BRPI0903797A2 - method for diagnosis and monitoring of alpha-synucleinopathies by determining levels of mirnas in biological samples - Google Patents
method for diagnosis and monitoring of alpha-synucleinopathies by determining levels of mirnas in biological samples Download PDFInfo
- Publication number
- BRPI0903797A2 BRPI0903797A2 BRPI0903797-7A BRPI0903797A BRPI0903797A2 BR PI0903797 A2 BRPI0903797 A2 BR PI0903797A2 BR PI0903797 A BRPI0903797 A BR PI0903797A BR PI0903797 A2 BRPI0903797 A2 BR PI0903797A2
- Authority
- BR
- Brazil
- Prior art keywords
- disease
- alpha
- parkinson
- synucleinopathies
- mirnas
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000003745 diagnosis Methods 0.000 title claims abstract description 20
- 208000032859 Synucleinopathies Diseases 0.000 title claims abstract description 17
- 239000012472 biological sample Substances 0.000 title claims abstract description 8
- 108091070501 miRNA Proteins 0.000 title claims description 16
- 238000012544 monitoring process Methods 0.000 title abstract description 7
- 208000018737 Parkinson disease Diseases 0.000 claims abstract description 36
- 108700011259 MicroRNAs Proteins 0.000 claims abstract description 21
- 210000004369 blood Anatomy 0.000 claims abstract description 7
- 239000008280 blood Substances 0.000 claims abstract description 7
- 239000013060 biological fluid Substances 0.000 claims abstract 2
- 239000002679 microRNA Substances 0.000 claims description 23
- 238000011282 treatment Methods 0.000 claims description 11
- 238000003753 real-time PCR Methods 0.000 claims description 5
- 108091028080 MiR-132 Proteins 0.000 claims description 4
- 108091046841 MiR-150 Proteins 0.000 claims description 4
- 108091033433 MiR-191 Proteins 0.000 claims description 4
- 108091062170 Mir-22 Proteins 0.000 claims description 4
- 108091028606 miR-1 stem-loop Proteins 0.000 claims description 4
- 108091084619 miR-125b-1 stem-loop Proteins 0.000 claims description 4
- 108091027943 miR-16 stem-loop Proteins 0.000 claims description 4
- 108091056204 miR-16-2 stem-loop Proteins 0.000 claims description 4
- 108091023402 miR-26a-2 stem-loop Proteins 0.000 claims description 4
- 108091088477 miR-29a stem-loop Proteins 0.000 claims description 4
- 108091029716 miR-29a-1 stem-loop Proteins 0.000 claims description 4
- 108091092089 miR-29a-2 stem-loop Proteins 0.000 claims description 4
- 108091066559 miR-29a-3 stem-loop Proteins 0.000 claims description 4
- 108091043187 miR-30a stem-loop Proteins 0.000 claims description 4
- 108091062429 miR-487b stem-loop Proteins 0.000 claims description 4
- 238000011002 quantification Methods 0.000 claims description 4
- -1 miRIOO Proteins 0.000 claims description 3
- 238000004393 prognosis Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 2
- 238000009830 intercalation Methods 0.000 claims description 2
- 108091073704 let-7c stem-loop Proteins 0.000 claims description 2
- 230000004048 modification Effects 0.000 claims description 2
- 238000012986 modification Methods 0.000 claims description 2
- 239000012620 biological material Substances 0.000 claims 1
- 230000002596 correlated effect Effects 0.000 claims 1
- 239000007850 fluorescent dye Substances 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 210000005087 mononuclear cell Anatomy 0.000 claims 1
- 230000000079 pharmacotherapeutic effect Effects 0.000 claims 1
- 230000004044 response Effects 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 2
- 238000001356 surgical procedure Methods 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 description 18
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 18
- 230000014509 gene expression Effects 0.000 description 14
- 239000000523 sample Substances 0.000 description 11
- 208000024891 symptom Diseases 0.000 description 9
- 230000007170 pathology Effects 0.000 description 7
- 238000012360 testing method Methods 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 210000004556 brain Anatomy 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 4
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 4
- 229960004502 levodopa Drugs 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 102100026882 Alpha-synuclein Human genes 0.000 description 3
- 101000834898 Homo sapiens Alpha-synuclein Proteins 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 206010034010 Parkinsonism Diseases 0.000 description 3
- 206010044565 Tremor Diseases 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 210000003169 central nervous system Anatomy 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 239000013068 control sample Substances 0.000 description 3
- 230000007850 degeneration Effects 0.000 description 3
- 210000005064 dopaminergic neuron Anatomy 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 206010006100 Bradykinesia Diseases 0.000 description 2
- 102100022207 E3 ubiquitin-protein ligase parkin Human genes 0.000 description 2
- 208000003098 Ganglion Cysts Diseases 0.000 description 2
- 101000619542 Homo sapiens E3 ubiquitin-protein ligase parkin Proteins 0.000 description 2
- 208000006083 Hypokinesia Diseases 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 206010052904 Musculoskeletal stiffness Diseases 0.000 description 2
- 208000012902 Nervous system disease Diseases 0.000 description 2
- 206010033799 Paralysis Diseases 0.000 description 2
- 208000027089 Parkinsonian disease Diseases 0.000 description 2
- 108010032428 Protein Deglycase DJ-1 Proteins 0.000 description 2
- 102000007659 Protein Deglycase DJ-1 Human genes 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 208000005400 Synovial Cyst Diseases 0.000 description 2
- 108010005656 Ubiquitin Thiolesterase Proteins 0.000 description 2
- 102000005918 Ubiquitin Thiolesterase Human genes 0.000 description 2
- 230000002567 autonomic effect Effects 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 description 2
- 235000012754 curcumin Nutrition 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 108091079016 miR-133b Proteins 0.000 description 2
- 108091043162 miR-133b stem-loop Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000007310 pathophysiology Effects 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 230000001144 postural effect Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 229920002477 rna polymer Polymers 0.000 description 2
- 210000003523 substantia nigra Anatomy 0.000 description 2
- ZIUSSTSXXLLKKK-KOBPDPAPSA-N (1e,4z,6e)-5-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-3-one Chemical compound C1=C(O)C(OC)=CC(\C=C\C(\O)=C\C(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 ZIUSSTSXXLLKKK-KOBPDPAPSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- PLRACCBDVIHHLZ-UHFFFAOYSA-N 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Chemical compound C1N(C)CCC(C=2C=CC=CC=2)=C1 PLRACCBDVIHHLZ-UHFFFAOYSA-N 0.000 description 1
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- 208000007848 Alcoholism Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 241000796533 Arna Species 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 208000012661 Dyskinesia Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010018341 Gliosis Diseases 0.000 description 1
- 206010019196 Head injury Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 208000008771 Lymphadenopathy Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091028108 MiR-212 Proteins 0.000 description 1
- 108091062154 Mir-205 Proteins 0.000 description 1
- 101001135571 Mus musculus Tyrosine-protein phosphatase non-receptor type 2 Proteins 0.000 description 1
- 238000011887 Necropsy Methods 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 101150023114 RNA1 gene Proteins 0.000 description 1
- 206010071390 Resting tremor Diseases 0.000 description 1
- 235000004789 Rosa xanthina Nutrition 0.000 description 1
- 241000109329 Rosa xanthina Species 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 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 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 201000007930 alcohol dependence Diseases 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 210000004227 basal ganglia Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000006990 cholangiocarcinoma Diseases 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 229940109262 curcumin Drugs 0.000 description 1
- 239000004148 curcumin Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 230000007387 gliosis Effects 0.000 description 1
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 208000003906 hydrocephalus Diseases 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 201000007450 intrahepatic cholangiocarcinoma Diseases 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 108091033753 let-7d stem-loop Proteins 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108091092072 miR-100 stem-loop Proteins 0.000 description 1
- 108091053935 miR-212 stem-loop Proteins 0.000 description 1
- 108091028397 miR-212-1 stem-loop Proteins 0.000 description 1
- 108091028945 miR-212-2 stem-loop Proteins 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 238000002610 neuroimaging Methods 0.000 description 1
- 239000003176 neuroleptic agent Substances 0.000 description 1
- 230000000701 neuroleptic effect Effects 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 208000037821 progressive disease Diseases 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 201000002212 progressive supranuclear palsy Diseases 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
MéTODO PARA DIAGNóSTICO E MONITORAMENTO DE ALFA-SINUCLEINOPATIAS PELA DETERMINAçáO DE NìVEIS DE MIRNAS EM AMOSTRAS BIOLóGICAS. A presente invenção revela um método de diagnóstico/prognóstico de alfa-sinucleinopatias como a Doença de Parkinson. O método da invenção compreende a detecção do nível de microRNAs maduros específicos em amostras de fluidos biológicos como o sangue, sendo útil para o diagnóstico, monitoramento de resposta a tratamento químico ou cirúrgico.METHOD FOR DIAGNOSIS AND MONITORING OF ALPHA SYNUCLEINOPATHIES BY THE DETERMINATION OF MIRN LEVELS IN BIOLOGICAL SAMPLES. The present invention discloses a diagnostic / prognostic method of alpha synucleinopathies such as Parkinson's disease. The method of the invention comprises detecting the level of specific mature microRNAs in biological fluid samples such as blood and is useful for diagnosis, monitoring response to chemical or surgical treatment.
Description
Relatório Descritivo de Patente de InvençãoPatent Invention Descriptive Report
Método para diagnóstico ε monitoramento de alfa-sinucleinopatias peladeterminação de níveis de MlRNAS em amostras biológicasMethod for diagnosis and monitoring of alpha synucleinopathies by determining MlRNAS levels in biological samples
Campo da InvençãoField of the Invention
A presente invenção refere-se a métodos in vitro de diagnóstico e/ouprognóstico. Mais especificamente, a presente invenção proporciona ummétodo para o diagnóstico e/ou prognóstico de alfa-sinucleinopatias, como aDoença de Parkinson. O método da invenção compreende a quantificaçãocomparativa de miRNAs de amostras de indivíduos suspeitos ou acometidos dealfa-sinucleinopatias, proporcionando informação diagnostica/prognostica útilna definição de condutas clínicas.The present invention relates to in vitro diagnostic and / or prognostic methods. More specifically, the present invention provides a method for the diagnosis and / or prognosis of alpha synucleinopathies such as Parkinson's disease. The method of the invention comprises the comparative quantitation of miRNAs from samples of suspected or afflicted individuals with lymphadenopathies, providing useful diagnostic / prognostic information in defining clinical management.
Antecedentes da InvençãoBackground of the Invention
A Doença de Parkinson (DP) é uma doença neurodegenerativa crônicaque acomete homens e mulheres de diferentes raças e classes sociais.Estudos epidemiológicos têm demonstrado que a DP ocorre em 1.81-3.32% dosindivíduos com idade superior a 65 anos. Frente ao aumento da expectativa devida da população mundial torna-se evidente a tendência ao aumento donúmero de pessoas potencialmente acometidas pela DP e a importância doadequado diagnóstico nestes pacientes. Também tem sido demonstrado piorana qualidade de vida dos indivíduos com DP3"5 e de seus cuidadores4.Parkinson's disease (PD) is a chronic neurodegenerative disease that affects men and women of different races and social classes. Epidemiological studies have shown that PD occurs in 1.81-3.32% of individuals over 65 years of age. Due to the increase in the due expectation of the world population, it is evident the tendency to increase the number of people potentially affected by PD and the importance of proper diagnosis in these patients. Worsening quality of life of individuals with PD3 "5 and their caregivers4 has also been shown.
A Doença de Parkinson é uma alfa-sinucleinopatia caracterizada pelaocorrência de sintomas motores como rigidez muscular, tremor de repouso,bradicinesia e instabilidade postural6. Os sintomas não-motores da DP incluemdisfunções autonômicas, sensoriais e neuropsiquiátricas7.Parkinson's disease is an alpha-synucleinopathy characterized by the occurrence of motor symptoms such as muscle stiffness, resting tremor, bradykinesia and postural instability6. Nonmotor symptoms of PD include autonomic, sensory, and neuropsychiatric dysfunction7.
As características clínicas da DP e a sua progressão são muito variáveispara cada paciente. Existem diferentes propostas para os critérios a seremconsiderados no diagnóstico clínico da DP. Os critérios propostos pelo Bancode Cérebros de Londres6 têm sido utilizados e consistem em três etapas: (1)Na primeira etapa é avaliada a presença de sintomas compatíveis com asíndrome parkinsoniana, na qual é necessária a presença de bradicinesiaassociada a pelo menos uma das seguintes manifestações: rigidez muscular,tremor de repouso, instabilidade postural. (2) Na segunda etapa sãoconsiderados os critérios de exclusão para a DP, ou seja, o indivíduo não podeapresentar: história de acidentes vasculares encefálicos de repetição comprogressão em degraus de sintomas, história de traumas cranianos repetidos,antecedente comprovado de encefalite, crises oculógiras, uso de neurolépticodesde o início dos sintomas da doença, mais que um caso de acometimentofamiliar, remissão prolongada de sintomas, persistência de acometimentounilateral após três anos, paralisia ocular supranuclear, sinais cerebelares,acometimento autonômico precoce e acentuado, demência em fases iniciais dadoença, sinais piramidais, presença de lesões expansivas intra-cranianas(tumores, hidrocefalia à neuroimagem), exposição ao MPTP, má-respostaterapêutica a altas doses de levodopa. (3) Na terceira etapa estão os critériosdenominados como de sustentação para o diagnóstico da DP, sendonecessário três ou mais dos seguintes: início unilateral, acometimentoassimétrico; doença progressiva; assimetria persistente afetandoprincipalmente o lado de início da doença; resposta excelente à levodopa(melhora de 70 a 100%); resposta à levodopa por cinco anos ou mais;discinesia induzida pela terapia com levodopa; evolução clínica igual ousuperior a dez anos.The clinical characteristics of PD and its progression vary greatly for each patient. There are different proposals for the criteria to be considered in the clinical diagnosis of PD. The criteria proposed by the London Brain Bank6 have been used and consist of three stages: (1) In the first stage, the presence of symptoms compatible with Parkinson's syndrome is evaluated, in which the presence of bradykinesia associated with at least one of the following manifestations is required: muscle stiffness, tremor at rest, postural instability. (2) In the second stage, the exclusion criteria for PD are considered, that is, the individual cannot present: history of recurrent stroke, symptom regression, history of repeated head trauma, proven history of encephalitis, ocular seizures, neuroleptic use since the onset of disease symptoms, more than one case of familial involvement, prolonged remission of symptoms, persistence of bilateral involvement after three years, supranuclear ocular paralysis, cerebellar signs, early and severe autonomic involvement, dementia in early stages, pyramidal signs , presence of intra-cranial expansive lesions (tumors, neuroimaging hydrocephalus), MPTP exposure, poor therapeutic response to high doses of levodopa. (3) In the third stage are the criteria called support for the diagnosis of PD, requiring three or more of the following: unilateral onset, asymmetric involvement; progressive disease; persistent asymmetry affecting mainly the onset side of the disease; excellent response to levodopa (70 to 100% improvement); response to levodopa for five years or more levodopa therapy-induced dyskinesia; clinical course of more than ten years.
Atualmente, o diagnóstico definitivo para DP só é possível com aconfirmação pela necropsia. Os critérios propostos para confirmaçãohistopatológica da DP consistem em redução substancial de células nervosasna substância negra acompanhada de gliose; pelo menos um corpúsculo deLewy na substância negra ou no Iocus ceruleus (devem ser examinadas pelomenos quatro diferentes seções em cada uma dessas áreas antes de concluirque os corpúsculos de Lewy estão ausentes); nenhuma evidência patológicapara outra doença que produza parkinsonismo, por exemplo: paralisiasupranuclear progressiva, degeneração cortico-gânglio-basal, atrofia demúltiplos sistemas8. Ressalta-se, desta forma, a importância da diferenciaçãoentre DP e outras doenças que apresentem parkinsonismo como paralisiasupranuclear progressiva, degeneração cortico-gânglio-basal, atrofia demúltiplos sistemas.Currently, the definitive diagnosis for PD is only possible with confirmation by necropsy. The proposed criteria for histopathological confirmation of PD are substantial reduction of nerve cells in the substantia nigra accompanied by gliosis; at least one Lewy corpuscle in substantia nigra or Iocus ceruleus (at least four different sections should be examined in each of these areas before concluding that Lewy corpuscles are absent); no pathological evidence for another disease that produces parkinsonism, for example: progressive supranuclear palsy, cortico-ganglion-basal degeneration, multiple system atrophy8. Thus, the importance of differentiation between PD and other diseases that present parkinsonism as progressive paralysis, supranuclear, cortico-ganglion-basal degeneration, atrophy of multiple systems is emphasized.
Os sinais e sintomas motores da DP devem-se, primariamente, aalterações funcionais dos núcleos da base. De fato, a progressão dasdificuldades motoras na DP reflete a gradual degeneração dos neurôniosdopaminérgicos nigroestriatais. No entanto, ressalta-se que a partir dosachados descritos por Braak e cols (2002)9 foi destacado o envolvimento dediferentes estruturas cerebrais, além dos núcleos da base, na fisiopatologia daDP o que auxilia a compreender as diferentes manifestações motoras e nãomotoras da DP. Apesar dos avanços na compreensão da fisiopatologia da DP asua etiologia ainda não está completamente estabelecida. Deve-se destacarque o diagnóstico de DP permanece unicamente baseado em critérios clínicose na experiência da avaliação realizada pelo médico neurologista. Ressalta-seque diversos quadros clínicos cursam com a ocorrência tremor o que reforça anecessidade do adequado diagnóstico diferencial. Estudos atuais sugerem queé freqüente a presença de sintomas não motores antecedendo os sintomasmotores e desta forma o diagnóstico de DP10.The motor signs and symptoms of PD are primarily due to functional alterations of the base nuclei. In fact, the progression of motor difficulties in PD reflects the gradual degeneration of nigrostriatal dopaminergic neurons. However, it is noteworthy that from the findings described by Braak et al (2002) 9, the involvement of different brain structures, as well as the basal nuclei, in the pathophysiology of PD was highlighted, which helps to understand the different motor and non-motor manifestations of PD. Despite advances in understanding the pathophysiology of PD, its etiology is not yet fully established. It should be noted that the diagnosis of PD remains solely based on clinical criteria and in the experience of the evaluation performed by the neurologist. It is noteworthy that several clinical pictures are associated with tremor, which reinforces the need for adequate differential diagnosis. Current studies suggest that the presence of nonmotor symptoms is frequent preceding motor symptoms and thus the diagnosis of PD10.
Pesquisas em biologia molecular têm estudado e identificado diferentesmicroRNAs (miRNAs)11. As miRNAs são seqüências de aproximadamente 22nucleotídeos de RNA (ácido ribonucléico) não-codificante e tem sido propostoque regulem a expressão do gene através do emparelhamento base-específicocom RNAmensageiro (RNAm) alvo. Até o momento entende-se que a maioriado miRNAs, em espécies animais, atuem através da inibição da efetivatradução do RNAm de genes alvo através de um imperfeito pareamento debase com a região 3'UTR (untranslated region) dos RNAm alvo. Essemecanismo parece estar envolvido com a inibição do início da tradução12. Em2007, foi identificado um miRNA, miR-133b, que é expresso em neurôniosdopaminérgicos. O miR-133b regula a maturação e função de neurôniosdopaminérgicos e foi identificado como deficiente no cérebro de pacientes com DP13.A ocorrência de alterações na expressão de miRNA também foi descritaem células tumorais e tem sido proposto que tais alterações possam contribuirno desenvolvimento do câncer14. Também foi identificado modificação naexpressão de miRNA em líquido cérebro-espinhal relacionados à Doença deAlzheimer15.Research in molecular biology has studied and identified different microRNAs (miRNAs) 11. MiRNAs are sequences of approximately 22 non-coding RNA (ribonucleic acid) nucleotides and have been proposed to regulate gene expression through base-specific pairing with target messenger RNA (mRNA). So far, it is understood that most miRNAs in animal species act by inhibiting effective mRNA translation of target genes through imperfect pairing with the 3'UTR (untranslated region) of target mRNAs. This mechanism seems to be involved with inhibiting the onset of translation12. In 2007, an miRNA, miR-133b, which is expressed in dopaminergic neurons was identified. MiR-133b regulates the maturation and function of dopaminergic neurons and has been identified as deficient in the brain of patients with DP13. The occurrence of changes in miRNA expression has also been described in tumor cells and it has been proposed that such changes may contribute to cancer development14. Modification of miRNA expression in cerebrospinal fluid related to Alzheimer's disease has also been identified15.
Considerado o potencial da quantificação de miRNA para o diagnósticode diversas patologias a necessidade de utilizar tecido para essa dosagemdificulta a utilização desta medida em patologias neurológicas. No entanto, apossibilidade da quantificação de miRNAs em sangue venoso periféricopoderia ser considerado como uma ferramenta útil e de baixo risco secomparado a procedimentos como biopsia cerebral e punção de líquidocérebro-espinhal.Considering the potential of miRNA quantification for the diagnosis of various pathologies, the need to use tissue for this measurement makes it difficult to use this measure in neurological pathologies. However, the possibility of quantifying miRNAs in peripheral venous blood could be considered as a useful and low-risk tool compared to procedures such as brain biopsy and spinal fluid puncture.
A literatura científica que circunscreve os objetos da invenção, semcontudo antecipá-los ou sequer sugeri-los é apresentada a seguir.The scientific literature that circumscribes the objects of the invention, however anticipating or even suggesting them, is presented below.
1. De Rijk MC, Launer LJ, Berger K, Breteler MM, Dartigues JF1Baldereschi M, Fratiglioni L, Lobo A, Martinez-Lage J, Trenkwalder C,Hofman A. Prevalence of Parkinson's disease in Europe: A collaborativestudy of population^based cohorts. Neurologic Diseases in the ElderlyResearch Group. Neurology.2000·, 54(11) Suppl 5: S21-3.1. From Rijk MC, Launer LJ, Berger K, Breteler MM, Dartigues JF1Baldereschi M, Fratiglioni L, Wolf A, Martinez-Lage J, Trenkwalder C, Hofman A. Prevalence of Parkinson's disease in Europe: A collaborative study of population ^ based cohorts . Neurologic Diseases in the ElderlyResearch Group. Neurology 2000, 54 (11) Suppl 5: S21-3.
2. Barbosa MT, Caramelli P, Maia DP, Cunningham MCQ, GuerraHL, Lima-Costa MF, Cardoso F. Parkinsonism and Parkinson's disease in theelderly: a community-based survey in Brazil: (the Bambuí study). Mov Disord.2006; 21(6):800-8.2. Barbosa MT, Caramelli P, Maia DP, Cunningham MCQ, GuerraHL, Lima-Costa MF, Cardoso F. Parkinsonism and Parkinson's disease in theelderly: a community-based survey in Brazil: (the Bambuí study). Mov Disord.2006; 21 (6): 800-8.
3. Chrischilles EA, Rubensteib LM, Voelker MD, Wallace RB,Rodnitzky RL. The health burdens of Parkinson's disease. Mov Disord.1998;13(3):406-13.3. Chrischilles EA, Rubensteib LM, Voelker MD, Wallace RB, Rodnitzky RL. The health burdens of Parkinson's disease. Mov Disord.1998; 13 (3): 406-13.
4. Schestatsky P, Zanatto VC, Margis R, Chachamovich E1 RecheM, Batista RG, Ficke D, Rieder CRM. Quality of Iife in a Brazilian sample ofpatients with Parkinson's disease and their caregivers. Rev BrasPsiquiatr.2006] 28(3):209-11.5. Rahman S, Griffin HJ1 Quinn NP1 Jahanshahi Μ. Quality of Iifein Parkinson's Disease: the relative importance of the symptoms. Mov Disord.2008; 23(10),1428-34.4. Schestatsky P, Zanatto VC, Margis R, Chachamovich E1 RecheM, Batista RG, Ficke D, Rieder CRM. Quality of Iife in a Brazilian sample of patients with Parkinson's disease and their caregivers. Rev BrasPsiquiatr.2006] 28 (3): 209-11.5. Rahman S, Griffin HJ1 Quinn NP1 Jahanshahi Μ. Quality of Iifein Parkinson's Disease: the relative importance of the symptoms. Mov Disord.2008; 23 (10), 1428-34.
6. Hughes AJ1 Daniel SE1 Kilford L, Lees AJ. Accuracy of clinicaidiagnosis of idiopathic Parkinsosn's disease: a clinic-pathological study of100 cases. J Neurol Neurosurg PsychiatyrAQ92\ 55:181-4.6. Hughes AJ1 Daniel SE1 Kilford L, Lees AJ. Accuracy of clinical diagnosis of idiopathic Parkins' disease: a clinic-pathological study of 100 cases. J Neurol Neurosurg Psychiatyr AQ92 55: 181-4.
7. Poewe W. Non-motor symptoms in Parkinson's disease. Eur JNeuroi 2008; 15(Suppl 1): 14-20.7. Poewe W. Non-motor symptoms in Parkinson's disease. Eur JNeuroi 2008; 15 (Suppl 1): 14-20.
8. Gelb D; Oliver E; Gilman SAN. Diagnostic criteria for parkinsondisease. Archieves of Neurology(56): 33-39.8. Gelb D; Oliver E; Gilman SAN. Diagnostic criteria for parkinsondisease. Archieves of Neurology (56): 33-39.
9. Braak H, Ghebremedhin E1 Rüb U1 Bratzke H, Del Tredici K.Stages in the development of Parkinson's disease-related pathology. CellTissue Res.2004; 318:121-34.9. Braak H, Ghebremedhin E1 Rüb U1 Bratzke H, Del Tredici K. Stages in the development of Parkinson's disease-related pathology. CellTissue Res.2004; 318: 121-34.
10. Chaudhuri RK, Naidu, Y. Early Parkinson' disease and non-motor issues. J Neurol. 2008; 225(suppl5): 33-38.10. Chaudhuri RK, Naidu, Y. Early Parkinson's disease and non-motor issues. J Neurol. 2008; 225 (suppl5): 33-38.
11. Alvarez-Garcia I, Miska EA. MicroRNA functions in animaldevelopment and human disease. Development 2005, 132:4653-4662.11. Alvarez-Garcia I, Miska EA. MicroRNA functions in animaldevelopment and human disease. Development 2005, 132: 4653-4662.
12. Bartel DP. MicroRNAs: genomics, biogenesis.mecjhanism andfunction Cell 2004, 116: 281-297.12. Bartel DP. MicroRNAs: genomics, biogenesis.mecjhanism andfunction Cell 2004, 116: 281-297.
13. Kim J, Inoue K1 Ishii J1 Vanti WB et al. A microRNA feedbackcircuit in midbrain dopamine neurons. Science 2007; 317: 1220-1224.13. Kim J, Inoue K1 Ishii J1 Vanti WB et al. The feedbackcircuit microRNA in midbrain dopamine neurons. Science 2007; 317: 1220-1224.
14. Meltzer PS. Small RNAs with big impacts Nature. 2005;435:745-6.14. Meltzer PS. Small RNAs with big impacts Nature. 2005; 435: 745-6.
15. Cogswell JP1 Taylor IA1 Waters , Shi Y1 Cannon , Keinar K1Kemppainen J, Brown D1 Chen C1 Prinjha RK1 Richardson JC1 Saunders AM,Roses AD1 Riehards CA. Identification of miRNA changes in Alzheimer 'sdiseae brain and CSF yields putative biomarkers ad insights into diseasepathways. JAIzheimerDis. 2008;14(1):27-41.15. Cogswell JP1 Taylor IA1 Waters, Shi Y1 Cannon, Keinar K1Kemppainen J, Brown D1 Chen C1 Prinjha RK1 Richardson JC1 Saunders AM, Roses AD1 Riehards CA. Identification of miRNA changes in Alzheimer's brain and CSF yields putative biomarkers ad insights into disease pathways. JAIzheimerDis. 2008; 14 (1): 27-41.
A literatura patentária contempla documentos apenas parcialmenterelacionados aos objetos da presente invenção.O pedido internacional de patente WO 2009/009457 "ALZHElMER1SDISEASE-SPECIFIC MICRO-RNA MICROARRAY AND RELATEDMETHODS", depositado pela Fundação de Pesquisa da Universidade deLouisville, revela métodos de diagnóstico e/ou prognóstico de Alzheimer pelamedida da quantidade, em uma amostra biológica, de um ou mais micro-RNAsrelacionados a Alzheimer.The patent literature contemplates documents only partially related to the objects of the present invention.The international patent application WO 2009/009457 "ALZHElMER1SDISEASE-SPECIFIC MICRO-RNA MICROARRAY AND RELATEDMETHODS", filed by the Research Foundation of the University of Louisville, discloses diagnostic methods and / or Alzheimer's prognosis by measuring the amount, in a biological sample, of one or more Alzheimer's-related microRNAs.
O pedido internacional de patente WO 2008/153692 "MICRORNAEXPRESSION PROFILING OF CEREBROSPINAL FLUID", depositado porTHE BRIGHAM AND WOMEN1S HOSPITAL, INC., revela métodos dediagnóstico nos quais o nível de determinado microRNAs específicos sãodeterminados no fluido cérebro-espinhal de um indivíduo. O método pode serusado para o diagnóstico ou monitoramento de doenças neurológicas,especialmente tumores cerebrais.International patent application WO 2008/153692 "MICRORNAEXPRESSION PROFILING OF CEREBROSPINAL FLUID", filed by THE BRIGHAM AND WOMEN1S HOSPITAL, INC., Discloses diagnostic methods in which the level of certain specific microRNAs are determined in an individual's cerebrospinal fluid. The method may be used for the diagnosis or monitoring of neurological diseases, especially brain tumors.
O pedido internacional de patente WO 2007/107789 "TREATMENT OFCNS CONDITIONS", depositado por Silentis S.A., revela métodos ecomposições para o tratamento de condições patológicas do sistema nervosocentral, por meio da administração intranasal de uma composição que modula,por RNA interferência, a expressão e/ou atividade de genes envolvidos em taiscondições.International patent application WO 2007/107789 "TREATMENT OFCNS CONDITIONS", filed by Silentis SA, discloses methods and compositions for the treatment of pathological conditions of the central nervous system by intranasally administering a composition that modulates, by interference RNA, the expression and / or activity of genes involved in such conditions.
O pedido internacional de patente WO 2009/025852 "METHODS OFUSING miRNA FOR DETECTION OF IN VIVO CELL DEATH", depositado porXenomics, INC. revela métodos não-invasivos para a detecção de mortecelular, através da medida de miRNAs tecido-específicos. O método pode seraplicado na detecção de patologias causadas ou acompanhadas de mortecelular, efeitos citotóxicos induzidos por fatores químicos ou físicos e apresença de anormalidades fetais específicas.International Patent Application WO 2009/025852 "METHODS OFUSING MICRO FOR DETECTION OF IN VIVO CELL DEATH", filed by Xenomics, INC. discloses noninvasive methods for the detection of cell death by measuring tissue-specific miRNAs. The method can be applied to detect pathologies caused or accompanied by cell death, chemical or physical factor-induced cytotoxic effects, and the presence of specific fetal abnormalities.
O pedido internacional de patente WO 2009/026416 "SHORT-CONTROLLING NUCLEIC ACIDS USEFUL IN THE TREATMENT ANDDETECTION OF DISEASES", depositado por VDX LLC, revela métodos para autilização de seqüências curtas controladores (scRNAs) para triagem eidentificação de ácidos nucléicos alvo que se liguem a seqüências de scRNAsem loop. Também são revelados métodos para detectar scRNAs e métodospara diagnóstico e tratamento úteis de doenças nas quais scRNAs ou níveisaberrantes dos mesmos são expressos.International patent application WO 2009/026416 "SHORT-CONTROLLING NUCLEIC ACIDS USEFUL IN THE TREATMENT AND DETECTION OF DISEASES", filed by VDX LLC, discloses methods for the use of short scRNAs for screening and identification of targeting nucleic acids that bind the scRNA sequences without loop. Also disclosed are methods for detecting scRNAs and methods for useful diagnosis and treatment of diseases in which scRNAs or aberrant levels are expressed.
O pedido internacional de patente WO 05045034, "RNAINTERFERENCE MEDIATED TREATMENT OF PARKINSON DISEASEUSING SHORT INTERFERING NUCLEIC ACID (siRNA)", depositado porSIRNA Therapeutics1 INC., revela métodos e reagentes úteis na modulação degenes de Parkinson, por exemplo, a expressão gênica de PARK1 (SNCA),PARK2, PARK7, e/ou PARK5 em uma variedade de aplicações, incluindoterapêutica, diagnostica, validação de alvos e descobertas genômicas.Especificamente, são reveladas moléculas pequenas de ácido nucleico, comosiNA, siRNA, dsRNA, miRNA, e shRNA, como moléculas capazes de mediar aRNA interferência contra a expressão gênica e/ou atividade de SNCA. Asreferidas moléculas são propostas como úteis no diagnóstico e tratamento deDoença de Parkinson e a qualquer outra doença ou condição que responda àmodulação da expressão ou atividade de PARK1 (SNCA), PARK2, PARK7,e/ou PARK5.International patent application WO 05045034, "RNAINTERFERENCE MEDIATED TREATMENT OF PARKINSON DISEASEUSING SHORT INTERFERING NUCLEIC ACID (siRNA)", filed by SIRNA Therapeutics1 INC., Discloses methods and reagents useful in modulating Parkinson's degens, for example, the gene expression of PARK1 ( SNCA), PARK2, PARK7, and / or PARK5 in a variety of applications, including therapeutics, diagnostics, target validation, and genomic discovery. Specifically, small nucleic acid molecules, such as siRNA, dsRNA, miRNA, and shRNA, are disclosed. molecules capable of mediating aRNA interference against CNS gene expression and / or activity. Said molecules are proposed as useful in the diagnosis and treatment of Parkinson's disease and any other disease or condition that responds to modulation of the expression or activity of PARK1 (CNS), PARK2, PARK7, and / or PARK5.
Não foram encontrados no estado da arte antecedentes que antecipemintegralmente, ou sequer sugiram, mesmo que em combinação, a estratégia dapresente invenção.No prior art was found in the prior art that fully anticipates, or even suggests, even in combination, the strategy of the present invention.
Sumário da InvençãoSummary of the Invention
É um dos objetos da presente invenção proporcionar métodos dediagnóstico e/ou prognóstico de alfa-sinucleinopatias, como a Doença deParkinson.It is an object of the present invention to provide diagnostic and / or prognostic methods for alpha synucleinopathies such as Parkinson's Disease.
Em um aspecto, sendo, portanto, um dos objetos da invenção, éproporcionado um método que compreende a quantificação comparativa demiRNAs de amostras de indivíduos suspeitos ou acometidos de alfa-sinucleinopatias.In one aspect, therefore, being one of the objects of the invention, there is provided a method comprising comparative quantification of demiRNAs from samples of individuals suspected or afflicted with alpha-synucleinopathies.
É também um outro objeto da presente invenção proporcionar ummétodo diagnóstico/prognóstico que compreende a quantificação comparativade miRNAs (microRNAs) selecionados de um grupo que compreende: miR 1,miR16-2*, miR22*, miR26a2*, miR29a, miR30a, miR487b, let7c*, miR16,miR100, miR150, miR125-b1*, miR132 e miR191, ou ainda combinações dosmesmos.It is also another object of the present invention to provide a diagnostic / prognostic method comprising comparative quantitation of miRNAs (microRNAs) selected from a group comprising: miR 1, miR16-2 *, miR22 *, miR26a2 *, miR29a, miR30a, miR487b, let7c *, miR16, miR100, miR150, miR125-b1 *, miR132 and miR191, or combinations of the same.
Estes e outros da presente invenção serão melhor compreendidos evalorizados a partir da descrição detalhada da invenção e das reivindicaçõesanexas.These and others of the present invention will be better understood and appreciated from the detailed description of the invention and the appended claims.
Breve Descrição das FigurasBrief Description of the Figures
A figura 1 mostra o padrão de expressão de microRNAs com níveisalterados na comparação entre indivíduos controle (Cnt) e diagnosticadosclinicamente como apresentando a Doença de Parkinson (PD) - Grupo I (Lowtemperatura).Figure 1 shows the pattern of expression of microRNAs with altered levels in the comparison between control (Cnt) and clinically diagnosed individuals with Parkinson's Disease (PD) - Group I (Low Temperature).
A figura 2 mostra o padrão de expressão de microRNAs com níveisalterados na comparação entre indivíduos controle (Cnt) e diagnosticadosclinicamente como apresentando a Doença de Parkinson (PD) - Grupo Il (Hightemperature).Figure 2 shows the pattern of expression of altered level microRNAs in the comparison between control (Cnt) and clinically diagnosed individuals with Parkinson's Disease (PD) - Group II (Hightemperature).
Descrição Detalhada da InvençãoDetailed Description of the Invention
Os detalhes relatados a seguir visam facilitar a reprodução da invenção,devendo, portanto, ser compreendidos como meramente ilustrativos, sem comisso restringir o escopo da invenção.The following details are intended to facilitate the reproduction of the invention and should therefore be construed as merely illustrative without restricting the scope of the invention.
A presente invenção é baseada no fato de que alguns miRNAs sãoexpressos em diferentes níveis em sangue periférico, quando avaliadopacientes com Doença de Parkinson em relação a indivíduos sem a doença.Esta observação permite considerar a utilização de sangue periférico naavaliação desta patologia. A comparação pode ser feita entre os níveis demiRNA em sangue venoso de um determinado indivíduo em relação acontroles. A comparação pode ser feita de forma direta ou pelo cálculo darazão entre os microRNAs e a determinação da presença da patologia (e suaevolução ou remissão) se a relação exceder limites de 25, 50 ou 75% emrelação aos níveis observados em indivíduos controle, sem a doença.The present invention is based on the fact that some miRNAs are expressed at different levels in peripheral blood when Parkinson's Disease patients are evaluated in relation to individuals without the disease. This observation allows to consider the use of peripheral blood in the evaluation of this pathology. The comparison can be made between the demiRNA levels in venous blood of a particular individual in relation to controls. The comparison can be made directly or by calculating the ratio between microRNAs and determining the presence of the pathology (and its evolution or remission) if the ratio exceeds 25, 50 or 75% limits relative to the levels observed in control subjects without disease.
A invenção é direcionada para o diagnóstico e monitoramento de umaalfa-sinucleinopatia, a Doença de Parkinson, a partir de amostra de sanguevenoso em um determinado indivíduo. O termo diagnóstico se refere àdetecção da doença. O termo monitoramento se refere aos testes realizadosem pacientes com o diagnóstico prévio da referida doença tendo comoproposta medir a progressão da doença ou efeito do tratamento.The invention is directed to the diagnosis and monitoring of an alpha-synucleinopathy, Parkinson's Disease, from a blood sample in a given individual. The term diagnosis refers to the detection of the disease. The term monitoring refers to tests performed on patients with a previous diagnosis of the disease, with the purpose of measuring disease progression or treatment effect.
Em uma concretização preferencial, o método da invenção compreendea obtenção de uma amostra de sangue venoso periférico de um dado indivíduoe a análise dessa amostra para determinar a concentração ou quantidade deuma série de até quatorze microRNAs em análise. Os resultados obtidos sãocomparados com os obtidos de amostras controle. A amostra controle provémde indivíduos livres da doença. No caso em que o método for utilizado paramonitorar a doença, a amostra "controle" é o resultado do teste obtido dopróprio paciente na medida anterior. Ou seja, o paciente pode ser avaliado paramudanças nos níveis de miRNAs em diferentes momentos e estágios dadoença, assim como previamente e posteriormente a determinada intervençãoterapêutica (medicação ou cirurgia).In a preferred embodiment, the method of the invention comprises obtaining a peripheral venous blood sample from a given subject and analyzing that sample to determine the concentration or amount of a series of up to fourteen microRNAs under analysis. The results obtained are compared with those obtained from control samples. The control sample comes from disease free individuals. In case the method is used to monitor the disease, the "control" sample is the result of the test obtained from the patient in the previous measure. That is, the patient can be evaluated for changes in miRNA levels at different times and stages of the disease, as well as before and after a specific therapeutic intervention (medication or surgery).
Ressalta-se que não é necessário que a amostra controle seja obtida eanalisada no mesmo momento em que esteja sendo realizada a análise daamostra teste. Uma vez que os dados "controle" podem ser pré-estabelecidos,esses níveis poderão fornecer a base para comparação sem a necessidade derepetir uma nova análise do controle para cada teste a ser analisado. Acomparação entre as amostras teste e controle fornece a base para aconclusão quanto ao fato de o indivíduo ser acometido por uma alfa-sinucleinopatia (por exemplo, Doença de Parkinson) em análise no caso dométodo estar sendo utilizado para o diagnóstico ou se a doença estáprogredindo ou regredindo; no caso do método estar sendo utilizado paramonitorar a patologia. De um modo geral, quanto maior diferença entre aamostra teste e a amostra controle, mais intensa a indicação da presença dadoença.It is noteworthy that it is not necessary for the control sample to be obtained and analyzed at the same time as the analysis of the test sample. Since "control" data can be pre-established, these levels can provide the basis for comparison without having to repeat a new control analysis for each test to be analyzed. Comparison between test and control samples provides the basis for the conclusion that the individual is afflicted with an alpha-synucleinopathy (eg, Parkinson's disease) under consideration if the method is being used for diagnosis or if the disease is progressing or regressing; if the method is being used to monitor the pathology. In general, the greater the difference between the test sample and the control sample, the more intense the indication of the presence of the disease.
Os microRNAs empregados no método da invenção incluem: miR-1;miR16-2*; miR22*; miR26a2*;miR29a; miR30a; miR487b; Iet7c*; miR16,miRIOO, miR150, miR125-b1*, miR132 e miR191. As designações fornecidasestão associadas a seqüências específicas que podem ser encontradas noregistro de microRNA (http://microrna.sanger.ac.uk/sequences/). OsmicroRNAs referem-se a seqüências encontradas em humanos, ilustrada/os natabela 1.The microRNAs employed in the method of the invention include: miR-1; miR16-2 *; miR22 *; miR26a2 * miR29a; miR30a; miR487b; Iet7c *; miR16, miRIOO, miR150, miR125-b1 *, miR132 and miR191. The designations provided are associated with specific sequences that can be found in the microRNA register (http://microrna.sanger.ac.uk/sequences/). OsmicroRNAs refer to sequences found in humans, illustrated in Table 1.
Tabela 1: Seqüência nucleotídica dos microRNAs maduros, com os quais foidesenvolvido a metodologia para diagnóstico de alfa-sinucleinopatias.Table 1: Nucleotide sequence of mature microRNAs, with which the methodology for the diagnosis of alpha synucleinopathies was developed.
<table>table see original document page 11</column></row><table>Por vezes, há membros da família destes miRNAs que são reconhecidose os quais poderiam ser considerados como equivalentes a seqüênciaespecífica. Apesar das seqüências serem apresentadas como seqüências deRNA1 deve ser compreendido que se referindo à hibridização ou outros ensaiosas seqüências de DNA correspondente podem ser utilizadas da mesma forma.Por exemplo, seqüência de RNA pode ser transcrita ao reverso e amplificadausando a reação em cadeia da polimerase visando facilitar a detecção. Nessescasos será o DNA diretamente quantificado. Também deve ser esclarecido queo complemento da seqüência de DNA transcrita ao reverso pode ser analisadoao invés da seqüência em si. Nesse contexto, o termo complemento se refereao oligonucleotideo que apresenta a exata seqüência complementar.<table> table see original document page 11 </column> </row> <table> Sometimes there are members of the family of these miRNAs that are recognized and which could be considered as equivalent to specific sequence. Although the sequences are presented as RNA1 sequences it should be understood that referring to hybridization or other assaying corresponding DNA sequences may be used in the same way. For example, RNA sequence may be reverse transcribed and amplified using the polymerase chain reaction aiming at facilitate detection. Nessescous will be the DNA directly quantified. It should also be clarified that the complement of the reverse transcribed DNA sequence can be analyzed instead of the sequence itself. In this context, the term complement refers to oligonucleotide which has the exact complementary sequence.
Para análise dos microRNAs a partir da amostra de sangue é utilizado ométodo de PCR quantitativo, com o emprego de fluoróforos intercalantes comoo SYBR-green (ou equivalente) como descrito no trabalhos de Chen et al de2005 (Real-time quantification of microRNAs by stem-loop RT-PCR. NucleicAcids Research 33:e179) e de Jiang JM et al de 2005 (Real-time expressionprofiling of microRNA precursors in human câncer cell lines. Nucleic AcidsResearch 33: 5394-5403).For the analysis of microRNAs from the blood sample, the quantitative PCR method is used, using intercalating fluorophores such as SYBR-green (or equivalent) as described in the work of Chen et al de 2005 (Real-time quantification of microRNAs by stem- RT-PCR loop NucleicAcids Research 33: e179) and Jiang JM et al 2005 (Real-time expression profiling of microRNA precursors in human cancer cell lines. Nucleic AcidsResearch 33: 5394-5403).
Também podem ser utilizadas sondas marcadas com fluoróforosespecíficos (FAM, HEX, TAMRA ou outros) como descrito em trabalhoscorrelacionando níveis de miRNAs com o câncer (Chen et al. 2009. The role ofmicroRNA expression pattern in human intrahepatic cholangiocarcinoma.Joumal of Hepatology 50: 358-369; Childs et al. 2009. Low-Level Expression ofMicroRNAs let-7d and miR-205 Are Prognostic Markers of Head and NeckSquamous Cell Carcinoma. American Journal of Pathology 174: 736-745; Sunet al. 2008. Curcumin (diferuloylmethane) alters the expression profiles ofmicroRNAs in human pancreatic câncer cells. Molecular Câncer Therapeutics7: 464-473; Szafranska et al. 2008. Analysis of microRNAs in pancreatic fine-needle aspirates can classify benign and malignant tissues. Clinicai Chemistry54: 1716-1724; Tang et al. 2008. Effect of alcohol on miR-212 expression inintestinal epithelial cells and its potential role in alcoholic Iiver disease.Alcoholism-Clinical and Experimental Research 32: 355-364).Specific fluorophoresis-labeled probes (FAM, HEX, TAMRA, or others) may also be used as described in studies correlating miRNA levels with cancer (Chen et al. 2009. The role of microRNA expression pattern in human intrahepatic cholangiocarcinoma.Joumal of Hepatology 50: 358 -369; Childs et al 2009. Low-Level Expression of MicroRNAs let-7d and miR-205 are prognostic markers of head and neck squamous cell carcinoma American Journal of Pathology 174: 736-745; Sunet al 2008. Curcumin (diferuloylmethane) alters the expression profiles of microRNAs in human pancreatic cancer cells Molecular Cancer Therapeutics7: 464-473 Szafranska et al 2008. Analysis of microRNAs in pancreatic fine-needle aspirates can classify benign and malignant tissues Clinical Chemistry54: 1716-1724; 2008. Effect of alcohol on miR-212 epithelial cells intestinal expression and its potential role in alcoholic Iiver disease.Alcoholism-Clinical and Experimental Research 32: 355-364).
O nível de expressão dos microRNAs quantificada, tendo-se como basea metodologia de PCR em tempo real, será realizada em duas etapas. Na 1aetapa, uma série de até 14 oligonucleotídeos (multi-plex) contendo umaseqüência universal fusionada a uma seqüência microRNA específica de seisnucleotídeos será utilizado para a síntese de cDNA a partir do microRNA. Na 2aetapa, as reações de PCR em tempo real serão realizadas utilizando umoligonucleotídeo que é microRNA específico e um oligonucleotídeocomplementar a seqüência universal presente no oligonucleotídeo utilizadopara a síntese dos cDNAs.The quantified microRNA expression level based on real-time PCR methodology will be performed in two steps. In step 1, a series of up to 14 multi-plex oligonucleotides containing a universal sequence fused to a specific sixnucleotide microRNA sequence will be used for cDNA synthesis from the microRNA. In step 2, real-time PCR reactions will be performed using a microRNA that is microRNA specific and an oligonucleotide complementing the universal sequence present on the oligonucleotide used for cDNA synthesis.
As reações de PCR quantitativa em tempo real (qPCR) serão realizadasem equipamento padrão. De forma ilustrativa, considera-se que a PCRapresente etapas das reações que serão compostas de uma desnaturaçãoinicial a 94°C por 5 minutos, seguido por 40 ciclos de 10s a 94°C, 15s a 60°C e10s a 72°C. Após, as amostras serão aquecidas de 60 para 99°C com umaumento de 0,1°C/s para adquirir os dados produzidos pela curva dedesnaturação dos produtos amplificados. qRT-PCRs serão feitos em umvolume final de 20 μL composto de 10 μl de cada amostra de cDNA diluída de50 a 100 vezes em 2 μL de Platinum Taq 10x PCR buffer, 1,2 μL MgCI2 50 mM,0,4 μL dNTPs 5 mM, 0,4 μL do par de oligonucleotídeos a 10 μΜ, 3,95 μL H2O,2,0 μL SYBR green ou sonda marcada, e 0,05 μL Taq DNA polymerase (5U/μΙ-).Real-time quantitative PCR (qPCR) reactions will be performed on standard equipment. Illustratively, PCR is considered to have reaction steps that will be composed of an initial denaturation at 94 ° C for 5 minutes, followed by 40 cycles of 10s at 94 ° C, 15s at 60 ° C and 10s at 72 ° C. Afterwards, the samples will be heated from 60 to 99 ° C with an increase of 0.1 ° C / s to acquire the data produced by the denaturing curve of the amplified products. qRT-PCRs will be done in a final volume of 20 μL composed of 10 μL of each cDNA sample diluted 50 to 100 times in 2 μL Platinum Taq 10x PCR buffer, 1.2 μL 50 mM MgCl2, 0.4 μL 5 mM dNTPs , 0.4 μL of the 10 μΜ oligonucleotide pair, 3.95 μL H2O, 2.0 μL SYBR green or labeled probe, and 0.05 μL Taq DNA polymerase (5U / μΙ-).
Os versados na arte valorizarão imediatamente os importantesbenefícios decorrentes do uso da presente invenção. Variações na forma deconcretizar o conceito inventivo aqui exemplificado devem ser compreendidascomo dentro do espírito da invenção e das reivindicações anexas.Those skilled in the art will immediately appreciate the important benefits arising from the use of the present invention. Variations in the form of decoding the inventive concept exemplified herein must be understood within the spirit of the invention and the appended claims.
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| BRPI0903797A BRPI0903797B8 (en) | 2009-09-17 | 2009-09-17 | method for diagnosing and monitoring alpha-synucleinopathies by determining myrn levels in biological samples |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| BRPI0903797A BRPI0903797B8 (en) | 2009-09-17 | 2009-09-17 | method for diagnosing and monitoring alpha-synucleinopathies by determining myrn levels in biological samples |
Publications (4)
| Publication Number | Publication Date |
|---|---|
| BRPI0903797A2 true BRPI0903797A2 (en) | 2011-05-24 |
| BRPI0903797A8 BRPI0903797A8 (en) | 2017-06-06 |
| BRPI0903797B1 BRPI0903797B1 (en) | 2020-10-20 |
| BRPI0903797B8 BRPI0903797B8 (en) | 2021-07-27 |
Family
ID=44025391
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| BRPI0903797A BRPI0903797B8 (en) | 2009-09-17 | 2009-09-17 | method for diagnosing and monitoring alpha-synucleinopathies by determining myrn levels in biological samples |
Country Status (1)
| Country | Link |
|---|---|
| BR (1) | BRPI0903797B8 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020016437A1 (en) * | 2018-07-19 | 2020-01-23 | Fundació Institut D'investigació En Ciències De La Salut Germans Trias I Pujol | In vitro method for the diagnosis of synucleinopathies |
-
2009
- 2009-09-17 BR BRPI0903797A patent/BRPI0903797B8/en not_active IP Right Cessation
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020016437A1 (en) * | 2018-07-19 | 2020-01-23 | Fundació Institut D'investigació En Ciències De La Salut Germans Trias I Pujol | In vitro method for the diagnosis of synucleinopathies |
| US12065701B2 (en) | 2018-07-19 | 2024-08-20 | Fundació Institut D'investigació En Ciències De La Salut Germans Trias I Pujol | In vitro method for the diagnosis of synucleinopathies |
Also Published As
| Publication number | Publication date |
|---|---|
| BRPI0903797B8 (en) | 2021-07-27 |
| BRPI0903797B1 (en) | 2020-10-20 |
| BRPI0903797A8 (en) | 2017-06-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Crigna et al. | Cell-free nucleic acid patterns in disease prediction and monitoring—hype or hope? | |
| CA2931082C (en) | Methods of using mirnas from bodily fluids for detection and monitoring of parkinson's disease (pd) | |
| Yuan et al. | Circulating microRNAs as biomarkers for depression: many candidates, few finalists | |
| US8648017B2 (en) | Methods of using small RNA from bodily fluids for diagnosis and monitoring of neurodegenerative diseases | |
| JP5965481B2 (en) | MicroRNA profiling for diagnosis of cutaneous T-cell lymphoma (CTCL) | |
| US9556487B2 (en) | Methods of using miRNA from bodily fluids for early detection and monitoring of mild cognitive impairment (MCI) and alzheimer's disease (AD) | |
| ES2993025T3 (en) | Methods of using mirnas from bodily fluids for detection and differentiation of neurodegenerative diseases | |
| Hermansen et al. | MicroRNA biomarkers in glioblastoma | |
| US20120322680A1 (en) | Use of mirnas as biomarkers in glioma diagnosis | |
| Zadehbagheri et al. | Profiling of miRNAs in serum of children with attention-deficit hyperactivity disorder shows significant alterations | |
| US20190169690A1 (en) | Methods and compositions for diagnosis of alzheimer's disease | |
| CN105648088A (en) | AD or MCI detection marker and detection method thereof | |
| JP2010094122A (en) | Diagnosis-treatment option for head-and-neck tumor using micro-rna as biomarker | |
| BRPI0903797A2 (en) | method for diagnosis and monitoring of alpha-synucleinopathies by determining levels of mirnas in biological samples | |
| Ahmed | Importance of Micrornas in Human Cancer Development: A Molecular Analytical Approach | |
| Salama et al. | Biomarkers in Neurology | |
| CN119842905A (en) | Biomarker related to bladder cancer and application thereof | |
| JP2012000107A (en) | Diagnosis of tumor by using new small rna as biomarker | |
| Vachev et al. | Alterations of miR-320 family members as a novel diagnostic biomarkers in peripheral blood of schizophrenia patients | |
| JP2011072229A (en) | Method for performing diagnosis/choice of treatment of hypopharynx cancer by using microrna as biomarker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| B03A | Publication of a patent application or of a certificate of addition of invention [chapter 3.1 patent gazette] | ||
| B25C | Requirement related to requested transfer of rights |
Owner name: UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL (BR/RS) Free format text: A FIM DE ATENDER A TRANSFERENCIA, REQUERIDA ATRAVES DA PETICAO NO 860140083103 DE 29/05/2014, E NECESSARIO ESCLARECER A DIVERGENCIA ENTRE O NOME DA EMPRESA CEDENTE E O NOME QUE CONSTA NO DOCUMENTO DE CESSAO E APRESENTAR DOCUMENTO QUE COMPROVE QUE O PROCURADOR TEM PODERES PARA FAZER A CESSAO. |
|
| B25A | Requested transfer of rights approved |
Owner name: UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL (BR/RS) |
|
| B06F | Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette] | ||
| B07D | Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette] | ||
| B07G | Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette] | ||
| B06A | Patent application procedure suspended [chapter 6.1 patent gazette] | ||
| B09A | Decision: intention to grant [chapter 9.1 patent gazette] | ||
| B16A | Patent or certificate of addition of invention granted [chapter 16.1 patent gazette] |
Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 20/10/2020, OBSERVADAS AS CONDICOES LEGAIS. |
|
| B16C | Correction of notification of the grant [chapter 16.3 patent gazette] |
Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 17/09/2009, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO |
|
| B21F | Lapse acc. art. 78, item iv - on non-payment of the annual fees in time |
Free format text: REFERENTE A 15A ANUIDADE. |
|
| B24J | Lapse because of non-payment of annual fees (definitively: art 78 iv lpi, resolution 113/2013 art. 12) |
Free format text: EM VIRTUDE DA EXTINCAO PUBLICADA NA RPI 2792 DE 09-07-2024 E CONSIDERANDO AUSENCIA DE MANIFESTACAO DENTRO DOS PRAZOS LEGAIS, INFORMO QUE CABE SER MANTIDA A EXTINCAO DA PATENTE E SEUS CERTIFICADOS, CONFORME O DISPOSTO NO ARTIGO 12, DA RESOLUCAO 113/2013. |