectors comprising them will find utility in the delivery of one or more antisense oligonucleotides or polynucleotides for inhibiting the expression of a selected mammalian mRNA in a host cell that has been transformed with the construct.
[0116] In the art the letters, A, G, C, T, and U respectively indicate nucleotides in which the nucleoside is Adenosine (Ade), Guanosine (Gua), Cytidine (Cyt), Thymidine (Thy), and Uridine (Ura). As used in the specification and claims, compounds that are "antisense" to a particular PNA, DNA, or mRNA "sense" strand are nucleotide compounds that have a nucleoside sequence that is complementary to the sense strand. It will be understood by those skilled in the art that the present invention broadly includes oligonucleotide compounds that are capable of binding to the selected DNA or mRNA sense strand. It will also be understood that mRNA includes not only the ribonucleotide sequences encoding a protein, but also regions including the 5 '-untranslated region, the 3 '-untranslated region, the 5 '-cap region and the intron/exon junction regions.
[0117] The invention includes compounds that are not strictly antisense; the compounds of the invention also include those oligonucleotides that may have some bases that are not complementary to bases in the sense strand provided such compounds have sufficient binding affinity for the particular DNA or mRNA for which an inhibition of expression is desired. In addition, base modifications or the use of universal bases such as inosine in the oligonucleotides of the invention are contemplated within the scope of the subject invention.
[0118] The antisense compounds may have some or all of the phosphates in the nucleotides replaced by phosphorothioates (X = S) or methylphosphonates (X = CH3) or other C1-4 alkylphosphonates. The antisense compounds optionally may be further differentiated from native DNA by replacing one or both of the free hydroxy groups of the antisense molecule with C alkoxy groups (R = C1-4 alkoxy). As used herein, C1-4 alkyl means a branched or unbranched hydrocarbon having 1 to 4 carbon-atoms.
[0119] The disclosed antisense compounds also may be substituted at the 3'-and/or 5'-ends by a substituted-acridine derivative. As used herein, "substituted-acridine," means any acridine derivative capable of intercalating nucleotide strands such as DNA. Preferred substituted acridines are 2-methoxy-6-chloro-9-pentylaminoacridine, N-(6-chloro-2-methoxy acridinyl)-0-methoxydiisopropylamino-phosphinyl-3-aminopropanol, and N-(6-chloro-
2-methoxyacridinyl)-0-methoxydiisopropylamino-phosphinyl-5-aminopentanol. Other suitable acridine derivatives are readily apparent to persons skilled in the art. Additionally, as used herein "P(0)(0)-substituted acridine" means a phosphate covalently linked to a substitute acridine.
[0120] As used herein, the term "nucleotides" includes nucleotides in which the phosphate moiety is replaced by phosphorothioate or alkylphosphonate and the nucleotides may be substituted by substituted acridines.
[0121] In one embodiment, the antisense compounds of the invention differ from native DNA by the modification of the phosphodiester backbone to extend the life of the antisense molecule. For example, the phosphates can be replaced by phosphorothioates. The ends of the molecule may also be optimally substituted by an acridine derivative that intercalates nucleotide strands of DNA. PCT Intl. Pat. Appl. Publ. No. WO 98/13526 and U. S. Patent No. 5,849,902 (each of which is specifically incorporated herein in its entirety by express reference thereto) describe a method of preparing three component chimeric antisense compositions, and discuss many of the currently available methodologies for synthesis of substituted oligonucleotides having improved antisense characteristics and/or half-life.
[0122] The reaction scheme involves ^-tetrazole-catalyzed coupling of phosphoramidites to give phosphate intermediates that are subsequently reacted with sulfur in 2,6-lutidine to generate phosphate compounds. Oligonucleotide compounds are prepared by treating the phosphate compounds with thiophenoxide (1:2:2 thiophenol/triethylamine/tetrahydrofuran, room temperature, 1 hr). The reaction sequence is repeated until an oligonucleotide compound of the desired length has been prepared. The compounds are cleaved from the support by treating with ammonium hydroxide at room temperature for 1 hr and then are further deprotected by heating at about 50°C overnight to yield preferred antisense compounds.
[0123] Selection of antisense compositions specific for a given gene sequence is based upon analysis of the chosen target sequence and determination of secondary structure, Tm, binding energy, relative stability, and antisense compositions were selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell. Highly preferred target regions of the mRNA, are those that are at or near the AUG translation initiation codon, and those sequences that were substantially complementary to 5'-regions of the mRNA. These secondary structure analyses and target site selection considerations were performed using v.4 of the OLIGO primer analysis software (Rychlik and Rhodes, 1989) and the BLASTN 2.0.5 algorithm software (Altschul et al, 1997).
EXEMPLARY DEFINITIONS
[0124] In accordance with the present invention, polynucleotides, nucleic acid segments, nucleic acid sequences, and the like, include, but are not limited to, DNAs (including and not limited to genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.
[0125] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Dictionary of Biochemistry and Molecular Biology, (2nd Ed.) J. Stenesh (Ed.), Wiley-Interscience (1989); Dictionary of Microbiology and Molecular Biology (3rd Ed.), P. Singleton and D. Sainsbury (Eds.), Wiley-Interscience (2007); Chambers Dictionary of Science and Technology (2nd Ed.), P. Walker (Ed.), Chambers (2007); Glossary of Genetics (5th Ed.), R. Rieger et al. (Eds.), Springer- Verlag (1991); and The HarperCollins Dictionary of Biology, W.G. Hale and J.P. Margham, (Eds.), HarperCollins (1991).
[0126] Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, and compositions are described herein. For purposes of the present invention, the following terms are defined below for sake of clarity and ease of reference:
[0127] In accordance with long standing patent law convention, the words "a" and "an," when used in this application, including the claims, denote "one or more."
[0128] The terms "about" and "approximately" as used herein, are interchangeable, and should generally be understood to refer to a range of numbers around a given number, as well as to all numbers in a recited range of numbers (e.g., "about 5 to 15" means "about 5 to about 15" unless otherwise stated). Moreover, all numerical ranges herein should be understood to include each whole integer within the range.
[0129] As used herein, an "antigenic polypeptide" or an "immunogenic polypeptide" is a polypeptide which, when introduced into a vertebrate, reacts with the vertebrate's immune system molecules, i.e., is antigenic, and/or induces an immune response in the vertebrate, i.e., is immunogenic.
[0130] The term "biologically-functional equivalent" is well understood in the art, and is further defined in detail herein. Accordingly, sequences that have about 85% to about 90%; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99%; of
nucleotides that are identical or functionally-equivalent to one or more of the nucleotide sequences provided herein are particularly contemplated to be useful in the practice of the methods and compositions set forth in the instant application.
[0131] As used herein, the term "buffer" includes one or more compositions, or aqueous solutions thereof, that resist fluctuation in the pH when an acid or an alkali is added to the solution or composition that includes the buffer. This resistance to pH change is due to the buffering properties of such solutions, and may be a function of one or more specific compounds included in the composition. Thus, solutions or other compositions exhibiting buffering activity are referred to as buffers or buffer solutions. Buffers generally do not have an unlimited ability to maintain the pH of a solution or composition; rather, they are typically able to maintain the pH within certain ranges, for example from a pH of about 5 to 7.
[0132] As used herein, the term "carrier" is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert (s), or such like, or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal or acceptable for a therapeutic or diagnostic purpose, as applicable.
[0133] As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments that have been isolated away from, or purified free from, total genomic DNA of the particular species from which they are obtained. Included within the term "DNA segment," are DNA segments and smaller fragments of such segments, as well as recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
[0134] The term "effective amount," as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.
[0135] The term "for example" or "e.g. " as used herein, is used merely by way of example, without limitation intended, and should not be construed as referring only those items explicitly enumerated in the specification.
[0136] As used herein, a "heterologous" is defined in relation to a predetermined referenced nucleic acid sequence. For example, with respect to a structural gene sequence, a heterologous promoter is defined as a promoter which does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation. Likewise, a heterologous
gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements.
[0137] As used herein, "homologous" means, when referring to polynucleotides, sequences that have the same essential nucleotide sequence, despite arising from different origins. Typically, homologous nucleic acid sequences are derived from closely related genes or organisms possessing one or more substantially similar genomic sequences. By contrast, an "analogous" polynucleotide is one that shares the same function with a polynucleotide from a different species or organism, but may have a significantly different primary nucleotide sequence that encodes one or more proteins or polypeptides that accomplish similar functions or possess similar biological activity. Analogous polynucleotides may often be derived from two or more organisms that are not closely related (e.g., either genetically or phylogenetically).
[0138] As used herein, the term "homology" refers to a degree of complementarity between two or more polynucleotide or polypeptide sequences. The word "identity" may substitute for the word "homology" when a first nucleic acid or amino acid sequence has the exact same primary sequence as a second nucleic acid or amino acid sequence. Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods may be used to assess whether a given sequence is identical or homologous to another selected sequence.
[0139] The terms "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of ordinary skill) or by visual inspection.
[0140] As used herein, the phrase "in need of treatment" refers to a judgment made by a caregiver such as a physician or veterinarian that a patient requires (or will benefit in one or more ways) from treatment. Such judgment may made based on a variety of factors that are in the realm of a caregiver's expertise, and may include the knowledge that the patient is ill as the result of a disease state that is treatable by one or more compound or pharmaceutical compositions such as those set forth herein.
[0141] The phrases "isolated" or "biologically pure" refer to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state. Thus, isolated polynucleotides or polypeptides in accordance with the present disclosure
preferably do not contain materials normally associated with those polynucleotides or polypeptides in their natural, or in situ, environment.
[0142] As used herein, the term "kit" may be used to describe variations of the portable, self- contained enclosure that includes at least one set of reagents, components, or pharmaceutically- formulated compositions of the present invention. Optionally, such kit may include one or more sets of instructions for use of the enclosed compositions, such as, for example, in a laboratory or clinical application.
[0143] "Link" or "join" refers to any method known in the art for functionally connecting one or more proteins, peptides, nucleic acids, or polynucleotides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, electrostatic bonding, and the like.
[0144] The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of man in a laboratory is naturally- occurring. As used herein, laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally-occurring animals.
[0145] As used herein, the term "nucleic acid" includes one or more types of: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D- ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including abasic sites). The term "nucleic acid," as used herein, also includes polymers of ribonucleosides or deoxyribonucleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like. "Nucleic acids" include single- and double- stranded DNA, as well as single- and double- stranded RNA. Exemplary nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small temporal RNA (stRNA), and the like, and any combination thereof.
[0146] The term "operably linked," as used herein, refers to that the nucleic acid sequences being linked are typically contiguous, or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.
[0147] As used herein, the term "patient" (also interchangeably referred to as "recipient" "host" or "subject") refers to any host that can serve as a recipient for one or more of the vascular access devices as discussed herein. In certain aspects, the recipient will be a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being). In certain embodiments, a "patient" refers to any animal host, including but not limited to, human and non-human primates, avians, reptiles, amphibians, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, racines, vulpines, and the like, including, without limitation, domesticated livestock, herding or migratory animals or birds, exotics or zoological specimens, as well as companion animals, pets, and any animal under the care of a veterinary practitioner.
[0148] The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human, and in particular, when administered to the human eye. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or as suspensions. Alternatively, they may be prepared in solid form suitable for solution or suspension in liquid prior to injection.
[0149] As used herein, "pharmaceutically-acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include, but are not limited to, acid-addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic (embonic) acid, alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; salts formed with an organic cation formed from Ν,Ν'-dibenzylethylenediamine or ethylenediamine; and combinations thereof.
[0150] As used herein, the term "plasmid" or "vector" refers to a genetic construct that is composed of genetic material (i.e., nucleic acids). Typically, a plasmid or a vector contains an origin of replication that is functional in bacterial host cells, e.g., Escherichia coli, and selectable markers for detecting bacterial host cells including the plasmid. Plasmids and vectors of the present invention may include one or more genetic elements as described herein arranged such
that an inserted coding sequence can be transcribed and translated in a suitable expression cells. In addition, the plasmid or vector may include one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments that are obtained from or derived from one or more natural and/or artificial sources.
[0151] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and includes any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to "peptide," "dipeptide," "tripeptide," "protein," "enzyme," "amino acid chain," and "contiguous amino acid sequence" are all encompassed within the definition of a "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with, any of these terms. The term further includes polypeptides that have undergone one or more post-translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids. Conventional nomenclature exists in the art for polynucleotide and polypeptide structures.
[0152] For example, one-letter and three-letter abbreviations are widely employed to describe amino acids: Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; He), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys). Amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form may be substituted for L-amino acid residues provided the desired properties of the polypeptide be retained.
[0153] As used herein, the terms "prevent," "preventing," "prevention," "suppress," "suppressing," and "suppression" as used herein refer to administering a compound either alone or as contained in a pharmaceutical composition prior to the onset of clinical symptoms of a disease state so as to prevent any symptom, aspect or characteristic of the disease state. Such preventing and suppressing need not be absolute to be deemed medically useful.
[0154] "Protein" is used herein interchangeably with "peptide" and "polypeptide," and includes both peptides and polypeptides produced synthetically, recombinantly, or in vitro and peptides and polypeptides expressed in vivo after nucleic acid sequences are administered into a host animal or human subject. The term "polypeptide" is preferably intended to refer to any amino acid chain length, including those of short peptides from about two to about 20 amino acid residues in length, oligopeptides from about 10 to about 100 amino acid residues in length, and
longer polypeptides including from about 100 amino acid residues or more in length. Furthermore, the term is also intended to include enzymes, i.e., functional biomolecules including at least one amino acid polymer. Polypeptides and proteins of the present invention also include polypeptides and proteins that are or have been post-translationally-modified, and include any sugar or other derivative(s) or conjugate(s) added to the backbone amino acid chain.
[0155] "Purified," as used herein, means separated from many other compounds or entities. A compound or entity may be partially purified, substantially purified, or pure. A compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e. , is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure. A partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the material with which it is naturally found, e.g., cellular material such as cellular proteins and/or nucleic acids.
[0156] The term "recombinant" indicates that the material (e.g., a polynucleotide or a polypeptide) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within or removed from, its natural environment, or native state. Specifically, e.g., a promoter sequence is "recombinant" when it is produced by the expression of a nucleic acid segment engineered by the hand of man. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis; a "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein which is produced by expression of a recombinant nucleic acid; and a "recombinant virus," e.g., a recombinant AAV virus, is produced by the expression of a recombinant nucleic acid.
[0157] The term "regulatory element," as used herein, refers to a region or regions of a nucleic acid sequence that regulates transcription. Exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and such like.
[0158] The term "RNA segment" refers to an RNA molecule that has been isolated free of total cellular RNA of a particular species. Therefore, RNA segments can refer to one or more RNA segments (either of native or synthetic origin) that have been isolated away from, or purified free from, other RNAs. Included within the term "RNA segment," are RNA segments and smaller fragments of such segments.
[0159] The term "a sequence essentially as set forth in SEQ ID NO:X" means that the sequence substantially corresponds to a portion of SEQ ID NO:X and has relatively few
nucleotides (or amino acids in the case of polypeptide sequences) that are not identical to, or a biologically functional equivalent of, the nucleotides (or amino acids) of SEQ ID NO:X. The term "biologically functional equivalent" is well understood in the art, and is further defined in detail herein. Accordingly, sequences that have about 85% to about 90%; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99%; of nucleotides that are identical or functionally equivalent to one or more of the nucleotide sequences provided herein are particularly contemplated to be useful in the practice of the invention.
[0160] Suitable standard hybridization conditions for nucleic acids for use in the present invention include, for example, hybridization in 50% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 μg/mL of denatured salmon sperm DNA at 42°C for 16 hr followed by 1 hr sequential washes with O.lx SSC, 0.1% SDS solution at 60°C to remove the desired amount of background signal. Lower stringency hybridization conditions for the present invention include, for example, hybridization in 35% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS and 100 μg/mL denatured salmon sperm DNA or E. coli DNA at 42°C for 16 hr followed by sequential washes with 0.8x SSC, 0.1% SDS at 55°C. Those of ordinary skill in the art will recognize that such hybridization conditions can be readily adjusted to obtain the desired level of stringency for a particular application.
[0161] As used herein, the term "structural gene" is intended to generally describe a polynucleotide, such as a gene, that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.
[0162] The term "subject," as used herein, describes an organism, including mammals such as primates, to which treatment with the compositions according to the present invention can be provided. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, apes; chimpanzees; orangutans; humans; monkeys; domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.
[0163] The term "substantially complementary," when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to all or a portion of the selected sequence, and thus will specifically bind to a portion of an mRNA encoding the selected sequence. As such, typically the sequences will be highly complementary to the mRNA "target" sequence, and will have no more than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 or so base mismatches throughout the complementary portion of the sequence. In many instances, it may be desirable for the sequences to be exact matches, i.e., be completely complementary to the
sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch. As such, highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product.
[0164] Substantially complementary nucleic acid sequences will be greater than about 80 percent complementary (or "% exact-match") to a corresponding nucleic acid target sequence to which the nucleic acid specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds. In certain aspects, as described above, it will be desirable to have even more substantially complementary nucleic acid sequences for use in the practice of the invention, and in such instances, the nucleic acid sequences will be greater than about 90 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and even up to and including about 96%, about 97%, about 98%, about 99%, and even about 100% exact match complementary to all or a portion of the target sequence to which the designed nucleic acid specifically binds.
[0165] Percent similarity or percent complementary of any of the disclosed nucleic acid sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
[0166] Naturally, the present invention also encompasses nucleic acid segments that are complementary, essentially complementary, and/or substantially complementary to at least one or more of the specific nucleotide sequences specifically set forth herein. Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to one
or more of the specific nucleic acid segments disclosed herein under relatively stringent conditions such as those described immediately above.
[0167] As used herein, the term "substantially free" or "essentially free" in connection with the amount of a component preferably refers to a composition that contains less than about 10 weight percent, preferably less than about 5 weight percent, and more preferably less than about 1 weight percent of a compound. In preferred embodiments, these terms refer to less than about 0.5 weight percent, less than about 0.1 weight percent, or less than about 0.01 weight percent.
[0168] Probes and primers for use in the present invention may be of any suitable length. By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, etc. , an algorithm defining all probes or primers contained within a given sequence can be proposed:
[0169] n to n + y, where n is an integer from 1 to the last number of the sequence and y is the length of the probe or primer minus one, where n + y does not exceed the last number of the sequence. Thus, for a 25-basepair probe or primer (i.e., a "25-mer"), the collection of probes or primers correspond to bases 1 to 25, bases 2 to 26, bases 3 to 27, bases 4 to 28, and so on over the entire length of the sequence. Similarly, for a 35-basepair probe or primer (i.e., a "35-mer), exemplary primer or probe sequence include, without limitation, sequences corresponding to bases 1 to 35, bases 2 to 36, bases 3 to 37, bases 4 to 38, and so on over the entire length of the sequence. Likewise, for 40-mers, such probes or primers may correspond to the nucleotides from the first basepair to bp 40, from the second bp of the sequence to bp 41, from the third bp to bp 42, and so forth, while for 50-mers, such probes or primers may correspond to a nucleotide sequence extending from bp 1 to bp 50, from bp 2 to bp 51, from bp 3 to bp 52, from bp 4 to bp 53, and so forth.
[0170] The term "substantially corresponds to," "substantially homologous," or "substantial identity," as used herein, denote a characteristic of a nucleic acid or an amino acid sequence, wherein a selected nucleic acid or amino acid sequence has at least about 70 or about 75 percent sequence identity as compared to a selected reference nucleic acid or amino acid sequence. More typically, the selected sequence and the reference sequence will have at least about 76, 77, 78, 79, 80, 81, 82, 83, 84 or even 85 percent sequence identity, and more preferably, at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity. More preferably still, highly homologous sequences often share greater than at least about 96, 97, 98, or 99 percent sequence identity between the selected sequence and the reference sequence to which it was compared.
[0171] The term "therapeutically-practical period" means the period of time that is necessary for one or more active agents to be therapeutically effective. The term "therapeutically-effective"
refers to reduction in severity and/or frequency of one or more symptoms, elimination of one or more symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and the improvement or a remediation of damage.
[0172] A "therapeutic agent" may be any physiologically or pharmacologically active substance that may produce a desired biological effect in a targeted site in a subject. The therapeutic agent may be a chemotherapeutic agent, an immunosuppressive agent, a cytokine, a cytotoxic agent, a nucleolytic compound, a radioactive isotope, a receptor, and a pro-drug activating enzyme, which may be naturally occurring, produced by synthetic or recombinant methods, or a combination thereof. Drugs that are affected by classical multidrug resistance, such as vinca alkaloids (e.g., vinblastine and vincristine), the anthracyclines (e.g., doxorubicin and daunorubicin), RNA transcription inhibitors (e.g., actinomycin-D) and microtubule stabilizing drugs (e.g., paclitaxel) may have particular utility as the therapeutic agent. Cytokines may be also used as the therapeutic agent. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. A cancer chemotherapy agent may be a preferred therapeutic agent. For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician's Desk Reference and Hardman and Limbird (2001).
[0173] As used herein, a "transcription factor recognition site" and a "transcription factor binding site" refer to a polynucleotide sequence(s) or sequence motif(s), which are identified as being sites for the sequence-specific interaction of one or more transcription factors, frequently taking the form of direct protein-DNA binding. Typically, transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, and the like, and/or can be predicted based on known consensus sequence motifs, or by other methods known to those of ordinary skill in the art.
[0174] "Transcriptional regulatory element" refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences. A transcriptional regulatory element can, for example, comprise one or more promoters, one or more response elements, one or more negative regulatory elements, and/or one or more enhancers.
[0175] "Transcriptional unit" refers to a polynucleotide sequence that comprises at least a first structural gene operably linked to at least a first ds-acting promoter sequence and optionally linked operably to one or more other cw-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequences, and at least a first distal regulatory element as may be required for the appropriate tissue-specific and developmental transcription of the structural gene sequence operably positioned under the control of the promoter and/or enhancer elements, as
well as any additional cis- sequences that are necessary for efficient transcription and translation (e.g., polyadenylation site(s), mRNA stability controlling sequence(s), etc.
[0176] As used herein, the term "transformation" is intended to generally describe a process of introducing an exogenous polynucleotide sequence (e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule) into a host cell or protoplast in which the exogenous polynucleotide is incorporated into at least a first chromosome or is capable of autonomous replication within the transformed host cell. Transfection, electroporation, and "naked" nucleic acid uptake all represent examples of techniques used to transform a host cell with one or more polynucleotides.
[0177] As used herein, the term "transformed cell" is intended to mean a host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into that cell.
[0178] As used herein, the terms "treat," "treating," and "treatment" refer to the administration of one or more compounds (either alone or as contained in one or more pharmaceutical compositions) after the onset of clinical symptoms of a disease state so as to reduce, or eliminate any symptom, aspect or characteristic of the disease state. Such treating need not be absolute to be deemed medically useful. As such, the terms "treatment," "treat," "treated," or "treating" may refer to therapy, or to the amelioration or the reduction, in the extent or severity of disease, of one or more symptom thereof, whether before or after its development afflicts a patient. "Treating" may include any administration or application of a compound or composition of the invention to a subject for purposes such as curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of a disease, disorder, or condition or one or more symptoms or manifestations of a disease, disorder, or condition. In certain aspects, the compositions of the present invention may also be administered prophylactically, i.e. , before development of any symptom or manifestation of the condition, where such prophylaxis is warranted. Typically, in such cases, the subject will be one that has been diagnosed for being "at risk" of developing such a disease or disorder, either as a result of familial history, medical record, or the completion of one or more diagnostic or prognostic tests indicative of a propensity for subsequently developing such a disease or disorder.
[0179] The tern "vector," as used herein, refers to a nucleic acid molecule (typically comprised of DNA) capable of replication in a host cell and/or to which another nucleic acid segment can be operatively linked so as to bring about replication of the attached segment. A plasmid, cosmid, or a virus is an exemplary vector.
[0180] In certain embodiments, it will be advantageous to employ one or more nucleic acid segments of the present invention in combination with an appropriate detectable marker (i.e., a "label,"), such as in the case of employing labeled polynucleotide probes in determining the presence of a given target sequence in a hybridization assay. A wide variety of appropriate indicator compounds and compositions are known in the art for labeling oligonucleotide probes, including, without limitation, fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, etc., which are capable of being detected in a suitable assay. In particular embodiments, one may also employ one or more fluorescent labels or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally less- desirable reagents. In the case of enzyme tags, colorimetric, chromogenic, or fluorogenic indicator substrates are known that can be employed to provide a method for detecting the sample that is visible to the human eye, or by analytical methods such as scintigraphy, fluorimetry, spectrophotometry, and the like, to identify specific hybridization with samples containing one or more complementary or substantially complementary nucleic acid sequences. In the case of so- called "multiplexing" assays, where two or more labeled probes are detected either simultaneously or sequentially, it may be desirable to label a first oligonucleotide probe with a first label having a first detection property or parameter (for example, an emission and/or excitation spectral maximum), which also labeled a second oligonucleotide probe with a second label having a second detection property or parameter that is different (i.e., discreet or discernible from the first label. The use of multiplexing assays, particularly in the context of genetic amplification/detection protocols are well-known to those of ordinary skill in the molecular genetic arts.
[0181] The section headings used throughout are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application (including, but not limited to, patents, patent applications, articles, books, and treatises) are expressly incorporated herein in their entirety by express reference thereto. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.
EXAMPLES
[0182] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the
practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1 - SUPPRESSION OF TUMOR METASTASIS BY THE RIBONUCLEASE REGNASE-1
[0183] A microfluidic chip approach has been utilized to examine behaviors of metastatic (MDA-MB-231, SUM-159) vs. non-metastatic (MDA-MB-468, MCF7) breast cancer cell lines (FIG. 7 A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E-1, FIG. 7E-2, FIG. 7E-3, FIG. 7E-4, and FIG. 7E- 5). MDA-MB-231 and SUM-159 cells belong to basal B type and have higher metastatic ability as compared to MCF7 and MDA-MB-468 cells. The microfluidic chips were fabricated in a way that cancer cells were precisely seeded in the microchips in desired numbers (2-5 cells per chamber) (FIG. 7A and FIG. 16A), and as a result, cells could be monitored in real time. As shown in FIG. 7B-FIG. 7E-5, striking differences in the clustering ability were observed between metastatic and non-metastatic cancer cells. As compared to non-metastatic MCF7 and MDA-MB- 468 cancer cells, which are randomly distributed in the microchips at all times, the highly metastatic breast cancer cells MDA-MB-231 and SUM-159 readily clustered together (FIG. 7B), and within 2 hrs, more than 80% individual MDA-MB-231 and SUM-159 cells form cell clusters. In contrast, less than 30% single MCF7 and MDA-MB-468 cells formed clusters (FIG. 7C). More than 50% metastatic cells formed clusters within 1 hr (FIG. 7D). Two representative wells containing 4 cells for each cell type further indicated the apparent clustering difference between metastatic and non-metastatic cells (FIG. 7E-1-FIG. 7E-5). B 16 melanoma cells, which are highly metastatic, were also included as a control; these cells exhibited strong tendency in forming cell clusters (FIG. 12B, FIG. 12C, and FIG. 12D). Taken together, these results demonstrated that the clustering ability of cancer cells is a unique feature, and one that is closely associated with their metastatic potential.
[0184] Potential mechanisms were explored that could explain the differential behaviors of metastatic and non-metastatic breast cancer cells. Regnase-1 expression was quantitated at both the transcriptional (mRNA) and the translational (protein) levels. Functionally, Regnase-1 is a cytosolic ribonuclease that degrades a set of mRNAs, and is initially found in innate and adaptive immune cells. Regnase-1, however, is also expressed in other tissues and cell types with unknown function.
[0185] In this Example, it is shown that at the transcriptional level, Regnase-1 -specific mRNA was expressed by all of the cancer cells studied (i.e., MDA-MB-231, and SUM-159, MCF7, MDA-MB-468) (FIG. 8A). The metastatic cancer cells, MDA-MB-231 and SUM-159, however, showed much higher levels of Regnase-1 -specific mRNA (FIG. 8B). Interestingly, immunoblot analyses revealed that in the highly-metastatic MDA-MB-231 and SUM-159 cancer cells, Regnase-1 was undetectable at the protein level (FIG. 8B). In contrast, Regnase-1 protein was readily detected in the non-metastatic cell lines, MCF7 and MDA-MB-468. In these studies, 293T cells were included as controls, wherein Regnase-1 was expressed at high levels (FIG. 8B).
[0186] Based on these results, it was reasoned that Regnase-1 was likely post-translationally regulated. Indeed, the proteasome inhibitor MG132 could rescue the protein level of Regnase-1 in SUM-159 and MDA-MB-231 cells (FIG. 8C). None of the other inhibitors that were examined, however, rescued Regnase-1, including the autophagy inhibitor-Bafilomycin Al, MALT1 inhibitor MI2 and Mepazine, IKK inhibitor IKKII, and TAK1 inhibitor 5Z7 (FIG. 8C). MG132 had no effect on Regnase-1 expression levels in MCF7 cells (FIG. 8C). The effect of MG132 on the level of Regnase-1 protein was shown to be dose-dependent (FIG. 8C), which indicated that highly-metastatic cells could introduce a signal to trigger the degradation of Regnase-1.
[0187] To further understand the mechanism, MCF7 cells were co-cultured with differing numbers of MDA-MB-231 or SUM-159 cells. Co-cultures with MDA-MB-231 or SUM-159 cells could not trigger the degradation of Regnase-1 in MCF7 cells (FIG. 8D). Similar results were also observed in MDA-MB-468 cells (FIG. 8D), which suggested that the post-translational inactivation of Regnase-1 was intrinsic in highly-metastatic cancer cells, but not due to cytokine(s) or the microenvironment. To further confirm that Regnase-1 was also inactivated in breast tissue, the protein level of Regnase-1 was measured in breast cancer samples. The protein level of Regnase-1 in breast cancer with metastasis was significantly lower than it was in breast cancers without metastasis (FIG. 8E and FIG. 8F). Together, these data suggested that Regnase-1 activity was regulated at the post-translational level, and was inactivated in metastatic cancer cells through proteasome-mediated degradation.
[0188] Next, the question of whether Regnase-1 was involved in cell clustering and cancer metastasis was explored. To that end, a series of Regnase-1 mutants were constructed, including a mutant that contained an Aspartic Acid to Alanine substitution (Asp— >Ala) at amino acid residue 225 (D225A), which lacked Regnase enzymatic activity. Wild-type Regnase-1 or the D225A mutant was stably introduced into MCF7, MDA-MB-231, SUM-159, and B 16 cells. Re- introduction of Regnase-1 into these cells, however, did not change cell morphology, cell proliferation, or affect attachment-dependent colony formation in vitro (FIG. 13A, FIG. 13B, and
FIG. 13C). However, re-introduction of Regnase-1 could inhibit cell attachment independent colony formation, cell migration and cell invasion (FIG. 14A, FIG. 14B, and FIG. 14C), but this effect was not solely dependent on the enzymatic activity of Regnase-1, and not solely observed in metastatic cells (FIG. 14A, FIG. 14B, and FIG. 14C). These results suggested that the enzymatic activity-dependent and metastatic cell-specific functions of Regnase-1 could not be explained by conventional assays.
[0189] Thus, it was next determined whether Regnase-1 could inhibit cell clustering in an established microchip-based assay. Surprisingly, Regnase-1 completely inhibited metastatic cell clustering (FIG. 9A and FIG. 9B). Since cell clustering ability is strongly associated with metastatic potential, the function of Regnase-1 in cancer metastasis in vivo was also examined. SUM- 159 cells that expressed either wild-type Regnase-1, or the enzymatically-inactive mutant, D225A, were injected into Rag2_/~ rC_/~ knockout mice via the tail vein, and the resulting tumor nodules in the lungs were examined. Surprisingly, stable expression of wild-type Regnase-1 completely inhibited lung metastasis, but the D225A mutant protein could not (FIG. 9C). While 30 visible lung metastases were observed in mice injected with SUM-159 cells transduced with control vector or D225A mutant, no visible lung metastases were observed in mice injected with SUM-159 cells that were stably transduced with wild-type Regnase-1 (FIG. 9C and FIG. 9D).
[0190] Lung metastases were confirmed in mice injected with Regnase-1 -transduced SUM-159 cells by H&E staining (FIG. 9C). Expression of Regnase-1 in B 16-F10 cells almost completely inhibited cell clustering (FIG. 9E and FIG. 9F). Next, the in vivo metastasis of B16-F10 cells that stably expressed either wild- type or mutant Regnase-1 was examined. Stable expression of the wild-type Regnase-1 in B 16 cells completely inhibited lung metastases in B6 mice (FIG. 9G and FIG. 9H). Conversely, mice injected with B 16 cells transduced with either the vector alone or the enzymatically-inactive mutant, D225A, showed extensive metastatic nodules in the lungs (-150 metastases). Overexpression of wild-type Regnase-1 in B16 cells, however, completely prevented their in vivo metastasis in lung (FIG. 9G and FIG. 9H), which was confirmed by H&E staining (FIG. 91). B6 mice transferred with B 16-Vector or B16-D225A mutant cells all died within three weeks (FIG. 9J). B6 mice transformedwith B16 cells transduced with wild-type Regnase-1, however, were all alive 4 weeks later (FIG. 9J). Similar results were obtained using the immune deficient Ragl KO mice injected with B 16 cells (FIG. 16A and FIG. 16B). It was shown that B 16-F10 cells could form primary tumors following subcutaneous injection (FIG. 17A and FIG. 17B), and although overexpression of Regnase-1 could almost completely inhibit lung metastasis, it only slightly inhibited primary tumor growth. Furthermore, the inhibitory effect was dependent upon the ribonuclease activity of Regnase-1 protein, as overexpression of wild-type or
the D225A mutant showed similar effects on primary tumor growth (FIG. 11 A). Collectively, these data demonstrated that Regnase-1, with its intact ribonuclease activity, was critically important in suppressing both cancer metastasis in vivo and cell clustering in vitro.
[0191] To understand the molecular mechanisms by which Regnase-1 suppresses cancer metastasis by controlling cell clustering, mRNA microarray analyses were performed on the B 16 vector, B 16-Regl-WT and B16-Regl-D225A cells. Comparison of genes known to be associated with tumor cell clustering with the results obtained from array analysis identified two SI 00 A family members, S100A4 and S100A6, that were strongly down-regulated in B 16-Regl-WT cells. These results were confirmed by real-time RT-PCR (FIG. 10A). Expression of S100A6, but not S100A4, was much higher in MDA-MB-231 and SUM- 159 cells than that in MCF7 and MDA- MB-468 cells - both at the mRNA and protein level (FIG. 10B, FIG. IOC and FIG. 18A). That expression pattern was inversely correlated with the level of Regnase-1 protein in these cells (FIG. 8B and FIG. 10B). Furthermore, overexpression of wild-type Regnase-1, but not vector alone or the D225A mutant, inhibited S100A6 expression in both MDA-MB-231 and SUM- 159 cells (FIG. 10D and FIG. 10E). Overexpression of wild-type Regnase-1 in MDA-MB-231 and SUM-159 cells, however, had a minimal effect on the level of S100A4 (FIG. 18B-1, FIG. 18B-2, and FIG. 18B-3). Furthermore, transient expression of wild-type Regnase-1 in 293T cells could inhibit S100A6, but not S100A4 expression (FIG. 18C-1 and FIG. 18C-2). The protein level of S100A6, but not S100A4, was negatively correlated with the protein levels of Regnase-1 in MCF7, MDA-MB-231, SUM-159 and MDA-MB-468 cells (FIG. 8B and FIG. IOC).
[0192] It is known that Regnase-1 could recognize the "stem-loop" structure at 3'UTR to degrade targeting mRNAs. By comparing the 3'UTR sequence of S100A6 between human and mouse, a conserved RNA sequence was found (FIG. 10F). This conserved RNA sequence could form a typical "stem- loop" structure (FIG. 10G), but no similar structure was found in the 3'UTR of S100A4 mRNA. To further confirm that S100A6 is a direct mRNA target of Regnase-1, the 3'UTR sequence was cloned into the end of GFP coding sequencing (CDS). Overexpression Regnase-1 wild-type, but not vector alone or the D225A mutant, almost completely inhibited the expression of GFP encoded by GFP-CDS fused with 3'UTR from S100A6 (FIG. lOH-1, FIG. 10H-2, and FIG. 10H-3). However, overexpression of wild-type Regnase-1 had minimal effects on the expression of GFP encoded by GFP CDS or GFP CDS fused with the 3'UTR of S100A4 (FIG. 19A-1, FIG. 19A-2, FIG. 19A-3, FIG. 19B-1, FIG. 19B-2, and FIG. 19B-3). These data suggested that S100A6 was a direct target of Regnase-1 and as such, has an important role in the control of cell clustering and cancer metastasis.
[0193] To examine the relationship between S100A6 and Regnase-1 in cell clustering and cancer metastasis, the following study was undertaken. First, it was determined whether whether stable expression of S100A6 could rescue the inhibitory function of Regnase-1 on cell clustering. To do this, the Regnase-1 WT gene was stably introduced into SUM- 159 cell by puromycin selection. Then the S100A6 gene was further introduced into puromycin-resistant SUM- 159 cells by G418 selection. In this manner, it was determined that overexpression of S100A6 (but not S100A4) in SUM-159-Regl-WT cells could partially reverse the cell-clustering ability (FIG. 11A and FIG. 11B). Overexpression of S100A6 also could partially rescue the metastatic ability of SUM-159-Regl-WT cells in vivo (FIG. 11C and FIG. 11D). By using B 16 cell Regl-WT cells, similar results were obtained showing that S100A6 (but not S100A4) could reverse the cell- clustering ability in vitro (FIG. HE and FIG. 11F). Overexpression of S100A6 also could partially rescue the metastatic ability in vivo (FIG. 11G and FIG. 11H). The number of metastatic foci in mice transferred with B 16-Regl-WT-S100A6 cells is significantly higher than it in mice transferred with B 16-Regl-WT- vector or B 16-Regl-WT-S100A4 cells (FIG. 11G). B6 mice injected with B16 cells will normally die within 3 weeks (FIG. 11J). However, mice injected with B 16 cells overexpressing wild- type Regnase-1 survived for more than 7 weeks. Additional expression of S100A6 in B 16-Regl-WT cells rendered the cells aggressive again, and upon in vivo injection, all recipient mice died within 5 weeks (FIG. 11 J). These results suggested that S100A6 was one of the targets for Regnase-1, and as such, it plays a critical role in cell clustering, and consequentially, in cancer metastasis.
[0194] In summary, these data provide evidence that Regnase-1 is a critical checkpoint in suppression of cancer metastasis through its regulation of the clustering ability of metastatic cancer cells. The data also suggest that the calcium binding protein S100A6 is a key mediator of cancer cell clustering and is a direct target of Regnase-1 activity. While extensive efforts have been devoted to the identification of oncogenes and genetic mechanisms in cancers, these results demonstrate the importance of metastatic-specific post-translational mechanisms, especially the proteasome-mediated degradation of Regnase-1 in metastatic cancer cells in promoting cancer metastasis. MATERIALS AND METHODS
[0195] Cell Lines, Plasmids, Reagents and Mice. Wild-type C57BL/6 mice, Ragl_/~ and Nu(NCr)-Foxnl mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Rag2_/" rc_/" mice were obtained from Taconic Biosciences, Inc. (Hudson, NY, USA). The human embryonic kidney 293T cells, human breast cancer MCF7, SUM-159, MDA-MB-231, and MDA-
MB-468 cells, as well as mouse B 16-F10 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). cDNA encoding Regnase-1 was purchased from GE Healthcare Dharmacon, Inc. (Lafayette, CO, USA). A Regnase-1 -specific antibody (MAB7875) used in Western hybridization analyses was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). The S100A4- and SI 00 A6- specific antibodies (ab27957 and abl81975, respectively) were purchased from Abeam, PLC (Cambridge, MA, USA). The anti-FLAG antibody, F1804, and the β-actin-specific antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-C-Myc antibody, SC-40, was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-mouse (7076), and anti-rabbit (7074) secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).
[0196] The breast cancer tissue array (BC081120a) was purchased from US BioMax, Inc. (Rockville, MD, USA). MG132 was purchased from EMD Millipore (474780) (Darmstadt, GERMANY). Bafilomycin Al (tlrl-bafl) was purchased from Invivogen (San Diego, CA, USA). MI2 (4848) was purchased from Tocris Bioscience (Bristol, UNITED KINGDOM). The TAK1 inhibitor (5Z)-7-Oxozeaenol (499610) was purchased from EMD Millipore. Mepazine hydrochloride 500500 was purchased from Calbiochem (San Diego, CA, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). SU-8 2100 photoresist was purchased from Rohm and Haas Electronic Materials, LLC (Marlborough, MA, USA). PDMS (GE 615 RTV) was purchased from Thermo Fisher Scientific. Trimethylchlorosilane (TMCS) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
[0197] The full-length Regnase-1 gene was amplified by PCR and ligated into pMYs-Puro vectors using the restriction enzymes Bglll and EcoRI. The ribonuclease-inactive Regnase-1 (Reg ) mutant, D225A, was generated through two-step PCR. The expression vectors encoding S100A4 and S100A6 were purchased from OriGene Technologies (Rockville, MD, USA). The coding sequence (CDS) of green-fluorescent protein (GFP) was amplified by PCR and ligated into pMYs-Puro vector using the restriction enzymes BamHI and EcoRI. The 3'UTRs of the S100A6 and S100A4 genes were amplified by PCR and ligated to the end of the GFP CDS by EcoRI and Xbal digestion.
[0198] To establish the stable Regnase-1 expression cell lines, plasmids encoding either wild- type (WT) or the D225A Reg" mutant were transfected into MCF7, MDA-MB-231, B 16-F10 and SUM- 159 cells. The transfected cells were selected by puromycin for two weeks. After puromycin selection, the Regnase-1 WT cells were further transfected with plasmids encoding S100A4 or S100A6. These transfected cells were further selected under G418 for two weeks.
[0199] Real-Time PCR. Real-time PCR. Total cellular RNA was isolated by using RNeasy mini kit (Qiagen, Inc., Chatsworth, CA, USA), and cDNA was prepared using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturers' protocols. One microgram of RNA was used for cDNA synthesis. Quantitative real-time PCR was performed using the following primers that are specific for ZC3H12A:
Forward Primer: 5'-CGTCAATGACAAGTTTATGC-3' (SEQ ID NO: 1)
Reverse Primer: 5'-ATTTCCTTCCATAGGGACAC-3' (SEQ ID NO:2)
Data were normalized to the housekeeping gene, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), and the relative abundance of transcripts was calculated using Q models.
[0200] Western blotting. Cells were washed with ice cold PBS and lysed using cold Pierce™ IP Lysis Buffer 87787. After measuring the protein concentrations using Bradford reagent (Bio- Rad, Hercules, CA, USA), whole cell lysates were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Protein was transferred to poly(vinylidene) fluoride (PVDF) membrane (Bio-Rad), and then probed with the indicated primary antibodies overnight at 4°C. The membrane was further incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies against mouse for 1 hr at room temperature (25°C). The expression levels of interested proteins were visualized using the ECL-Plus Western detection system (GE Health Care, Buckinghamshire, UNITED KINGDOM).
[0201] In Vitro Scratch Assay. Selected cells were suspended through twice times of PBS washing and trypsin digestion. The total cell number was determined by using a hemocytometer. Cells were plated onto 6-well dishes to create a confluent monolayer. To allow cells to adhere and spread on the dish completely, the dishes were incubated overnight at 37°C. The cell monolayer was scraped into a straight line to create a "scratch" with a p200 pipet tip. The debris was removed and the edge of the scratch was smoothed by washing the cells once with growth medium. The scraped dishes were allowed to grow for another day. Then, the images were captured by a phase-contrast microscope.
[0202] Trans-Well Migration Assay. The migration and invasion ability of interested cells was measured by using a 24-well Transwell system (Corning, Inc., Corning, NY, USA). Briefly, selected cells were harvested and counted using a hemocytometer. Then, cells were suspended in culture medium and 2 x 105 cells were added into the upper chamber of the trans- well insert. The same culture medium was added into the lower chamber. Cells were incubated at 37°C for 24 hrs. The non-migrated cells in the upper chamber were removed by wiping with a cotton swab. Migrated cells were then fixed with 4% formaldehyde in PBS, and stained with 2% crystal violet
in 2% ethanol. The migrated cells, which were on the lower surface of membrane, were counted using light microscopy.
[0203] CFSE Proliferation Assays. Established stable cell lines were seeded into 12-well plates and further incubate at 37°C overnight. Cells were washed twice with pre-warmed PBS and incubated with CFSE dye at a final concentration of 5 μΜ. After 20 min, the remaining culture medium was washed twice to remove excess CFSE dye. Labeled cells were allowed to grow for several other days. At different days, cells were harvested and analyzed by flow cytometry.
[0204] Cell clustering assay using microfluidic chips. Microstructure patterns were first designed in AutoCAD software and fabricated using standard photolithography and molding processes. The masters were prepared by spin coating SU-8 2100 negative photoresist onto a silicon wafer and UV crosslinking for 30 min. Subsequently, the designed pattern was developed using SU-8 developer (MicroChem Corp., Westborough, MA, USA) and cleaned with isopropyl alcohol under nitrogen gas. The silicon masters were baked at 150°C for 30 min and treated with the anti-adhesive agent, TMCS, via vapor reaction for 4 hr. Next, the structure on the silicon wafer was used to fabricate the PDMS layer. The mold and PDMS layers were then baked at 80°C for 4 hr, and the cured PDMS was cut and peeled off. After treatment with oxygen plasma, the PDMS layer was reacted immediately with PEG5000 for 30 min at 150°C. Finally, the device was clean with PBS. Suspended cells in the concentration of 1 x 106/mL were dropped into a microfluidic chip. Chip channels containing 2-5 cells were monitored by camera to determine their clustering ability.
[0205] Experimental Metastasis. Cells at 70% confluent were washed by PBS for two times. Cells were detached by 0.05% trypsin, and the trypsin activity was quenched by FBS containing medium. The cell number was counted by using a hemocytometer. Cells were further washed in PBS for additional two times and then suspended in PBS at a final concentration of 5 x 106/mL. The large cell aggregates were excluded by filter the cell solution through a Falcon 70 μιη cell strainer. The cells were injected into mice via the tail vein. For B 16-F10 cells, 0.5 x 106 cells in 100 μΐ. cell solution were injected into C57B6 mice or Ragl_/". For SUM-159 cells, 1 x 106 cells in 200 μL· cell solution were injected into the Rag2_/~ rc_/~ double-knockout or Nu(NCr)-Foxnl mice. Typically after two weeks, the mice that received B 16-F10 cells were sacrificed and dissected. Mice that received SUM-159 B 16-Regnase-1-WT-S100A6 were sacrificed and dissected at 4 weeks. Mice that received SUM-159-Regnase-l WT-S100A6 were sacrificed and dissected at 8 weeks. Images of lungs with metastasis were recorded, and the extent of lung metastasis was observed under a dissecting microscope. The number of detectable metastases on
both sides were counted, with the number of lung metastases on all the four lobes added together for the total number of lung metastases.
[0206] Subcutaneous Tumor Formation. Cell preparation was performed as the same as performed at the experimental metastasis. Subcutaneous injections of 1 x 106 cells were performed into each flank and the interscapular region of each mouse. Tumor formation was evaluated regularly by palpation of injection sites. When tumor reached the designated diameters, mice were sacrificed and subcutaneous tumors were dissected for imaging.
[0207] Immunohistochemistry. For antigen retrieval, the breast cancer tissue array was heated in citrate buffer, pH 6.0 for 30 min at 93 °C. The tissue sections were de-waxed. The tissue array was incubated with 1/200 mouse anti-Regnase-1 antibody at 4°C overnight. After rinsing three times in IX PBS for 5 min each, sections were incubated with anti-mouse immunoglobulin for 30 min, then with horseradish peroxidase- strep tavidin complex for 30 min. The bound antibody complexes were visualized as a brown stain by incubating with diaminobenzidine. The lung tissue with metastasis was also fixed and stained with H&E. All sections were viewed and captured on a Nikon Eclipse E600 microscope.
EXAMPLE 2 -- REGNASE-1 COGNATES AND ZC3H12A-LIKE PROTEINS
[0208] The discovery of Regnase-1 in cancer metastasis opens a new avenue of investigation. To dissect key mechanisms regarding the regulation and function of Regnase-1, as well as potential targets of Regnase-1 in tumor cells, whole genomic shRNA and CRISP/Cas9 techniques may be employed to screen molecules that cause instability of Regnase- 1 post-translationally. To identify enzymatic targets of Regnase-1, mutagenesis techniques may also be employed to analyze functional fragments of Regnase-1, as well as the effects of modification of specific amino acids. RNA sequencing and array techniques may be used to dissect the downstream Regnase-1 target genes that play critical role in cancer metastasis.
[0209] By using an online homology search tool, eight other proteins have been identified that also have a "Zc3hl2a-like ribonuclease NYN domain." Thus, the whole family includes nine members: ZC3H12A, ZC3H12B, ZC3H12C, ZC3H12D, KHNYN, NYNRIN, ELOVL5, KCNK12 and N4BP1 ; their protein sequences are as follows:
[0210]
H. sapiens ZC3H12A (Regnase-1) NP_079355.2 ribonuclease (SEQ ID NO:3):
MSGPCGEKPVLEASPTMSLWEFEDSHSRQGTPRPGQELAAEEASALELQMKVDFFRKLGYSSTE IHSVLQ KLGVQADTNTVLGELVKHGTATERERQTSPDPCPQLPLVPRGGGTPKAPNLEPPLPEEEKEGSDLRPWI DGSNVAMSHGNKEVFSCRGI LLAVNWFLERGHTD I TVFVP SWRKEQPRPDVP I TDQHI LRELEKKKI LVF
TPSRRVGGKRWCYDDRFIVKLAYESDGIWSNDTYRDLQGERQEWKRFIEERLLMYSFVNDKFMPPDDP LGRHGPSLDNFLRKKPLTLEHRKQPCPYGRKCTYGIKCRFFHPERPSCPQRSVADELRANALLSPPRAPS KDKNGRRPSPSSQSSSLLTESEQCSLDGKKLGAQASPGSRQEGLTQTYAPSGRSLAPSGGSGSSFGPTDW LPQTLDSLPYVSQDCLDSGIGSLESQMSELWGVRGGGPGEPGPPRAPYTGYSPYGSELPATAAFSAFGRA MGAGHFSVPADYPPAPPAFPPREYWSEPYPLPPPTSVLQEPPVQSPGAGRSPWGRAGSLAKEQASVYTKL CGVFPPHLVEAVMGRFPQLLDPQQLAAEILSYKSQHPSE
[0211]
H. sapiens ZC3H12B NP_001010888.3 ribonuclease ZC3H12B (SEQ ID NO:4):
MTATAEVETPKMEKSASKEEKQQPKQDSTEQGNADSEEWMSSESDPEQISLKSSDNSKSCQPRDGQLKKK EMHSKPHRQLCRSPCLDRPSFSQSSILQDGKLDLEKEYQAKMEFALKLGYAEEQIQSVLNKLGPESLIND VLAELVRLGNKGDSEGQINLSLLVPRGPSSREIASPELSLEDEIDNSDNLRPWIDGSNVAMSHGNKEEF SCRGIQLAVDWFLDKGHKDITVFVPAWRKEQSRPDAPITDQDILRKLEKEKILVFTPSRRVQGRRWCYD DRFIVKLAFDSDGI IVSNDNYRDLQVEKPEWKKFIEERLLMYSFVNDKFMPPDDPLGRHGPSLENFLRKR PIVPEHKKQPCPYGKKCTYGHKCKYYHPERANQPQRSVADELRISAKLSTVKTMSEGTLAKCGTGMSSAK GEITSEVKRVAPKRQSDPSIRSVAMEPEEWLSIARKPEASSVPSLVTALSVPTIPPPKSHAVGALNTRSA SSPVPGSSHFPHQKASLEHMASMQYPPILVTNSHGTPISYAEQYPKFESMGDHGYYSMLGDFSKLNINSM HNREYYMAEVDRGVYARNPNLCSDSRVSHTRNDNYSSYNNVYLAVADTHPEGNLKLHRSASQNRLQPFPH GYHEALTRVQSYGPEDSKQGPHKQSVPHLALHAQHPSTGTRSSCPADYPMPPNIHPGATPQPGRALVMTR MDSISDSRLYESNPVRQRRPPLCREQHASWDPLPCTTDSYGYHSYPLSNSLMQPCYEPVMVRSVPEKMEQ LWRNPWVGMCNDSREHMIPEHQYQTYKNLCNIFPSNIVLAA/MEKNPHTADAQQLAALIVAKLRA
[0212]
H. sapiens ZC3H12C NP_203748.1 probable ribonuclease ZC3H12C (SEQ ID NO:5):
MPGGGSQEYGVLCIQEYRKNSKVESSTRNNFMGLKDHLGHDLGHLYVESTDPQLSPAVPWSTVENPSMDT VNVGKDEKEASEENASSGDSEENTNSDHESEQLGSISVEPGLITKTHRQLCRSPCLEPHILKRNEILQDF KPEESQTTSKEAKKPPDWREYQTKLEFALKLGYSEEQVQLVLNKLGTDALINDILGELVKLGNKSEADQ TVSTINTITRETSSLESQRSESPMQEIVTDDGENLRPIVIDGSNVAMSHGNKEVFSCRGIKLAVDWFLER GHKDITVFVPAWRKEQSRPDALITDQEILRKLEKEKILVFTPSRRVQGRRWCYDDRFIVKLAFESDGII VSNDNYRDLANEKPEWKKFIDERLLMYSFVNDKFMPPDDPLGRHGPSLDNFLRKKPIVPEHKKQPCPYGK KCTYGHKCKYYHPERGSQPQRSVADELRAMSRNTAAKTANEGGLVKSNSVPCSTKADSTSDVKRGAPKRQ SDPSIRTQVYQDLEEKLPTKNKLETRSVPSLVSIPATSTAKPQSTTSLSNGLPSGVHFPPQDQRPQGQYP SMMMATKNHGTPMPYEQYPKCDSPVDIGYYSMLNAYSNLSLSGPRSPERRFSLDTDYRISSVASDCSSEG SMSCGSSDSYVGYNDRSYVSSPDPQLEENLKCQHMHPHSRLNPQPFLQNFHDPLTRGQSYSHEEPKFHHK PPLPHLALHLPHSAVGARSSCPGDYPSPPSSAHSKAPHLGRSLVATRIDSISDSRLYDSSPSRQRKPYSR QEGLGSWERPGYGIDAYGYRQTYSLPDNSTQPCYEQFTFQSLPEQQEPAWRIPYCGMPQDPPRYQDNREK IYINLCNIFPPDLVRIVMKRNPHMTDAQQLAAAILVEKSQLGY
[0213]
H. sapiens ZC3H12D NP_997243.2 probable ribonuclease ZC3H12D (SEQ ID NO:6):
MEHPSKMEFFQKLGYDREDVLRVLGKLGEGALVNDVLQELIRTGSRPGALEHPAAPRLVPRGSCGVPDSA QRGPGTALEEDFRTLASSLRPIVIDGSNVAMSHGNKETFSCRGIKLAVDWFRDRGHTYIKVFVPSWRKDP PRADTPIREQHVLAELERQAVLVYTPSRKVHGKRLVCYDDRYIVKVAYEQDGVIVSNDNYRDLQSENPEW KWFIEQRLLMFSFVNDRFMPPDDPLGRHGPSLSNFLSRKPKPPEPSWQHCPYGKKCTYGIKCKFYHPERP HHAQLAVADELRAKTGARPGAGAEEQRPPRAPGGSAGARAAPREPFAHSLPPARGSPDLAALRGSFSRLA FSDDLGPLGPPLPVPACSLTPRLGGPDWVSAGGRVPGPLSLPSPESQFSPGDLPPPPGLQLQPRGEHRPR DLHGDLLSPRRPPDDPWARPPRSDRFPGRSVWAEPAWGDGATGGLSVYATEDDEGDARARARIALYSVFP RDQVDRVMAAFPELSDLARLILLVQRCQSAGAPLGKP
[0214]
H. sapiens ΚΗΝΥΝ NP_056114.1 ΚΗΝΥΝ protein, isoform 1 (SEQ ID NO:7):
MPTWGARPASPDRFAVSAEAENKVREQQPHVERIFSVGVSVLPKDCPDNPHIWLQLEGPKENASRAKEYL KGLCSPELQDEIHYPPKLHCIFLGAQGFFLDCLAWSTSAHLVPRAPGSLMISGLTEAFVMAQSRVEELAE RLSWDFTPGPSSGASQCTGVLRDFSALLQSPGDAHREALLQLPLAVQEELLSLVQEASSGQGP GALAS WE GRSSALLGAQCQGVRAPPSDGRESLDTGSMGPGDCRGARGDTYAVEKEGGKQGGPREMDWGWKELPGEEA WEREVALRPQSVGGGARESAPLKGKALGKEEIALGGGGFCVHREPPGAHGSCHRAAQSRGASLLQRLHNG NASPPRVPSPPPAPEPPWHCGDRGDCGDRGDVGDRGDKQQGMARGRGPQWKRGARGGNLVTGTQRFKEAL QDPFTLCLANVPGQPDLRHIVIDGSNVAMVHGLQHYFSSRGIAIAVQYFWDRGHRDITVFVPQWRFSKDA KVRESHFLQKLYSLSLLSLTPSRVMDGKRISSYDDRFMVKLAEETDGI IVSNDQFRDLAEESEKWMAI IR ERLLPFTFVGNLFMVPDDPLGRNGPTLDEFLKKPARTQGSSKAQHPSRGFAEHGKQQQGREEEKGSGGIR KTRETERLRRQLLEVFWGQDHKVDFILQREPYCRDINQLSEALLSLNF
[0215]
H. sapiens NP_079357.2 NYNREN protein (SEQ ID NO: 8):
MLLSGGDPPAQEWFMVQTKSKPRVQRQRLQVQRIFRVKLNAFQSRPDTPYFWLQLEGPRENMGKAKEYLK GLCSPELWKEVRYPPILHCAFLGAQGLFLDCLCWSTLAYLVPGPPGSLMVGGLTESFIMTQNWLEELVGR LRWGPAPLLTPRGIWEAEVTRAFGALVWIRGDQHAGDLLQLPPAVQELLLSLVRDAAGKEDI IEWLSRFG ISDSHSDPEVLICPPQQQKEAPAMVSVGESPGPFVDMGTLQNRGPENSKRLSSLGATGSLITAQSTPQEA ANQLVRVGSNNQDGMDSAQEEGTVQATSSQDSTNHTQALLKQRQVQKIEDKLLFQPPVSALGVCPPWKAW TPGPAFGPLWPGAIAATFWRINELHSLHLAWLLSQACFNFPFWQRPLGPIQLKLPGQNPLPLNLEWKQKE LAPLPSAESPAGRPDGGLGGEAALQNCPRPEISPKVTSLLWPGSSDVKDKVSSDLPQIGPPLTSTPQLQ AGGEPGDQGSMQLDFKGLEEGPAPVLPTGQGKPVAQGGLTDQSVP GAQTVPETLKVPMAAAVPKAENPSR TQVPSAAPKLPTSRMMLAVHTEPAAPEVPLAPTKPTAQLMATAQKTWNQPVLVAQVEPTTPKTPQAQKM PVAKTSPAGPKTPKAQAGPAATVSKAPAASKAPAAPKVPVTPRVSRAPKTPAAQKVPTDAGPTLDVARLL SEVQPTSRASVSLLKGQGQAGRQGPQSSGTLALSSKHQFQMEGLLGAWEGAPRQPPRHLQANSTVTSFQR YHEALNTPFELNLSGEPGNQGLRRWIDGSSVAMVHGLQHFFSCRGIAMAVQFFWNRGHREVTVFVPTWQ LKKNRRVRESHFLTKLHSLKMLSITPSQLENGKKITTYDYRFMVKLAEETDGI IVTNEQIHILMNSSKKL MVKDRLLPFTFAGNLFMVPDDPLGRDGPTLDEFLKKPNRLDTDIGNFLKVWKTLPPSSASVTELSDDADS GPLESLPNMEEVREEKEERQDEEQRQGQGTQKAAEEDDLDSSLASVFRVECPSLSEEILRCLSLHDPPDG ALDIDLLPGAASPYLGIPWDGKAPCQQVLAHLAQLTIPSNFTALSFFMGFMDSHRDAIPDYEALVGPLHS LLKQKPDWQWDQEHEEAFLALKRALVSALCLMAPNSQLPFRLEVTVSHVALTAILHQEHSGRKHPIAYTS KPLLPDEESQGPQSGGDSPYAVAWALKHFSRCIGDTPWLDLSYASRTTADPEVREGRRVSKAWLIRWSL LVQDKGKRALELALLQGLLGENRLLTPAASMPRFFQVLPPFSDLSTFVCIHMSGYCFYREDEWCAGFGLY VLSPTSPPVSLSFSCSPYTPTYAHLAAVACGLERFGQSPLPWFLTHCNWIFSLLWELLPLWRARGFLSS DGAPLPHPSLLSYI ISLTSGLSSLPFIYRTSYRGSLFAVTVDTLAKQGAQGGGQWWSLPKDVPAPTVSPH AMGKRPNLLALQLSDSTLADIIARLQAGQKLSGSSPFSSAFNSLSLDKESGLLMFKGDKKPRVWWPTQL RRDLIFSVHDIPLGAHQRPEETYKKLRLLGWWPGMQEHVKDYCRSCLFCIPRNLIGSELKVIESPWPLRS TAPWSNLQIEWGPVTISEEGHKHVLIVADPNTRWVEAFPLKPYTHTAVAQVLLQHVFARWGVPVRLEAA QGPQFARHVLVSCGLALGAQVASLSRDLQFPCLTSSGAYWEFKRALKEFIFLHGKKWAASLPLLHLAFRA SSTDATPFKVLTGGESRLTEPLWWEMSSANIEGLKMDVFLLQLVGELLELHWRVADKASEKAENRRFKRE SQEKEWNVGDQVLLLSLPRNGSSAKWVGPFYIGDRLSLSLYRIWGFPTPEKLGCIYPSSLMKAFAKSGTP LSFKVLEQ
[0216]
H. sapiens ELOVL5 NP_001229757.1 Elongation of VLC FA protein 5, isoform 2 (SEQ ID NO:9): MEHFDASLSTYFKALLGPRDTRVKGWFLLDNYIPTFICSVIYLLIVWLGPKYMRNKQPFSCRGILWYNL GLTLLSLYMFCESKREQPRRSACASRTDPSTQQQLPENRLVTGVWEGKYNFFCQGTRTAGESDMKI IRVL WWYYFSKLIEFMDTFFFILRKNNHQITVLHVYHHASMLNIWWFVMNWVPCGHSYFGATLNSFIHVLMYSY YGLSSVPSMRPYLWWKKYITQGQLLQFVLTI IQTSCGVIWPCTFPLGWLYFQIGYMISLIALFTNFYIQT
YNKKGASRRKDHLKDHQNGSMAAVNGHTNSFSPLENNVKPRKLRKD
[0217]
H. sapiens KCNK12 NP_071338.1 potassium channel subfamily K member 12 (SEQ ID NO:10):
MSSRSPRPPPRRSRRRLPRPSCCCCCCRRSHLNEDTGRFVLLAALIGLYLVAGATVFSALESPGEAEARA RWGATLRNFSAAHGVAEPELRAFLRHYEAALAAGVRADALRPRWDFPGAFYFVGTWSTIGFGMTTPATV GGKAFLIAYGLFGCAGTILFFNLFLERI ISLLAFIMRACRERQLRRSGLLPATFRRGSALSEADSLAGWK PSVYHVLLILGLFAVLLSCCASAMYTSVEGWDYVDSLYFCFVTFSTIGFGDLVSSQHAAYRNQGLYRLGN FLFILLGVCCIYSLFNVISILIKQVLNWMLRKLSCRCCARCCPAP GAPLARRNAITPGSRLRRRLAALGA DPAARDSDAEGRRLSGELISMRDLTASNKVSLALLQKQLSETANGYPRSVCA/ TRQNGFSGGVGALGIMN NRLAETSASR
[0218]
H. sapiens N4BP1 NP_694574.3 NEDD4-binding protein 1 (SEQ ID NO:ll):
MAARAVLDEFTAPAEKAELLEQSRGRIEGLFGVSLAVLGALGAEEPLPARIWLQLCGAQEAVHSAKEYIK GICEPELEERECYPKDMHCIFVGAESLFLKSLIQDTCADLCILDIGLLGIRGSAEAWMARSHIQQFVKL FENKENLPSSQKESEVKREFKQFVEAHADNYTMDLLILPTSLKKELLTLTQGEENLFETGDDEVIEMRDS QQTEFTQNAATGLNISRDETVLQEEARNKAGTPVSELTKQMDTVLSSSPDVLFDPINGLTPDEEALSNER ICQKRRFSDSEERHTKKQFSLENVQEGEILHDAKTLAGNVIADLSDSSADSENLSPDIKETTEEMEYNIL VNFFKTMGYSQEIVEKVIKVYGPSTEPLLLLEEIEKENKRFQEDREFSAGTVYPETNKTKNKGVYSSTNE LTTDSTPKKTQAHTQQNMVEKFSQLPFKVEAKPCTSNCRINTFRTVPIEQKHEVWGSNQNYICNTDPETD GLSPSVASPSPKEVNFVSRGASSHQPRVPLFPENGLHQQPEPLLPNNMKSACEKRLGCCSSPHSKPNCST LSPPMPLPQLLPSVTDARSAGPSDHIDSSVTGVQRFRDTLKIPYKLELKNEPGRTDLKHIVIDGSNVAIT HGLKKFFSCRGIAIAVEYFWKLGNRNITVFVPQWRTRRDPNVTEQHFLTQLQELGILSLTPARMVFGERI ASHDDRFLLHLADKTGGI IVTNDNFREFVNESVSWREI ITKRLLQYTFVGDIFMVPDDPLGRSGPRLEEF LQKEVCLRDMQPLLSALPNVGMFDPSFRVPGTQAASTSHQPPTRIQGAPSSHWLPQQPHFPLLPALPSLQ QNLPMPAQRSSAETNELREALLKIFPDSEQRLKIDQILVAHPYMKDLNALSAMVLD
SUMMARY
[0219] Cancer metastasis is a leading cause of cancer-related death due to poorly defined molecular mechanisms, and our treatment strategies are also limited. Here, it was shown that the immune regulator Regnase-1, which is regulated by post-translational mechanisms, is critically involved in cancer metastasis. Stable expression of Regnase-1 in cancer cells could dramatically inhibit metastasis through its ribonuclease activity, and tumors with lower levels of Regnase- 1 are much more aggressive in metastasis than those with higher levels of Regnase-1. The antiproliferative and anti-inflammatory activities of Regnase- 1 were shown not to be responsible for its effects in suppressing tumor metastasis. Thus, Regnase-1 clearly employs new mechanisms in the control of cancer metastasis and acts as a critical checkpoint in cancer treatment. Given extensive efforts in identifying genetic mechanisms in cancer metastasis, these results provide new evidence on post-translational mechanisms, especially at the level of protein stability, in the control of cancer metastasis. The components targeting the signaling networks that control Regnase-1 stability as well as molecular targets that are controlled by Regnase-1 are novel and ideal therapeutic targets for prevention of cancer metastasis.
REFERENCES:
[0220] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference in their entirety :zz
[0221] ACETO, N et al, "Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis," Cell, 158(5): 1110- 1122 (Aug. 2014).
[0222] ADAMS, J. and M. KAUFFMAN, "Development of the proteasome inhibitor Velcade (Bortezomib), " Cancer Invest, 22(2):304-311 (2004).
[0223] ALTSCHUL, SF et al, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucl. Acids &&,25(Γ7):338 -3 02 (1997).
[0224] BALK, SP et al., "Biology of prostate-specific antigen," /. Clin. Oncol., 21(2):383-391 (2003).
[0225] CHAFFER, CL and WEINBERG, RA, "A perspective on cancer cell metastasis, " Science, 331(6024): 1559-1564 (Mar. 2011).
[0226] CHO, EH et al., "Characterization of circulating tumor cell aggregates identified in patients with epithelial tumors," Phys. Biol, 9(1):016001 (Feb. 2012).
[0227] CIFUENTES, RA et al, "ZC3H12A (MCPIP1): molecular characteristics and clinical implications," Clin. Chim. Acta, 411(23-24): 1862-1868 (Aug. 2010).
[0228] DANOVA, M et al, "Tissue and soluble biomarkers in breast cancer and their applications: ready to use?" /. Natl. Cancer Inst. Monogr., 2011(43):75-78 (2011).
[0229] DUFFY, MJ, "Serum tumor markers in breast cancer: are they of clinical value?" Clin. Chem., 52(3):345-351 (2006).
[0230] FERNANDEZ, Y et al, "Inhibition of apoptosis in human breast cancer cells: role in tumor progression to the metastatic state," Int. J. Cancer, 101(4):317-326 (Oct. 2002).
[0231] FIDLER, IJ, "The relationship of embolic homogeneity, number, size and viability to the incidence of experimental metastasis," Eur. J. Cancer, 9(3):223-227 (Mar. 1973).
[0232] GRIBSKOV, M, and BURGESS, RR, "Sigma factors from E. coli, B. subtilis, phage SPOl, and phage T4 are homologous proteins," Nucleic Acids Res., 14(16):6745-6763 (Aug. 1986).
[0233] HALE, WG, and MARGHAM, JP, "HARPER COLLINS DICTIONARY OF BIOLOGY," HarperPerennial, New York (1991).
[0234] HARDMAN, JG, and LIMBIRD, LE, (Eds.), "GOODMAN AND OILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS" 10th Edition, McGraw-Hill, New York (2001).
[0235] HARRIS, L et al, "American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer," /. Clin. Oncol, 25(33):5287- 5312 (2007).
[0236] HARRIS, MP et al, "Adenovirus -mediated p53 gene transfer inhibits growth of human tumor cells expressing mutant p53 protein, " Cancer Gene Ther., 3(2): 121-130 (Mar. 1996).
[0237] IWASAKI, H et al, "The IkappaB kinase complex regulates the stability of cytokine- encoding mRNA induced by TLR-IL-1R by controlling degradation of Regnase-1, " Nat. Immunol, 12(12): 1167-1175 (Oct. 2011).
[0238] KACINSKI, BM et al, "The cytokine CSF-1 (M-CSF) expressed by endometrial carcinomas in vivo and in vitro, may also be a circulating tumor marker of neoplastic disease activity in endometrial carcinoma patients," Int. J. Radiat. Oncol. Biol. Phys., 19(3):619-626 (1990).
[0239] KIMBUNG, S et al, "Clinical and molecular complexity of breast cancer metastases," Semin. Cancer Biol, 35:85-95 (Dec. 2015).
[0240] KYTE, J, and DOOLITTLE, RF, "A simple method for displaying the hydropathic character of a protein," /. Mol. Biol , 157(1): 105-132 (1982)
[0241] LIANG, CC et al, "In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro, " Nat. Protocols, 2(2):329-333 (Apr. 2007).
[0242] MARINO, N et al, "Breast cancer metastasis: issues for the personalization of its prevention and treatment," Am. J. Pathol, 183(4): 1084- 1095 (Jul. 2013).
[0243] MATSUSHITA, K et al, "Zc3hl2a is an RNase essential for controlling immune responses by regulating mRNA decay, " Nature, 458(7242): 1185-90 (Mar. 2009).
[0244] MOHANTY, S and XU, L, "Experimental metastasis assay, " /. Vis. Exp., 42: 1942 (Aug. 2010).
[0245] MOLNAR, B et al, "Circulating tumor cell clusters in the peripheral blood of colorectal cancer patients," Clin. Cancer Res., 7(12):4080-4085 (Dec. 2001).
[0246] NEEDLEMAN, SB and WUNSCH, CD, "A general method applicable to the search for similarities in the amino acid sequence of two proteins," /. Mol. Biol , 48(3):443-453 (Mar. 1970).
[0247] NEVE, RM et al. , "A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes," Cancer Cell, 10(6):515-527 (Dec. 2006).
[0248] RAAMANATHAN, A et al, "Programmable bio-nano-chip systems for serum CA125 quantification: toward ovarian cancer diagnostics at the point-of-care." Cancer Prev. Res. (Phila), 5(5):706-716 (2012).
[0249] RYCHLIK, W and RHOADS, RE, "A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA," Nucl. Acids Res., 17:8543-8551 (1989).
[0250] SCHRODINGER 2013. Schrodinger, LLC: New York, NY (2013).
[0251] SINGLETON, P and SAINSBURY, D, "DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY," 2nd Ed., John Wiley and Sons, New York (1987).
[0252] UEHATA, T et al, "Maltl -induced cleavage of Regnase-1 in CD4(+) helper T cells regulates immune activation, " Cell, 153(5): 1036- 1049 (May 2013).
[0253] VANHARANTA, S and MASSAGUE, J, "Origins of metastatic traits," Cancer Cell, 24(4):410-421 (Oct. 2013).
[0254] VASSILEV, LT et al., "In vivo activation of the p53 pathway by small-molecule antagonists of MDM2, " Science, 303(5659):844-848 (Feb. 2004).
[0255] WAN, L et al, "Tumor metastasis: moving new biological insights into the clinic," Nat. Med, 19(11): 1450-1464 (Nov. 2013).
[0256] WANG, Y et al, "Specific expression of the human voltage-gated proton channel Hvl in highly metastatic breast cancer cells, promotes tumor progression and metastasis, " Biochem. Biophys. Res. Commun., 412(2):353-359 (Jul. 2011).
[0257] WU, CC et al, "Cancer cell-secreted proteomes as a basis for searching potential tumor markers: nasopharyngeal carcinoma as a model," Proteomics, 5(12):3173-3182 (2005).
[0258] YAN, J et al, "Metastasis suppressor genes, " Histol Histopathol, 28(3):285-292 (Mar.
2013).
[0259] ZHANG, RD et al, "Relative malignant potential of human breast carcinoma cell lines established from pleural effusions and a brain metastasis," Invasion Metastasis, 11(4):204-215 (Apr. 1991).
[0260] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. All references (including publications, patent applications and patents) cited herein are incorporated herein by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein. Recitation of ranges of values herein are merely intended to
serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.
[0261] The description herein of any aspect or embodiment of the invention using terms such as "comprising," "having," "including," or "containing," with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of," "consists essentially of," or "substantially comprises," that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).
[0262] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the invention have been described herein in terms of illustrative embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods, and/or the steps or the sequence of steps of the methods without departing from the spirit, scope, and concept of the invention. More specifically, it will be apparent that certain compounds, which are chemically- and/or physiologically-related, may be substituted for one or more of the compounds described herein, while still achieving the same or similar results. All such substitutions and/or modifications, as apparent to one or more of ordinary skill in the relevant arts, are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.
Claims
1. A composition comprising: a therapeutically-effective amount of a first isolated mammalian Regnase-1 peptide, a Regnase-1 polypeptide, a Regnase-1 activator, or a combination thereof.
2. The composition in accordance with claim 1, further comprising a second distinct therapeutic agent, one or more agents for determining the metastatic potential of a mammalian cancer cell, or a combination thereof.
3. The composition in accordance with claim 1 or claim 2, formulated for mammalian administration, and preferably, for human administration.
4. The composition in accordance with any preceding claim, further comprising a first chemotherapeutic agent, a first diagnostic agent, a first imaging agent, or any combination thereof.
5. The composition in accordance with any preceding claim, comprised within a kit that includes at least a first set of instructions for administration of the composition to a mammal in need thereof.
6. The pharmaceutical composition in accordance with any preceding claim, for use in the diagnosis, the prophylaxis, the therapy, or the amelioration of one or more symptoms of a cancer or cancer metastasis in a mammal.
7. The composition in accordance with any preceding claim, wherein the Regnase-1 peptide or the Regnase-1 polypeptide comprises an at least 50 amino acid sequence that is at least 95% identical to an at least 50 contiguous amino acid sequence from any one of SEQ ID NO:4 to SEQ ID NO: 11.
8. The composition in accordance with any preceding claim, wherein the Regnase-1 polypeptide comprises an at least 100 amino acid sequence that is at least 95% identical to
an at least 100 contiguous amino acid sequence from any one of SEQ ID NO:4 to SEQ ID NO: 11.
9. The composition in accordance with any preceding claim, wherein the Regnase-1 polypeptide comprises an at least 250 amino acid sequence that is at least 95% identical to an at least 250 contiguous amino acid sequence from any one of SEQ ID NO:4 to SEQ ID NO: 11.
10. The composition in accordance with any preceding claim, wherein the Regnase-1 polypeptide consists of an amino acid sequence that is at least 95% identical to any one of
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: 11.
11. The composition in accordance with any preceding claim, wherein the Regnase-1 peptide or the Regnase-1 polypeptide is produced by a gene therapy vector comprising a nucleic acid sequence that expresses the peptide or the polypeptide in a mammalian cell.
12. The composition in accordance with any preceding claim, wherein the Regnase-1 peptide or the Regnase-1 polypeptide is encoded by an adenoviral vector system that operably expresses the peptide or the polypeptide in a population of mammalian cells transformed with the vector system.
13. The composition in accordance with any preceding claim, admixed with one or more pharmaceutically-acceptable carriers, buffers, diluents, vehicles, or excipients.
14. The composition in accordance with any preceding claim, 1) formulated with a population of liposomes, nanoparticles, or microparticles; or 2) admixed with one or more surfactants, niosomes, ethosomes, transferosomes, phospholipids, sphingosomes, or any combination thereof.
15. The composition in accordance with any preceding claim, formulated for systemic administration to a mammal, and preferably, for intravenous administration to a human.
16. A composition in accordance with any preceding claim, adapted and configured as part of a therapeutic kit that comprises the composition, and at least a first set of instructions for administration of the composition to a human in need thereof.
17. A composition in accordance with any preceding claim, for use in diagnosis, therapy, or a combination thereof.
18. The composition in accordance with any preceding claim, for use in therapy, prophylaxis, or amelioration of one or more symptoms of a mammalian disease, disorder, dysfunction, deficiency, defect, trauma, injury, or abnormal condition.
19. An isolated population of mammalian cells comprising the composition in accordance with any preceding claim.
20. The isolated population of mammalian cells in accordance with claim 19, characterized as human cancer cells.
21. Use of a composition in accordance with any one of claims 1 to 18, in the manufacture of a medicament for diagnosing, treating, or ameliorating one or more symptoms of cancer in a mammal.
22. Use in accordance with claim 21, wherein the mammalian subject is a human, a non- human primate, a companion animal, an exotic, or livestock.
23. Use of a composition in accordance with any one of claims 1 to 18, in the manufacture of a medicament for treating cancer, and preferably, metastatic cancer, in a human.
24. Use of a composition in accordance with any one of claims 1 to 18, in the manufacture of a diagnostic reagent for determining the metastatic potential of one or more cancers in a mammalian subject.
25. A kit comprising: 1) the composition in accordance with any one of claims 1 to 18; and 2) instructions for administering the composition to a mammal in need thereof, as part of a
regimen for the prevention, diagnosis, treatment, or amelioration of one or more symptoms of a disease, a dysfunction, an abnormal condition, or a trauma in the mammal.
26. A method of diagnosing, treating, or ameliorating one or more symptoms of cancer in a mammal, the method comprising administering to the animal an effective amount of a composition in accordance with any one of claims 1 to 18, for a time sufficient to diagnose, treat, or ameliorate the one or more symptoms of cancer in the mammal.
27. The method in accordance with claim 26, wherein the mammal is a human at risk for developing one or more cancer metastases.
28. The method in accordance with claim 26 or claim 27, wherein the cancer is diagnosed as, or is identified as, a refractory, a metastatic, a relapsed, or a treatment-resistant cancer.
29. The method in accordance with any one of claims 26 to claim 28, wherein the method further comprises administering a therapeutically-effective amount of at least one anticancer agent or one anti-metastatic agent to the mammal.
30. The method in accordance with any one of claims 26 to 29, wherein the composition is administered to the mammal in a single administration, or in a series of successive administrations over a period of time.
31. The method in accordance with claim 30, wherein the period of time is one or more days, one or more weeks, or one or more months.
32. The method in accordance with any one of claims 26 to 28, wherein the composition is administered substantially concurrently with the administration of one or more additional therapeutic agents.
33. The method in accordance with any one of claims 26 to 31, wherein the composition comprises a population of adenoviral vectors suitable for administration to a human.
34. The method in accordance with any one of claims 26 to 33, wherein the mammal is human.
35. The method in accordance with any one of claims 26 to 34, wherein the method further comprises administering a therapeutically-effective amount of radiation or an additional chemotherapeutic to the mammal.
36. A method of administering a diagnostic, therapeutic, or prophylactic agent to one or more cells, tissues, organs, or systems of a mammalian subject in need thereof, comprising administering to the subject an effective amount of the composition of in accordance with any one of claims 1 to 18.
37. The method in accordance with claim 36, wherein the cells are identified as human cancer cells.
38. A method of altering or reducing the metastatic potential of a cancer cell in a mammal, comprising administering to a mammalian subject in need thereof, an amount of (a) the composition in accordance with any one of claims 1 to 18, or (b) a composition comprising a population of adenoviral vectors that express the peptide or polypeptide of any one of claims 1 to 18, and for a time effective to alter or reduce the metastatic potential of the cancer cell in the mammal.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662287188P | 2016-01-26 | 2016-01-26 | |
US62/287,188 | 2016-01-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2017132420A1 true WO2017132420A1 (en) | 2017-08-03 |
Family
ID=59398634
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2017/015198 WO2017132420A1 (en) | 2016-01-26 | 2017-01-26 | Compositions and methods for suppression and diagnosis of cancer metastasis |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2017132420A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113750218A (en) * | 2021-08-10 | 2021-12-07 | 南通市肿瘤医院 | Application of polypeptide ZC3H12A and mutant thereof in preparation of anti-liver cancer drugs |
WO2023008337A1 (en) * | 2021-07-26 | 2023-02-02 | ピューロテックバイオ株式会社 | Anti-hepatitis b virus agent targeting host factor lipg |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120070452A1 (en) * | 2009-02-27 | 2012-03-22 | Shizuo Akira | Immunoadjuvant composition and use thereof |
WO2014023808A2 (en) * | 2012-08-08 | 2014-02-13 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for evaluating a cancer patient for propensity to respond to a therapy |
WO2016167957A1 (en) * | 2015-04-15 | 2016-10-20 | Saint Louis University | Tumor suppression by mcpip1 |
-
2017
- 2017-01-26 WO PCT/US2017/015198 patent/WO2017132420A1/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120070452A1 (en) * | 2009-02-27 | 2012-03-22 | Shizuo Akira | Immunoadjuvant composition and use thereof |
WO2014023808A2 (en) * | 2012-08-08 | 2014-02-13 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for evaluating a cancer patient for propensity to respond to a therapy |
WO2016167957A1 (en) * | 2015-04-15 | 2016-10-20 | Saint Louis University | Tumor suppression by mcpip1 |
Non-Patent Citations (3)
Title |
---|
AKIRA: "Regnase-1, a ribonuclease involved in the regulation of immune responses", SYMPOSIA ON QUANTITATIVE BIOLOGY, vol. LXXVIII, 2013, pages 51 - 60, XP055403306 * |
LIU ET AL.: "Abstract LB-042: MCPIP1 suppresses breast tumor progression by targeting anti-apoptosis pathway", AMERICAN ASSOCIATION FOR CANCER RESEARCH (AACR) 106TH ANNUAL MEETING, 18 April 2015 (2015-04-18), Philadelphia, XP055404731 * |
SKALNIAK ET AL.: "Proteasome inhibitor MG -132 induces MCPIP1 expression", THE FEBS JOURNAL, vol. 280, 2013, pages 2665 - 2674 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023008337A1 (en) * | 2021-07-26 | 2023-02-02 | ピューロテックバイオ株式会社 | Anti-hepatitis b virus agent targeting host factor lipg |
CN113750218A (en) * | 2021-08-10 | 2021-12-07 | 南通市肿瘤医院 | Application of polypeptide ZC3H12A and mutant thereof in preparation of anti-liver cancer drugs |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Burnier et al. | Type IV collagen-initiated signals provide survival and growth cues required for liver metastasis | |
US11971402B2 (en) | Methods and reagents for determination and treatment of organotropic metastasis | |
Nagel et al. | Comprehensive analysis of homeobox genes in Hodgkin lymphoma cell lines identifies dysregulated expression of HOXB9 mediated via ERK5 signaling and BMI1 | |
AU2009209415A1 (en) | Methods and compositions relating to carcinoma stem cells | |
CN107083433B (en) | Application of lncRNA in diagnosis and treatment of liver cancer | |
US20110177967A1 (en) | Methods and compositions using splicing regulatory proteins involved in tumor suppression | |
US11857563B2 (en) | Inhibition of expansion and function of pathogenic age-associated B cells and use for the prevention and treatment of autoimmune disease | |
CN111278469A (en) | Methods for treating triple negative breast cancer | |
CA2925922C (en) | Inhibition of thymine dna glycosylase in the treatment of cancer | |
US20220112498A1 (en) | Methods for diagnosing and treating metastatic cancer | |
Zhou et al. | A novel role of TGFBI in macrophage polarization and macrophage-induced pancreatic cancer growth and therapeutic resistance | |
WO2016042114A1 (en) | Cxcl14 as a biomarker of hedgehog pathway activity for the diagnosis, prognosis and treatment of idiopathic pulmonary fibrosis | |
WO2017132420A1 (en) | Compositions and methods for suppression and diagnosis of cancer metastasis | |
EP2169077A1 (en) | Methods and compositions for diagnosing an adenocarcinoma | |
CN107267616B (en) | Application of non-coding gene biomarker in liver cancer | |
US20170218036A1 (en) | Innate immune system modification for anticancer therapy | |
JP5887413B2 (en) | Methods of reducing cancer cell radiation resistance and proliferation, metastasis and invasion by modulating TM4SF4 expression or activity in non-small cell lung cancer | |
CN106995857B (en) | Application of biomarker ENSG00000267416 in cancer | |
JP2024500064A (en) | How to determine resistance to checkpoint inhibitor therapy | |
US10865415B2 (en) | Prevention, diagnosis and treatment of cancer overexpressing GPR160 | |
CN111534587A (en) | Molecular marker 5-tRF-His, breast cancer detection kit and application thereof | |
US9574240B2 (en) | Gene amplification of coactivator CoAA and uses thereof | |
Heath et al. | The Neurodevelopmental Protein POGZ Suppresses Metastasis in Triple Negative Breast Cancer by Attenuating TGFβ Signaling | |
He et al. | Induction of CX3CL1 expression by LPS and its impact on invasion and migration in oral squamous cell carcinoma | |
Guarnaccia | DEVELOPMENT AND VALIDATION OF TARGET THERAPIES FOR PATIENTS WITH BRAIN CANCER, THROUGH THE MODULATION OF ANGIOGENESIS, INVASIVENESS, AND PHARMACOLOGICAL SENSITIVITY/RESISTANCE, IN THE ERA OF PRECISION MEDICINE |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17744919 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 17744919 Country of ref document: EP Kind code of ref document: A1 |