WO2024042158A1 - Cellules souches à tropisme cérébral - Google Patents
Cellules souches à tropisme cérébral Download PDFInfo
- Publication number
- WO2024042158A1 WO2024042158A1 PCT/EP2023/073225 EP2023073225W WO2024042158A1 WO 2024042158 A1 WO2024042158 A1 WO 2024042158A1 EP 2023073225 W EP2023073225 W EP 2023073225W WO 2024042158 A1 WO2024042158 A1 WO 2024042158A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- brain
- cox7b
- population
- disease
- Prior art date
Links
- 210000004556 brain Anatomy 0.000 title claims abstract description 150
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 65
- 230000010415 tropism Effects 0.000 title claims abstract description 35
- 210000004027 cell Anatomy 0.000 claims abstract description 538
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 101
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 79
- 208000014644 Brain disease Diseases 0.000 claims abstract description 52
- 238000000034 method Methods 0.000 claims abstract description 31
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 10
- 230000001939 inductive effect Effects 0.000 claims abstract description 4
- 230000037396 body weight Effects 0.000 claims description 147
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 claims description 28
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 24
- 201000010099 disease Diseases 0.000 claims description 23
- 239000003814 drug Substances 0.000 claims description 22
- 201000011240 Frontotemporal dementia Diseases 0.000 claims description 20
- 208000001089 Multiple system atrophy Diseases 0.000 claims description 20
- 230000001225 therapeutic effect Effects 0.000 claims description 20
- 238000011282 treatment Methods 0.000 claims description 20
- 210000001178 neural stem cell Anatomy 0.000 claims description 19
- 230000004770 neurodegeneration Effects 0.000 claims description 17
- 229940124597 therapeutic agent Drugs 0.000 claims description 16
- 206010012289 Dementia Diseases 0.000 claims description 15
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 12
- 208000029028 brain injury Diseases 0.000 claims description 11
- 208000000609 Pick Disease of the Brain Diseases 0.000 claims description 10
- 208000030886 Traumatic Brain injury Diseases 0.000 claims description 10
- 208000031237 olivopontocerebellar atrophy Diseases 0.000 claims description 10
- 230000002265 prevention Effects 0.000 claims description 9
- 230000009529 traumatic brain injury Effects 0.000 claims description 9
- 208000024827 Alzheimer disease Diseases 0.000 claims description 8
- 102000000634 Cytochrome c oxidase subunit IV Human genes 0.000 claims description 8
- 108090000365 Cytochrome-c oxidases Proteins 0.000 claims description 8
- 208000015122 neurodegenerative disease Diseases 0.000 claims description 8
- 208000018737 Parkinson disease Diseases 0.000 claims description 7
- 201000006417 multiple sclerosis Diseases 0.000 claims description 7
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 6
- 208000023105 Huntington disease Diseases 0.000 claims description 6
- 206010003591 Ataxia Diseases 0.000 claims description 5
- 206010010904 Convulsion Diseases 0.000 claims description 5
- 208000011990 Corticobasal Degeneration Diseases 0.000 claims description 5
- 208000010859 Creutzfeldt-Jakob disease Diseases 0.000 claims description 5
- 206010021143 Hypoxia Diseases 0.000 claims description 5
- 208000009829 Lewy Body Disease Diseases 0.000 claims description 5
- 201000002832 Lewy body dementia Diseases 0.000 claims description 5
- 208000026072 Motor neurone disease Diseases 0.000 claims description 5
- 208000037658 Parkinson-dementia complex of Guam Diseases 0.000 claims description 5
- 208000009106 Shy-Drager Syndrome Diseases 0.000 claims description 5
- 208000006011 Stroke Diseases 0.000 claims description 5
- 206010002022 amyloidosis Diseases 0.000 claims description 5
- 208000013968 amyotrophic lateral sclerosis-parkinsonism-dementia complex Diseases 0.000 claims description 5
- 208000014450 amyotrophic lateral sclerosis-parkinsonism/dementia complex 1 Diseases 0.000 claims description 5
- 206010015037 epilepsy Diseases 0.000 claims description 5
- 210000005260 human cell Anatomy 0.000 claims description 5
- 208000036546 leukodystrophy Diseases 0.000 claims description 5
- 208000005264 motor neuron disease Diseases 0.000 claims description 5
- 201000002212 progressive supranuclear palsy Diseases 0.000 claims description 5
- 208000003755 striatonigral degeneration Diseases 0.000 claims description 5
- 208000006096 Attention Deficit Disorder with Hyperactivity Diseases 0.000 claims description 4
- 208000020925 Bipolar disease Diseases 0.000 claims description 4
- 208000004020 Brain Abscess Diseases 0.000 claims description 4
- 208000001408 Carbon monoxide poisoning Diseases 0.000 claims description 4
- 206010010254 Concussion Diseases 0.000 claims description 4
- 206010020772 Hypertension Diseases 0.000 claims description 4
- 208000013200 Stress disease Diseases 0.000 claims description 4
- 201000004810 Vascular dementia Diseases 0.000 claims description 4
- 208000029560 autism spectrum disease Diseases 0.000 claims description 4
- 208000028683 bipolar I disease Diseases 0.000 claims description 4
- 208000010877 cognitive disease Diseases 0.000 claims description 4
- 230000009514 concussion Effects 0.000 claims description 4
- 206010014599 encephalitis Diseases 0.000 claims description 4
- 230000007954 hypoxia Effects 0.000 claims description 4
- 208000027061 mild cognitive impairment Diseases 0.000 claims description 4
- 201000000980 schizophrenia Diseases 0.000 claims description 4
- 208000020431 spinal cord injury Diseases 0.000 claims description 4
- 208000006379 syphilis Diseases 0.000 claims description 4
- 231100000331 toxic Toxicity 0.000 claims description 4
- 230000002588 toxic effect Effects 0.000 claims description 4
- 208000003926 Myelitis Diseases 0.000 claims description 3
- 208000012902 Nervous system disease Diseases 0.000 claims description 3
- 208000025966 Neurological disease Diseases 0.000 claims description 3
- 208000026106 cerebrovascular disease Diseases 0.000 claims description 3
- 210000004962 mammalian cell Anatomy 0.000 claims description 3
- 230000002018 overexpression Effects 0.000 abstract description 29
- 101000957492 Homo sapiens Cytochrome c oxidase subunit 7B, mitochondrial Proteins 0.000 abstract description 13
- 102100038835 Cytochrome c oxidase subunit 7B, mitochondrial Human genes 0.000 abstract description 6
- 230000014509 gene expression Effects 0.000 description 60
- 239000002609 medium Substances 0.000 description 37
- 239000007924 injection Substances 0.000 description 32
- 238000002347 injection Methods 0.000 description 32
- 210000001130 astrocyte Anatomy 0.000 description 30
- 241000699666 Mus <mouse, genus> Species 0.000 description 28
- 239000005090 green fluorescent protein Substances 0.000 description 27
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 26
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 26
- 241000699670 Mus sp. Species 0.000 description 26
- 239000013598 vector Substances 0.000 description 26
- 206010028980 Neoplasm Diseases 0.000 description 20
- 206010006187 Breast cancer Diseases 0.000 description 19
- 230000001965 increasing effect Effects 0.000 description 19
- 239000013612 plasmid Substances 0.000 description 19
- 208000026310 Breast neoplasm Diseases 0.000 description 18
- 206010061289 metastatic neoplasm Diseases 0.000 description 18
- 201000011510 cancer Diseases 0.000 description 17
- 230000002503 metabolic effect Effects 0.000 description 17
- 230000001394 metastastic effect Effects 0.000 description 17
- 239000008103 glucose Substances 0.000 description 16
- 108020004999 messenger RNA Proteins 0.000 description 16
- 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 15
- 206010027476 Metastases Diseases 0.000 description 15
- 239000002773 nucleotide Substances 0.000 description 14
- 125000003729 nucleotide group Chemical group 0.000 description 14
- 108060001084 Luciferase Proteins 0.000 description 13
- 239000005089 Luciferase Substances 0.000 description 13
- 230000005012 migration Effects 0.000 description 13
- 238000013508 migration Methods 0.000 description 13
- 238000011529 RT qPCR Methods 0.000 description 12
- 230000001590 oxidative effect Effects 0.000 description 12
- 108020005004 Guide RNA Proteins 0.000 description 11
- 210000004369 blood Anatomy 0.000 description 11
- 239000008280 blood Substances 0.000 description 11
- 230000030279 gene silencing Effects 0.000 description 11
- 210000003494 hepatocyte Anatomy 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 150000007523 nucleic acids Chemical class 0.000 description 11
- 230000004083 survival effect Effects 0.000 description 11
- 101000979735 Homo sapiens NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8, mitochondrial Proteins 0.000 description 10
- 102100024975 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8, mitochondrial Human genes 0.000 description 10
- 238000003384 imaging method Methods 0.000 description 10
- 230000009401 metastasis Effects 0.000 description 10
- 230000002438 mitochondrial effect Effects 0.000 description 10
- 108020004707 nucleic acids Proteins 0.000 description 10
- 102000039446 nucleic acids Human genes 0.000 description 10
- 238000001543 one-way ANOVA Methods 0.000 description 10
- 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 10
- 238000001262 western blot Methods 0.000 description 10
- 108020005196 Mitochondrial DNA Proteins 0.000 description 9
- 150000001413 amino acids Chemical group 0.000 description 9
- 238000001727 in vivo Methods 0.000 description 9
- 210000005155 neural progenitor cell Anatomy 0.000 description 9
- 210000002569 neuron Anatomy 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 102100027278 4-trimethylaminobutyraldehyde dehydrogenase Human genes 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 241001559542 Hippocampus hippocampus Species 0.000 description 8
- 101000836407 Homo sapiens 4-trimethylaminobutyraldehyde dehydrogenase Proteins 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 230000029918 bioluminescence Effects 0.000 description 8
- 238000005415 bioluminescence Methods 0.000 description 8
- 239000000446 fuel Substances 0.000 description 8
- 102000052726 human COX7B Human genes 0.000 description 8
- 239000002243 precursor Substances 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 8
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 7
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 7
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000002158 endotoxin Substances 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 229920006008 lipopolysaccharide Polymers 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 7
- 230000002441 reversible effect Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- 230000002407 ATP formation Effects 0.000 description 6
- 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 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 6
- 241000713666 Lentivirus Species 0.000 description 6
- 108091093105 Nuclear DNA Proteins 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 230000012292 cell migration Effects 0.000 description 6
- 210000000349 chromosome Anatomy 0.000 description 6
- -1 coatings Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000003306 harvesting Methods 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 238000010149 post-hoc-test Methods 0.000 description 6
- 239000013603 viral vector Substances 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 206010055113 Breast cancer metastatic Diseases 0.000 description 5
- 101150018934 COX7B gene Proteins 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 230000030833 cell death Effects 0.000 description 5
- 238000002659 cell therapy Methods 0.000 description 5
- 210000003169 central nervous system Anatomy 0.000 description 5
- 235000014113 dietary fatty acids Nutrition 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- 229930195729 fatty acid Natural products 0.000 description 5
- 239000000194 fatty acid Substances 0.000 description 5
- 150000004665 fatty acids Chemical class 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 238000003364 immunohistochemistry Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 5
- 239000007925 intracardiac injection Substances 0.000 description 5
- 230000016273 neuron death Effects 0.000 description 5
- 239000000546 pharmaceutical excipient Substances 0.000 description 5
- 229950010131 puromycin Drugs 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 4
- 206010002091 Anaesthesia Diseases 0.000 description 4
- 108091092878 Microsatellite Proteins 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 229930193140 Neomycin Natural products 0.000 description 4
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 description 4
- 230000002411 adverse Effects 0.000 description 4
- 230000037005 anaesthesia Effects 0.000 description 4
- 230000002715 bioenergetic effect Effects 0.000 description 4
- 230000008499 blood brain barrier function Effects 0.000 description 4
- 210000001218 blood-brain barrier Anatomy 0.000 description 4
- 230000006931 brain damage Effects 0.000 description 4
- 231100000874 brain damage Toxicity 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 230000003412 degenerative effect Effects 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000001976 improved effect Effects 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 208000014674 injury Diseases 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229960004927 neomycin Drugs 0.000 description 4
- 210000003061 neural cell Anatomy 0.000 description 4
- 238000011580 nude mouse model Methods 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000012188 paraffin wax Substances 0.000 description 4
- 108091033319 polynucleotide Proteins 0.000 description 4
- 102000040430 polynucleotide Human genes 0.000 description 4
- 239000002157 polynucleotide Substances 0.000 description 4
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 4
- 238000011476 stem cell transplantation Methods 0.000 description 4
- 239000013589 supplement Substances 0.000 description 4
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 4
- 208000022679 triple-negative breast carcinoma Diseases 0.000 description 4
- 238000010200 validation analysis Methods 0.000 description 4
- 239000003981 vehicle Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102100039075 Aldehyde dehydrogenase family 1 member A3 Human genes 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- 206010051290 Central nervous system lesion Diseases 0.000 description 3
- 208000030808 Clear cell renal carcinoma Diseases 0.000 description 3
- 241000701022 Cytomegalovirus Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101000959046 Homo sapiens Aldehyde dehydrogenase family 1 member A3 Proteins 0.000 description 3
- 101001072881 Homo sapiens Phosphoglucomutase-like protein 5 Proteins 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 238000011786 NMRI nude mouse Methods 0.000 description 3
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 102100036635 Phosphoglucomutase-like protein 5 Human genes 0.000 description 3
- 238000003559 RNA-seq method Methods 0.000 description 3
- 241000712907 Retroviridae Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000000692 Student's t-test Methods 0.000 description 3
- 102000003970 Vinculin Human genes 0.000 description 3
- 108090000384 Vinculin Proteins 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- 239000012298 atmosphere Substances 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 210000000424 bronchial epithelial cell Anatomy 0.000 description 3
- 206010073251 clear cell renal cell carcinoma Diseases 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 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 3
- 238000010362 genome editing Methods 0.000 description 3
- 230000034659 glycolysis Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 238000007914 intraventricular administration Methods 0.000 description 3
- 230000009545 invasion Effects 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 210000005240 left ventricle Anatomy 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 208000020816 lung neoplasm Diseases 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 108010082117 matrigel Proteins 0.000 description 3
- 238000002493 microarray Methods 0.000 description 3
- 210000000274 microglia Anatomy 0.000 description 3
- 238000010232 migration assay Methods 0.000 description 3
- 210000003470 mitochondria Anatomy 0.000 description 3
- 210000001700 mitochondrial membrane Anatomy 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 230000001537 neural effect Effects 0.000 description 3
- 230000003959 neuroinflammation Effects 0.000 description 3
- 238000010899 nucleation Methods 0.000 description 3
- 210000004248 oligodendroglia Anatomy 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 230000029058 respiratory gaseous exchange Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 230000009885 systemic effect Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 230000008733 trauma Effects 0.000 description 3
- 241000701242 Adenoviridae Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 description 2
- 102000016911 Deoxyribonucleases Human genes 0.000 description 2
- 241000710781 Flaviviridae Species 0.000 description 2
- 108010036781 Fumarate Hydratase Proteins 0.000 description 2
- 102100036160 Fumarate hydratase, mitochondrial Human genes 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000598279 Homo sapiens NADH-ubiquinone oxidoreductase chain 5 Proteins 0.000 description 2
- 239000007760 Iscove's Modified Dulbecco's Medium Substances 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 206010059282 Metastases to central nervous system Diseases 0.000 description 2
- 102100036971 NADH-ubiquinone oxidoreductase chain 5 Human genes 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 238000010459 TALEN Methods 0.000 description 2
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 101150063416 add gene Proteins 0.000 description 2
- 108700010877 adenoviridae proteins Proteins 0.000 description 2
- 230000001919 adrenal effect Effects 0.000 description 2
- 210000004504 adult stem cell Anatomy 0.000 description 2
- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 description 2
- 230000000172 allergic effect Effects 0.000 description 2
- 239000002543 antimycotic Substances 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000004709 cell invasion Effects 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000002975 chemoattractant Substances 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 239000002612 dispersion medium Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000005462 in vivo assay Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 208000028867 ischemia Diseases 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 210000005244 lower chamber Anatomy 0.000 description 2
- 238000009593 lumbar puncture Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000002025 microglial effect Effects 0.000 description 2
- 230000006686 mitochondrial oxygen consumption Effects 0.000 description 2
- 210000004498 neuroglial cell Anatomy 0.000 description 2
- 230000004112 neuroprotection Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000002496 oximetry Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 230000030786 positive chemotaxis Effects 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000003252 repetitive effect Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229940054269 sodium pyruvate Drugs 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000002459 sustained effect Effects 0.000 description 2
- 238000012385 systemic delivery Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012384 transportation and delivery Methods 0.000 description 2
- 238000007492 two-way ANOVA Methods 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- DZLOHEOHWICNIL-QGZVFWFLSA-N (2R)-2-[6-(4-chlorophenoxy)hexyl]-2-oxiranecarboxylic acid ethyl ester Chemical compound C=1C=C(Cl)C=CC=1OCCCCCC[C@]1(C(=O)OCC)CO1 DZLOHEOHWICNIL-QGZVFWFLSA-N 0.000 description 1
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 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
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-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
- MDJIPXYRSZHCFS-UHFFFAOYSA-N 2-phenyl-n-[5-[2-[2-[5-[(2-phenylacetyl)amino]-1,3,4-thiadiazol-2-yl]ethylsulfanyl]ethyl]-1,3,4-thiadiazol-2-yl]acetamide Chemical compound N=1N=C(CCSCCC=2SC(NC(=O)CC=3C=CC=CC=3)=NN=2)SC=1NC(=O)CC1=CC=CC=C1 MDJIPXYRSZHCFS-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 101710196118 Aldehyde dehydrogenase 9 Proteins 0.000 description 1
- 229930183010 Amphotericin Natural products 0.000 description 1
- QGGFZZLFKABGNL-UHFFFAOYSA-N Amphotericin A Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCCC=CC=CC(C)C(O)C(C)C(C)OC(=O)CC(O)CC(O)CCC(O)C(O)CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 QGGFZZLFKABGNL-UHFFFAOYSA-N 0.000 description 1
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 241000712892 Arenaviridae Species 0.000 description 1
- 239000012583 B-27 Supplement Substances 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 241000776207 Bornaviridae Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102100027943 Carnitine O-palmitoyltransferase 1, liver isoform Human genes 0.000 description 1
- 101710120614 Carnitine O-palmitoyltransferase 1, liver isoform Proteins 0.000 description 1
- 206010007953 Central nervous system lymphoma Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 102000013701 Cyclin-Dependent Kinase 4 Human genes 0.000 description 1
- 108010025464 Cyclin-Dependent Kinase 4 Proteins 0.000 description 1
- 102100030497 Cytochrome c Human genes 0.000 description 1
- 101710140482 Cytochrome c oxidase subunit 7B, mitochondrial Proteins 0.000 description 1
- 108010075031 Cytochromes c Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000007702 DNA assembly Methods 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 208000016192 Demyelinating disease Diseases 0.000 description 1
- 206010012305 Demyelination Diseases 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- 108010089760 Electron Transport Complex I Proteins 0.000 description 1
- 102000008013 Electron Transport Complex I Human genes 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000711950 Filoviridae Species 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 101710184006 Glutaminase 1 Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241000700739 Hepadnaviridae Species 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 241000700586 Herpesviridae Species 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 101100440795 Homo sapiens COX7B gene Proteins 0.000 description 1
- 108010020056 Hydrogenase Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- 229910025794 LaB6 Inorganic materials 0.000 description 1
- 101710128836 Large T antigen Proteins 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 229940121875 Mitochondrial pyruvate carrier inhibitor Drugs 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- UOZODPSAJZTQNH-UHFFFAOYSA-N Paromomycin II Natural products NC1C(O)C(O)C(CN)OC1OC1C(O)C(OC2C(C(N)CC(N)C2O)OC2C(C(O)C(O)C(CO)O2)N)OC1CO UOZODPSAJZTQNH-UHFFFAOYSA-N 0.000 description 1
- 241000701945 Parvoviridae Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 241000150350 Peribunyaviridae Species 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 108010040201 Polymyxins Proteins 0.000 description 1
- 241001631648 Polyomaviridae Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 description 1
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 102000008579 Transposases Human genes 0.000 description 1
- 108010020764 Transposases Proteins 0.000 description 1
- 229930194936 Tylosin Natural products 0.000 description 1
- 239000004182 Tylosin Substances 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 241000021375 Xenogenes Species 0.000 description 1
- 108010084455 Zeocin 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
- 210000000579 abdominal fat Anatomy 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229940009444 amphotericin Drugs 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000001857 anti-mycotic effect Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 239000005667 attractant Substances 0.000 description 1
- 230000003376 axonal effect Effects 0.000 description 1
- 238000004676 ballistic electron emission microscopy Methods 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000006177 biological buffer Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 230000003925 brain function Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 201000008274 breast adenocarcinoma Diseases 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229960004203 carnitine Drugs 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 230000001364 causal effect Effects 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000003306 cell dissemination Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 239000000544 cholinesterase inhibitor Substances 0.000 description 1
- 230000006726 chronic neurodegeneration Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000005786 degenerative changes Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940090949 docosahexaenoic acid Drugs 0.000 description 1
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229950006213 etomoxir Drugs 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- 239000012213 gelatinous substance Substances 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 230000036433 growing body Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 210000001308 heart ventricle Anatomy 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 210000000688 human artificial chromosome Anatomy 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000000185 intracerebroventricular administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 208000028242 linear skin defects with multiple congenital anomalies 2 Diseases 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000001325 log-rank test Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000012577 media supplement Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 229960000951 mycophenolic acid Drugs 0.000 description 1
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 1
- 229960000210 nalidixic acid Drugs 0.000 description 1
- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000007996 neuronal plasticity Effects 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000003900 neurotrophic factor Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 229960000988 nystatin Drugs 0.000 description 1
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 230000003372 organotropic effect Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 238000006213 oxygenation reaction Methods 0.000 description 1
- UOZODPSAJZTQNH-LSWIJEOBSA-N paromomycin Chemical compound N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)O[C@@H]1CO UOZODPSAJZTQNH-LSWIJEOBSA-N 0.000 description 1
- 229960001914 paromomycin Drugs 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- JTJMJGYZQZDUJJ-UHFFFAOYSA-N phencyclidine Chemical compound C1CCCCN1C1(C=2C=CC=CC=2)CCCCC1 JTJMJGYZQZDUJJ-UHFFFAOYSA-N 0.000 description 1
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 208000016800 primary central nervous system lymphoma Diseases 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- ZYHMJXZULPZUED-UHFFFAOYSA-N propargite Chemical compound C1=CC(C(C)(C)C)=CC=C1OC1C(OS(=O)OCC#C)CCCC1 ZYHMJXZULPZUED-UHFFFAOYSA-N 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 239000003725 proteoliposome Substances 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 238000012950 reanalysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000003716 rejuvenation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 230000004202 respiratory function Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 125000003748 selenium group Chemical class *[Se]* 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- IHQKEDIOMGYHEB-UHFFFAOYSA-M sodium dimethylarsinate Chemical compound [Na+].C[As](C)([O-])=O IHQKEDIOMGYHEB-UHFFFAOYSA-M 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 229960000268 spectinomycin Drugs 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000009168 stem cell therapy Methods 0.000 description 1
- 238000009580 stem-cell therapy Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000008337 systemic blood flow Effects 0.000 description 1
- 230000001839 systemic circulation Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000013520 translational research Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- WBPYTXDJUQJLPQ-VMXQISHHSA-N tylosin Chemical compound O([C@@H]1[C@@H](C)O[C@H]([C@@H]([C@H]1N(C)C)O)O[C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CC=O)CO[C@H]1[C@@H]([C@H](OC)[C@H](O)[C@@H](C)O1)OC)CC)[C@H]1C[C@@](C)(O)[C@@H](O)[C@H](C)O1 WBPYTXDJUQJLPQ-VMXQISHHSA-N 0.000 description 1
- 229960004059 tylosin Drugs 0.000 description 1
- 235000019375 tylosin Nutrition 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 1
- 229960001600 xylazine Drugs 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/24—Antidepressants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
Definitions
- the present invention relates to the use of a population of cells overexpressing the cytochrome c oxidase subunit Vllb (COX7B) protein, for use in treating and/or preventing a brain disease.
- COX7B cytochrome c oxidase subunit Vllb
- Neurodegeneration is a pathological state that results in neural cell death. Although the causes of neurodegeneration may be diverse and not always ascertainable, a large number of brain diseases share neurodegeneration as a common pathological state. For example, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS) all cause chronic neurodegeneration, which is characterized by a slow, progressive neural cell death over a period of several years.
- ALS amyotrophic lateral sclerosis
- Acute neurodegeneration is characterized by a sudden onset of neural cell death as a result of ischemia, such as stroke, or trauma, such as traumatic brain injury, or as a result of axonal transection by demyelination or trauma caused, for example, by spinal cord injury or multiple sclerosis. Regardless of the underlying cause, a growing body of evidence indicates that, once neurodegeneration is triggered, the outcome for all these disorders is invariably the same - the ultimate death of neural cells.
- Neurodegeneration represents a particularly challenging biological environment for cell therapy.
- One problem in cell therapeutics is low cell survival (less than 5%) of the cells grafted, as the grafted cells tend to undergo significant cell death shortly after injection in vivo.
- the main issue is the targeting of the grafted cells: indeed, the brain is relatively isolated from the rest of the organism (notably by the bloodbrain barrier, BBB). This implies that the cells must be injected within the brain, which is a particularly heavy and difficult medical intervention: indeed, injecting stem cells in the brain faces two major issues. The first issue is the transplantation procedure itself, which raises safety, ethical and efficacy issues.
- injected cells agglomerate and form clusters at the site of injection in the brain parenchyma, and the grafting efficiency/survival rate of the injected cells is estimated to be less than 5%.
- Stem cells can also be delivered by lumbar puncture injection into the cerebrospinal fluid. Currently, this procedure is more efficient than intravenous administration, notably because more cells are found in the injured site.
- the Applicant herein surprisingly provides a solution for increasing the brain tropism of cells.
- the present invention relates to a population of cells overexpressing the cytochrome c oxidase subunit Vllb (COX7B) protein, for use in treating and/or preventing a brain disease or disorder in a subject in need thereof.
- COX7B cytochrome c oxidase subunit Vllb
- the cells are stem cells, preferably mesenchymal stem cells or neural stem cells.
- the cells are mammalian cells, preferably human cells.
- the COX7B protein is overexpressed at a level so as to induce the brain tropism of said cells.
- the cells stably overexpress COX7B protein.
- the COX7B protein is mammalian COX7B protein, preferably human COX7B protein.
- the cells further express at least one surface receptor which binds to a brain-specific ligand.
- the brain disease or disorder is selected from the group comprising or consisting of neurodegenerative diseases, neurological disorders, brain injuries and cerebrovascular diseases.
- the brain disease or disorder is selected from the group comprising or consisting of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, dementia, diffuse Lewy Body dementia, frontotemporal dementia (FTD), Louis body dementia, ataxia, motor neuron disease, amyotrophic lateral sclerosis (ALS), epilepsy and seizures, multiple system atrophy, multiple sclerosis, leukodystrophy, progressive supranuclear palsy, Olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, Striatonigral degeneration, cortico-basal ganglionic degeneration, Parkinson-ALS-dementia complex of Guam and Pick's disease, amyloidosis, Pick's disease, Lou Gehrig's disease, Creutzfeld-Jakob disease, mild cognitive impairment, syphilis, attention deficit
- the population of cells is to be administered in a therapeutic effective amount to a subj ect in need thereof.
- the therapeutic effective amount is from about 1,000 to about 10,000,000 million of said cells per kg of body weight.
- the population of cells is to be systemically administered to said subject, preferably intravenously injected.
- the present invention also relates to a pharmaceutical composition comprising a population of cells overexpressing COX7B protein, and a pharmaceutically acceptable vehicle, for use in the prevention and/or the treatment of a brain disease or disorder.
- a pharmaceutical composition comprising a population of cells overexpressing COX7B protein, and a pharmaceutically acceptable vehicle, for use in the prevention and/or the treatment of a brain disease or disorder.
- Another object of the present invention is a combination kit comprising (i) a population of cells overexpressing COX7B protein or a pharmaceutical composition comprising the same and (ii) another therapeutic agent, for use for the prevention and/or the treatment of a brain disease or disorder.
- the present invention further relates to a method for inducing the brain tropism of cells, preferably stem cells, comprising a step of overexpressing COX7B protein in said cells.
- identity refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity” per se has an art-recognized meaning and can be calculated using published techniques. Methods to determine identity and similarity are codified in computer programs. Computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package, the GAP program.
- a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include an average up to five point-mutations per each 100 nucleotides of the reference nucleotide sequence.
- a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
- These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
- pharmaceutically acceptable excipient refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
- a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
- preparations should meet sterility, pyrogenicity, general safety and purity standards as required by EMA or FDA Office of Biologies standards.
- treating refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the disease or condition, preferably the brain disease or disorder.
- Those in need of treatment include those already affected with the disease or condition, preferably the brain disease or disorder, as well as those prone to have the disease or condition, preferably the brain disease or disorder, or those in whom the disease or condition, preferably the brain disease or disorder, is to be prevented.
- a subject or mammal is successfully "treated" for the disease or condition, preferably the brain disease or disorder, if, after receiving a therapeutic amount of the population of cells for use according to the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the symptoms associated with the disease or condition, preferably the brain disease or disorder, and/or reduced morbidity and mortality, and/or improvement in quality of life issues.
- the above parameters for assessing successful treatment and improvement in the disease or condition, preferably the brain disease or disorder are readily measurable by routine procedures familiar to a physician.
- “Therapeutically effective amount” refers to the level or amount of an agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of the disease or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease or condition; (3) bringing about ameliorations of the symptoms of the disease or condition; (4) reducing the severity or incidence of the disease or condition; or (5) curing the disease or condition.
- a therapeutically effective amount may be administered prior to the onset of the disease or condition, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after initiation of the disease or condition, for a therapeutic action.
- stem cell refers to a progenitor cell capable of self-renewal, z'.e., can proliferate without differentiation, whereby the progeny of a stem cell or at least part thereof substantially retains the unspecialized or relatively less specialized phenotype, the differentiation potential, and the proliferation competence of the mother stem cell.
- the term encompasses stem cells capable of substantially unlimited self-renewal, z'.e., wherein the capacity of the progeny or part thereof for further proliferation is not substantially reduced compared to the mother cell, as well as stem cells which display limited selfrenewal, z'.e., wherein the capacity of the progeny or part thereof for further proliferation is demonstrably reduced compared to the mother cell.
- subject refers to an animal individual, preferably a mammalian individual, more preferably a human individual.
- an individual may be a mammalian individual.
- Mammalians include, but are not limited to, all primates (human and non-human), cattle (including cows), horses, pigs, sheep, goats, dogs, cats, and any other mammal which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease or condition, preferably a brain disease or disorder.
- an individual may be a “patient”, z'.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease or condition, preferably a brain disease or disorder.
- the individual is an adult (e.g., an individual above the age of 18).
- the individual is a child e.g., an individual below the age of 18).
- the individual is a male.
- the individual is a female.
- the present invention relates to a population of cells overexpressing the cytochrome c oxidase subunit Vllb (COX7B) protein, for use in treating and/or preventing a brain disease or disorder in a subject in need thereof
- COX7B cytochrome c oxidase subunit Vllb
- the cells are stem cells.
- stem cells are undifferentiated cells with very high proliferation capabilities, that are able to differentiate into multiple cell types.
- Stem cells may be totipotent, pluripotent or multipotent.
- the stem cells are preferably multipotent, z'.e., the cells can differentiate into multiple cell types in a specific cell lineage.
- the stem cells are mesenchymal stem cells or neural stem cells. In a preferred embodiment, the stem cells are neural stem cells. In certain embodiments, the neural stem cells will give rise to neuronal cells.
- neural stem cells and “neural progenitor cells” are used interchangeably.
- the stem cells are mesenchymal stem cells.
- the stem cells are adult stem cells or embryonic stem cells. In a preferred embodiment, the stem cells are adult stem cells. In certain embodiments, the stem cells are induced pluripotent stem cells (iPSC) or derive from iPSC.
- iPSC induced pluripotent stem cells
- the cells are mammalian cells, preferably human cells. In some embodiments, the cells are harvested from a mammalian donor, preferably a human donor.
- the stem cells are neurons precursors, oligodendrocyte precursors, astrocyte precursors or microglia precursors. In some embodiments, the stem cells are neuron precursors. In some embodiments, the stem cells are oligodendrocyte precursors. In some embodiments, the stem cells are astrocyte precursors. In some embodiments, the stem cells are microglia precursors.
- harvesting cells from donor subjects may be subject to respective legal and ethical norms.
- harvesting of cells from a living human donor may need to be compatible with sustenance of further life of the donor.
- a fraction of a cell population may typically be removed from a tissue or organ of a living human donor, e.g., using biopsy or resection, such that an adequate level of physiological functions is maintained in the donor.
- harvesting of cells from a non-human animal may be considered, but is not obligatorily need compatible with further survival of the non-human animal.
- the non- human animal may be humanely culled after harvesting of the cells.
- Cells may be obtained from a donor, preferably a human donor, who has sustained circulation, e.g, a beating heart, and sustained respiratory functions, e.g., breathing lungs or artificial ventilation.
- sustained circulation e.g, a beating heart
- sustained respiratory functions e.g., breathing lungs or artificial ventilation.
- Harvesting of cells from such donors is advantageous, since the tissue does not suffer substantial anoxia (lack of oxygenation), which usually results from ischemia (cessation of circulation).
- the cells originate from the subject to be treated. It is to be understood that the purpose of an autologous transplantation (i.e., the administration in an organism of cells from the same organism) is to minimize or abolish the risk of an immune response against the transplant, which would induce the reject of the transplant. The skilled person will appreciate the safety of the transplanted cells.
- the cells originate from a different individual than the subject, preferably and individual of the same species as the subject.
- the cells are ReNcell cells.
- the population of cells for use according to the invention may be homogenous or heterogenous, preferably homogenous.
- the population of cells for use according to the invention is homogenous.
- the population of cells for use according to the invention is substantially free of other cell types.
- the population of cells for use according to the invention is substantially free of cells that do not overexpress COX7B.
- substantially free means less than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or less.
- the population of cells for use according to the invention is heterogenous. In certain embodiments, the population of cells for use according to the invention comprises at least 2 cell types.
- the population of cells for use according to the invention may be frozen (z'.e., cryopreserved) for long term storage.
- the population of cells for use according to the invention may be frozen and stored in liquid nitrogen or at any temperature, preferably from about 0°C to about -196°C, more preferably from about -20°C to about -196°C, even more preferably from about -80°C°C to about -196°C so long as the cells are able to be used as stem cells after thawing therefrom.
- the population of cells for use according to the invention is contacted with at least one cryoprotectant agent, such as, e.g., glycerol, prior to freezing.
- the population of cells for use according to the invention may be thawed and expanded further to obtain fresh cells.
- the population of cells for use according to the invention is cultured, z'.e., seeded and expanded in vitro.
- the environment in which the cells are cultured may comprise at least a cell medium, typically a liquid medium, which supports the survival and/or growth of the cells.
- the term “cell medium” or “cell culture medium” or “medium” refers to an aqueous liquid or gelatinous substance comprising nutrients which can be used for maintenance or growth of cells.
- Cell culture media can contain serum or be serum-free.
- the medium comprises a basal medium formulation as known in the art.
- basal media formulations include Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Elam), Liebovitz L-15, DMEM/F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, Medium 199, Waymouth's MB 752/1, StemMacsTM from Miltenyi, Prime- XV from FUJIFILM Irvine Scientific or Williams Medium E, and modifications and/or combinations thereof Compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells cultured.
- the medium is a commercially available serum-free medium that supports the growth of stem cells.
- Such basal media formulations contain ingredients necessary for mammal cell development, which are known per se.
- these ingredients may include inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides, nucleosides and/or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g., glutathione) and sources of carbon (e.g., glucose, pyruvate, e.g., sodium pyruvate, acetate, e.g., sodium acetate), etc.
- physiological buffers e.g., HEPES, bicarbonate
- nucleotides e.g., nucleosides and/or nucleic acid bases
- ribose deoxyribose
- amino acids e.g., vitamins, antioxidants (e.g
- basal media can be supplied with one or more further components.
- additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion.
- Such supplements include insulin, transferrin, selenium salts, and combinations thereof.
- These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution.
- Further antioxidant supplements may be added, e.g., P-mercaptoethanol. While many basal media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution.
- a medium may be further supplied with antibiotic and/or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and/or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin.
- antibiotic and/or antimycotic compounds such as, typically, mixtures of penicillin and streptomycin, and/or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neo
- the cells are maintained in culture conditions so as not to induce differentiation of the cells. In some embodiments, the cells are maintained in culture conditions so as not to induce determination of the cells.
- the COX7B protein is a mammalian COX7B protein, preferably human COX7B protein.
- COX7B with Entrez Gene ID No. 1349, also refers non-limitatively to Cytochrome C Oxidase Subunit 7B; Cytochrome C Oxidase Subunit 7B, Mitochondrial; Cytochrome C Oxidase Polypeptide Vllb; Cytochrome C Oxidase Subunit Vllb; Cytochrome-C Oxidase Chain Vllb; LSDMCA2 or APLCC.
- the cytochrome c oxidase subunit 7B typically has the amino acid sequence as set forth in SEQ ID NO: 1 in humans.
- the COX7B protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with SEQ ID NO: 1.
- the COX7B protein is the wild type human COX7B. In some embodiments, the COX7B protein has the amino acid sequence of SEQ ID NO: 1.
- the COX7B protein is a mutant COX7B.
- the COX7B protein comprises at least one amino acid mutation compared to the sequence of SEQ ID NO: 1.
- “at least one” means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19 or 20.
- amino acid mutation comprises substitutions, deletions, insertions, inversions, and combination thereof.
- the mutations on COX7B do not produce any adverse effect on the cell, z'.e., the mutations do not alter the viability, proliferation and/or differentiation capacities of the cell. In some embodiments, the mutations are not oncogenic.
- COX7B protein was responsible for controlling the brain tropism of cells (see Example).
- the COX7B protein is overexpressed at a level so as to induce the brain tropism of the cells.
- induce the brain tropism means that, when administered to a subject, preferably in the systemic circulation of the subject, an increased proportion of the population of cells according to the present invention homes to the brain of the subject compared to the same type of cells without COX7B overexpression.
- the brain tropism of the population of cells according to the present invention is increased at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold or more compared to the same type of cells without COX7B overexpression.
- COX7B overexpression is necessary to induce brain tropism, z'.e., the same type of cells without COX7B overexpression have 0% of brain localization.
- At least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the population of cells according to the present invention localizes to the brain of the subject. In some embodiments, 100% of the population of cells according to the present invention localizes to the brain of the subject.
- the cells overexpress at least 1.2-fold the COX7B protein. It is to be understood that the at least 1.2-fold increase is with respect to COX7B expression levels before overexpression, or with respect to COX7B expression level in an identical population of cells that do not overexpress COX7B.
- “at least 1.2-fold” means 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400- fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 10,000-fold, 100,000- fold or more.
- Overexpression of COX7B can be controlled and/or measured by methods well known in the art.
- COX7B overexpression in the population of cells is measured prior to administration to a subject.
- COX7B overexpression is measured at the protein level.
- Non -limitative examples of methods that enable quantitation of a specific protein include Western-Blot, immunofluorescence, quantitative flow cytometry, and liquid chromatography-mass spectrometry (LC-MS).
- COX7B overexpression is measured at the RNA level, preferably the messenger RNA (mRNA) level.
- RNA level preferably the messenger RNA (mRNA) level.
- Non-limitative examples of methods that enable quantitation of a specific RNA include RNA-seq, quantitative reverse transcription polymerase chain reaction (RT- qPCR), quantitative polymerase chain reaction (or “real-time polymerase chain reaction”, qPCR), and fluorescent nucleic acid probes.
- the cells stably or transiently overexpress COX7B protein. In a preferred embodiment, the cells stably overexpress COX7B protein. In another embodiment, the cells transiently overexpress COX7B protein.
- the COX7B protein is encoded by an exogenous or endogenous nucleic acid.
- the exogenous or endogenous nucleic acid comprises the COX7B gene, preferably mammalian COX7B gene, more preferably human COX7B gene.
- the COX7B protein is encoded by an exogenous nucleic acid.
- the COX7B protein is encoded by an endogenous nucleic acid, typically the COX7B protein is encoded by at least 2 copies of the COX7B gene in the genome of the cell.
- the exogenous or endogenous nucleic acid further comprises a promoter, preferably a strong promoter.
- strong promoters include cytomegalovirus (CMV) promoter, phosphoglycerokinase (PGK) promoter, and cytomegalovirus/actin/p-globin (CAG) promoter.
- CMV cytomegalovirus
- PGK phosphoglycerokinase
- CAG cytomegalovirus/actin/p-globin
- COX7B overexpression is achieved by contacting the population of cells with a vector suitable for COX7B overexpression.
- the vector comprises at least one nucleic acid as defined herein.
- the vector comprises the human COX7B ORF cDNA sequence.
- the vector is an integrating vector or a non-integrating vector.
- the vector is an integrating vector.
- the integrating vector is selected from the group comprising or consisting of integrating virus, integrating plasmids, enzymes including engineered transposase or engineered integrase, or genome editing methods including CRISPR-Cas9.
- the integrating vector is an integrating virus selected from the group comprising or consisting of Retroviridae, Adenoviridae, Flaviviridae, Herpesviridae, Hepadnaviridae, Papillomaviridae, Polyomaviridae, Parvoviridae, Arenaviridae, Bornaviridae, Bunyaviridae, Filoviridae and Paramyxoviridae families of viruses, preferably selected from the group comprising or consisting of Retroviridae, Adenoviridae and Flaviviridae families of viruses.
- the integrating virus belongs to the family of Retroviridae family.
- the integrating virus is a lentivirus.
- a genome editing method is used to insert at least one copy of COX7B gene in the genome of the cells.
- Non limitative examples of genome editing methods include CRISPR-Cas9, zinc finger nucleases (ZFN), transcription activator-like effector nuclease (TALEN), and the like. Means to implement these methods are well known in the art.
- COX7B overexpression does not affect the function and/or viability of the cell.
- the vector is a non-integrating vector.
- the non-integrating vector is viral vector or a non-viral vector.
- the vector is a non-integrating viral vector selected from the group comprising or consisting of adenoviral vector, adeno-associated virus (AAV) vector, integration-deficient lentiviral vector (IDLVs), poxviral vector, herpes simplex virus vector.
- AAV adeno-associated virus
- IDLVs integration-deficient lentiviral vector
- poxviral vector herpes simplex virus vector.
- the vector is a non-integrating non-viral vector selected from the group comprising or consisting of plasmid, fosmid, cosmid, artificial chromosome (e.g., human artificial chromosome), nanoparticle (e.g., polymer-based nanoparticle, proteoliposome or lentiviral-like particle), minicircle DNA, and the like.
- the non-integrating non-viral vector is a plasmid or a nanoparticle.
- the non-integrating non-viral vector is a plasmid.
- the plasmid is a pCMV3 plasmid.
- the plasmid is pCMV3 encoding the human COX7B ORF cDNA sequence.
- COX7B overexpression is achieved by contacting the population of cells with a naked nucleic acid expressing COX7B.
- the naked nucleic acid enters the cell by a mean selected from the group comprising or consisting of electroporation, sonoporation, ballistic propulsion of DNA-coated particles, and injection.
- the cells further express at least one surface receptor which binds to a brain-specific ligand.
- the expression of the at least one surface receptor further increases the brain tropism induced by COX7B overexpression.
- brain disease or disorder refers to all kinds of diseases or conditions which occur in the brain of animals, preferably in the brain of humans.
- degenerative brain disease or “neurodegenerative disease” is a subset of brain diseases and refers to any brain disease occurring from a degenerative change in nerve cells of the nervous system. The causes of degenerative brain diseases are mostly unknown, and the diseases have slow onset and continuously progress via selective intrusion into associated nerve systems.
- the brain disease or disorder is selected from the group comprising or consisting of neurodegenerative diseases, neurological disorders, brain injuries and cerebrovascular diseases.
- the brain disease or disorder comprises neuronal cell death.
- the brain disease or disorder comprises or consists of increased neuronal cell death, wherein “increased neuronal cell death” refers to an increase of 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8- fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold or more compared to a healthy subject.
- the brain disease or disorder is selected from the group comprising or consisting of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, dementia, diffuse Lewy Body dementia, frontotemporal dementia (FTD), Louis body dementia, ataxia, motor neuron disease, amyotrophic lateral sclerosis (ALS), epilepsy and seizures, multiple system atrophy, multiple sclerosis, leukodystrophy, progressive supranuclear palsy, Olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, Striatonigral degeneration, cortico-basal ganglionic degeneration, Parkinson- ALS-dementia complex of Guam and Pick's disease, amyloidosis, Pick's disease, Lou Gehrig's disease, Creutzfeld-Jakob disease, mild cognitive impairment, syphilis, attention deficit hyperactivity disorder (ADHD), schizophrenia, depression, manic-depression, stress disorder, spinal cord injury, myelitis, Lugeric's disease
- ADHD attention deficit hyperactivity
- the brain disease or disorder is selected from the group comprising or consisting of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, dementia, diffuse Lewy Body dementia, frontotemporal dementia (FTD), Louis body dementia, ataxia, motor neuron disease, amyotrophic lateral sclerosis (ALS), epilepsy and seizures, multiple system atrophy, multiple sclerosis, leukodystrophy, progressive supranuclear palsy, Olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, Striatonigral degeneration, cortico-basal ganglionic degeneration, Parkinson- ALS-dementia complex of Guam and Pick's disease, amyloidosis, Pick's disease, Lou Gehrig's disease, Creutzfeld-Jakob disease, mild cognitive impairment, syphilis, attention deficit hyperactivity disorder (ADHD), schizophrenia, depression, manic-depression, stress disorder and the like.
- ADHD attention deficit hyperactivity disorder
- the brain disease or disorder is selected from the group comprising or consisting of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, dementia, diffuse Lewy Body dementia, frontotemporal dementia (FTD), Louis body dementia, ataxia, motor neuron disease, amyotrophic lateral sclerosis (ALS), epilepsy and seizures, multiple system atrophy, multiple sclerosis, leukodystrophy, progressive supranuclear palsy, Olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, Striatonigral degeneration, cortico-basal ganglionic degeneration, Parkinson- ALS-dementia complex of Guam and Pick's disease, amyloidosis, Pick's disease, Lou Gehrig's disease, Creutzfeld-Jakob disease and the like.
- ALS amyotrophic lateral sclerosis
- OPCA Olivopontocerebellar atrophy
- Shy-Drager syndrome Striatonigral degeneration
- the brain disease or disorder is selected from the group comprising or consisting of traumatic brain injury (TBI), concussion, vascular dementia, hypertension, toxic brain injury such as hypoxia or carbon monoxide poisoning, encephalitis, stroke, brain tumors, brain abscess, autism spectrum disorder and the like.
- TBI traumatic brain injury
- concussion vascular dementia
- hypertension toxic brain injury such as hypoxia or carbon monoxide poisoning
- encephalitis encephalitis
- stroke brain tumors
- brain abscess autism spectrum disorder and the like.
- the brain disease or disorder comprises an increase of the permeability of the blood brain barrier (BBB).
- BBB blood brain barrier
- the population of cells compensates the neuronal cell death. In some embodiments, the population of cells compensates at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the neuronal cell death induced by the brain disease or disorder.
- administering the population of cells in a subject suffering from a brain disease or disorder increases at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold or more the number of neuronal cells compared to an untreated subject with the same brain disease or disorder.
- the population of cells restores normal brain function in a subject suffering from a brain disease or disorder.
- the population of cells is to be administered in a therapeutic effective amount to a subject in need thereof.
- the therapeutic effective amount is from about 1,000 to about 10,000,000 million of cells per kg of body weight.
- the therapeutic effective amount is from about 2,000 to about 10,000,000 million of cells per kg of body weight, from about 3,000 to about 10,000,000 million of cells per kg of body weight, from about 4,000 to about 10,000,000 million of cells per kg of body weight, from about 5,000 to about 10,000,000 million of cells per kg of body weight, from about 6,000 to about 10,000,000 million of cells per kg of body weight, from about 7,000 to about 10,000,000 million of cells per kg of body weight, from about 8,000 to about 10,000,000 million of cells per kg of body weight, from about 9,000 to about 10,000,000 million of cells per kg of body weight, from about 10,000 to about 10,000,000 million of cells per kg of body weight, from about 20,000 to about 10,000,000 million of cells per kg of body weight, from about 30,000 to about 10,000,000 million of cells per kg of body weight, from about 40,000 to about 10,000,000 million of cells per kg of body weight, from about 50,000 to about 10,000,000 million of cells per kg of body weight, from about 60,000 to about 10,000,000 million of cells per kg of body weight, from about 70,000
- the therapeutic effective amount is from about 1,000 to about 9,000,000 million of cells per kg of body weight, from about 1,000 to about 8,000,000 million of cells per kg of body weight, from about 1,000 to about 7,000,000 million of cells per kg of body weight, from about 1,000 to about 6,000,000 million of cells per kg of body weight, from about 1,000 to about 5,000,000 million of cells per kg of body weight, from about 1,000 to about 4,000,000 million of cells per kg of body weight, from about 1,000 to about 3,000,000 million of cells per kg of body weight, from about 1,000 to about 2,000,000 million of cells per kg of body weight, from about 1,000 to about 1,000,000 million of cells per kg of body weight, from about 1,000 to about 900,000 million of cells per kg of body weight, from about 1,000 to about 800,000 million of cells per kg of body weight, from about 1,000 to about 700,000 million of cells per kg of body weight, from about 1,000 to about 600,000 million of cells per kg of body weight, from about 1,000 to about 500,000 million of cells per kg of body weight, from about 1,000 to about 400,000 million of cells per kg
- the therapeutic effective amount is from about 1,000 to about 10,000,000 cells per kg of body weight.
- the therapeutic effective amount is from about 2,000 to about 10,000,000 cells per kg of body weight, from about 3,000 to about 10,000,000 cells per kg of body weight, from about 4,000 to about 10,000,000 cells per kg of body weight, from about 5,000 to about 10,000,000 cells per kg of body weight, from about 6,000 to about 10,000,000 cells per kg of body weight, from about 7,000 to about 10,000,000 cells per kg of body weight, from about 8,000 to about 10,000,000 cells per kg of body weight, from about 9,000 to about 10,000,000 cells per kg of body weight, from about 10,000 to about 10,000,000 cells per kg of body weight, from about 20,000 to about 10,000,000 cells per kg of body weight, from about 30,000 to about 10,000,000 cells per kg of body weight, from about 40,000 to about 10,000,000 cells per kg of body weight, from about 50,000 to about 10,000,000 cells per kg of body weight, from about 60,000 to about 10,000,000 cells per kg of body weight, from about 70,000 to about 10,000,000 cells per kg of body weight, from about 80,000 to about 10,000,000 cells per kg of body weight, from about
- the therapeutic effective amount is from about 1,000 to about 9,000,000 cells per kg of body weight, from about 1,000 to about 8,000,000 cells per kg of body weight, from about 1,000 to about 7,000,000 cells per kg of body weight, from about 1,000 to about 6,000,000 cells per kg of body weight, from about 1,000 to about 5,000,000 cells per kg of body weight, from about 1,000 to about 4,000,000 cells per kg of body weight, from about 1,000 to about 3,000,000 cells per kg of body weight, from about 1,000 to about 2,000,000 cells per kg of body weight, from about 1,000 to about 1,000,000 cells per kg of body weight, from about 1,000 to about 900,000 cells per kg of body weight, from about 1,000 to about 800,000 cells per kg of body weight, from about 1,000 to about 700,000 cells per kg of body weight, from about 1,000 to about 600,000 cells per kg of body weight, from about 1,000 to about 500,000 cells per kg of body weight, from about 1,000 to about 400,000 cells per kg of body weight, from about 1,000 to about 300,000 cells per kg of body weight, from about 1,000 to about 200,000 cells per kg of body weight, from about 1,000 to about
- the therapeutic effective amount is from about 1,000 to about 100,000,000 cells.
- the therapeutic effective amount is from about 2,000 to about 100,000,000 cells, from about 10,000 to about 100,000,000 cells, from about 100,000 to about 100,000,000 cells, from about 1,000,000 to about 100,000,000 cells, or from about 10,000,000 to about 100,000,000 cells. In some embodiments, the therapeutic effective amount is from about 1,000 to about 10,000,000 cells, from about 1,000 to about 1,000,000 cells, from about 1,000 to about 100,000 cells, or from about 1,000 to about 10,000 cells.
- the population of cells is to be systemically administered to the subject, z'.e., injected in the bloodstream of the subject.
- the population of cells is to be administered by intravenous injection.
- the population of cells is to be administered by intra-arterial or intracardiac injection.
- the population of cells is to be administered by intramuscular, intradermal or subcutaneous injection.
- the population of cells is maintained in a suitable medium.
- the suitable medium enables the survival of the population of cells and is safe for mammalian administration, preferably for human administration.
- the medium provides nutrients to the population of cells.
- the medium maintains a suitable osmotic pressure for the population of cells.
- the population of cells is transferred to another medium, herein referred to as injection medium, prior to injection, wherein the other medium enables the survival of the population of cells and is safe for mammalian administration, preferably for human administration, but does not comprise nutrients.
- the injection medium is physiological serum.
- the injection medium is a biological buffer.
- the medium is sterilized by means known in the art such as, e.g., irradiation. In some embodiments, the sterilization eliminates 99.9%, 99.99%, 99.999% or 100% of microorganisms, preferably 100%. In some embodiments, the medium is sterile. As used herein, “sterile” means that the medium is devoid of bacteria, fungi, archaea and/or protozoa.
- the population of cells is to be administered in combination with another therapeutic agent.
- therapeutic agents include drugs, pharmaceutical agents, peptides, cells (such as cells different from the cells of the invention) or the likes.
- the other therapeutic agent is for treating at least one brain disease.
- Such therapeutic agents are known by the skilled in the art and are commonly used in medical practice.
- Non-limitative examples of brain disease treatments include, e.g, acetylcholinesterase inhibitors for the treatment of Alzheimer’s disease, anti-inflammatory molecules, antioxidants and the like.
- the other therapeutic agent is administered prior, concomitantly or after the population of cells for use according to the invention.
- the other therapeutic agent is administered prior to the population of cells for use according to the invention, preferably between about 1 month and about 1 minute prior to the population of cells for use according to the invention. In some embodiments, the other therapeutic agent is administered concomitantly with the population of cells for use according to the invention. In some embodiments, the other therapeutic agent is administered after the population of cells for use according to the invention, preferably between about 1 minute and about 1 month after the population of cells for use according to the invention.
- the present invention further relates to a pharmaceutical composition
- a pharmaceutical composition comprising a population of cells overexpressing COX7B protein, and a pharmaceutically acceptable vehicle, for use in the prevention and/or the treatment of a brain disease or disorder.
- the pharmaceutically acceptable vehicle is selected in a group comprising or consisting of a solvent, a diluent, a carrier, an excipient, a dispersion medium, a coating, and any combinations thereof.
- the carrier, diluent, solvent or excipient must be “acceptable” in the sense of being compatible with the cell population for use according to the invention, and not be deleterious upon being administered to an individual.
- the vehicle does not produce an adverse, allergic or other untoward reaction when administered to an individual, preferably a human individual.
- the pharmaceutical compositions should meet general safety and purity standards as required by regulatory offices, such as, for example, the Food and Drugs Administration (FDA) Office or the European Medicines Agency (EMA).
- FDA Food and Drugs Administration
- EMA European Medicines Agency
- the present invention further relates to a combination kit comprising (i) a population of cells overexpressing COX7B protein or a pharmaceutical composition comprising the same and (ii) another therapeutic agent, for use for the prevention and/or the treatment of a brain disease or disorder.
- a combination kit comprising (i) means for overexpressing COX7B protein in cells and (ii) another therapeutic agent, for use for the prevention and/or the treatment of a brain disease or disorder.
- the present invention relates to a combination kit comprising (i) a population of cells overexpressing COX7B protein or a pharmaceutical composition comprising the same or means for overexpressing COX7B protein in cells and (ii) another therapeutic agent, for use for the prevention and/or the treatment of a brain disease or disorder.
- Means and methods for overexpressing a protein in cells are known from the skilled person in the art.
- means for overexpressing COX7B protein in cells include, but are not limited to, vectors comprising at least one coding sequence for COX7B as described hereinabove and reagents for transfection of cells.
- the cells where COX7B is overexpressed are the cells of the subject to be treated (autologous cells).
- the cells are stem cells.
- the stem cells are mesenchymal stem cells or neural stem cells.
- the stem cells are mesenchymal stem cells.
- the stem cells are neural stem cells.
- the COX7B protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with SEQ ID NO: 1.
- the kit further comprises means to administer the population of cells to a subject in need thereof.
- the kit comprises means to transfect a nucleic acid molecule encoding the COX7B protein in cells, preferably stem cells.
- the present invention further relates to a method for treating and/or preventing a brain disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population of cells overexpressing COX7B protein or a pharmaceutical composition comprising the same.
- the cells are stem cells.
- the stem cells are mesenchymal stem cells or neural stem cells.
- the stem cells are mesenchymal stem cells.
- the stem cells are neural stem cells. Suitable cells are described hereinabove.
- the method comprises the steps of (i) harvesting a population of stem cells in a subject; (ii) overexpressing COX7B protein in the population of stem cells; and (iii) administering to the subject the population of stem cells overexpressing COX7B protein.
- Means to overexpress COX7B in a cell are known in the art and are described hereinabove.
- the COX7B protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with SEQ ID NO: 1.
- the therapeutic effective amount is from about 1,000 to about 10,000,000 million of cells per kg of body weight.
- the method comprises administering the population of cells to the subject by systemic injection, preferably by intravenous injection.
- cells are co-administered with a suitable carrier or solute.
- the method further comprises administering to the subject further therapeutic agent, preferably a therapeutic agent for treating a brain disease or disorder.
- the present invention further relates to a method for inducing the brain tropism of cells, preferably stem cells, comprising a step of overexpressing COX7B protein in the cells.
- a method for inducing the brain tropism of cells preferably stem cells
- a step of overexpressing COX7B protein in the cells comprising a step of overexpressing COX7B protein in the cells.
- Cells and overexpression of COX7B are described hereinabove.
- the COX7B protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with SEQ ID NO: 1.
- the present invention further relates to a population of cells overexpressing COX7B protein, for the manufacture of a medicament for the treatment and/or prevention of a brain disease or disorder in a subject in need thereof.
- Figure 1A-1F is a combination of schemes, dot plot, histograms and graph showing the validation of the brain tropism of MDA-MB-231 -derived brain-seeking variants.
- Fig. 1A shows a schematic representation of in vivo experiments, where 6-week- old female mice were injected intracardially with 100,000 luciferase- and green fluorescent protein (GFP)-expressing cancer cells on Day 0, imaged once a week to track metastases, and sacrificed at Week 4 for organ collection followed by ex vivo bioluminescence imaging and immunohistochemistry.
- Fig. IB shows ex vivo bioluminescence imaging of mouse brains at the end of the protocol illustrated in Fig. 1A.
- FIG. 2A-2M is a combination of histograms, graph and photograph showing that brain-seeking variants are more oxidative than parental MDA-MB-231 human breast cancer cells.
- Fig. 2D-2F glucose consumption (Fig.
- Figure 3A-3H is a set of histograms showing identification of cyclooxygenase 7b (COX7B) as a candidate protein for the brain tropism of human breast cancer.
- Figures 4A-4Q is a combination of histograms and photograph showing the cause-effect relationship between COX7B expression and the selective migration of human breast cancer cells towards astrocytes.
- Fig. 4A, n 9
- Fig. 4P shows COX7B silencing using CRISPR-Cas9 in 231-BR cells and 23 l-BR-2 cells.
- Fig. 4Q shows COX7B overexpression on MDA-MB-231 cells using a pCMV3 vector. Shown are western blots reporting in triplicate on COX7b expression. P-actin served as a loading control.
- Figure 5A-5N is a combination of graphs and histograms showing that COX7B expression drives the oxidative switch of brain-seeking variants.
- Fig. 5A-5D shows 231- BR brain-seeking variant cells expressing or not (KO using a CRISP-Cas9 strategy) COX7B.
- FIG. 5A Representative Seahorse traces are shown on Fig. 5A.
- Fig. 5M-5N show cell counting of 231-BR cells (Fig.
- COX7B silencing does not alter brain-seeking variant cell numbers in vitro.
- 231-BR and 23 l-BR-2 cells were either wild-type or silenced for COX7B using a CRISPR-Cas9 strategy.
- Figure 6A-6L is a combination of dot plots, histograms and graphs showing that COX7B drives the brain tropism of metastatic human breast cancer cells in mice.
- Fig. 6A-6I shows the brain tropism of 231-BR and 231-BR-2 brain-seeking variants expressing or not COX7B (KO using a CRISP-Cas9 strategy) and of parental MDA-MB- 231 human breast cancer cells overexpressing or not COX7B was assessed using the protocol depicted in Fig. 1A. The cells constitutively expressed luciferase and GFP.
- Fig. 6J-6L shows overall survival of patients with breast cancer (Fig. J, all types combined, 1090 patients), lung cancer (Fig. 6K, all types combined, 1925 patients) and renal clear cell carcinoma (RCC, Fig. 6L, 530 patients). Data are shown as individual values and medians (Fig. 6A-6C), means ⁇ SEM (Fig.
- Figure 7A-7C is a combination of histograms showing that COX7B silencing does not alter the metabolic plasticity of brain-seeking variant cells.
- 231-BR and 231-BR- 2 cells were either wild-type or silenced for COX7B using a CRISPR-Cas9 strategy.
- Fig. 7A-7C Mitochondrial fuel usage of parental MDA-MB-231 cells, 231-BR cells expressing or not COX7B and 23 l-BR-2 expressing or not COX7B.
- FIG. 8 is a photograph showing that ReNcell VM human neural progenitor cells can be engineered to overexpress human COX7B.
- ReNcell VM human neural progenitor cells were transfected or not with a pCMV3 plasmid encoding the human COX7B cDNA ORF.
- Parental cells are Rencell VM and the cells overexpressing COX7B were called ReNcell VM COX7B OE cells. Cells were allowed to recover 24 h after transfection in proliferation medium.
- ReNcell VM COX7B OE clones that were individually tested by western blotting for the expression of the COX7B protein (detected at 9 kD using western blotting).
- P-actin expression (detected at 42 kD) served as a loading control.
- the clones overexpressing COX7B (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23 and 24) compared to the parental cells were pooled together to create the ReNcell COX7B OE cell line.
- Figure 9A-9H is a combination of histograms and photographs showing that human stem cells overexpressing COX7B have a better homing to damaged brains after injection in the blood stream than the parental stem cells that do not express COX7B.
- 100,000 ReNcell VM human neural stem cells constitutively expressing GFP, 100,000 ReNcell VM COX7B OE human neural stem cells constitutively expressing GFP or no cells (control) were injected in the blood stream of nude mice previously treated with LPS to induce brain damage. Mice were sacrificed 3 days later by cervical dislocation under terminal anesthesia, and their brains were harvested, cut along the separation between right and left hemispheres, and embedded in paraffin. (Fig.
- FIG. 9A-9F Shown are representative images of brain sections immunostained for GFP. For visualization, each positive cell is represented by a black dot.
- FIG. 9G Graph showing the number of GFP-positive cells per brain hemisphere in control mice, mice injected with ReNcell VM cells in the blood stream, and mice injected with ReNcell VM COX7B OE cells in the blood stream.
- FIG. 9H Graph showing the number of GFP-positive cells per whole brain in control mice, mice injected with ReNcell VM cells in the blood stream, and mice injected with ReNcell VM COX7B OE cells in the blood stream.
- MDA-MB-231 human triple-negative breast adenocarcinoma cancer cells were from Caliper (catalogue #119369).
- MDA-MB-231 -derived brain-seeking variants 231-BR and 231-BR-2 were from the National Cancer Institute, Bethesda and Indiana University School of Medicine, Indianapolis, respectively.
- Cells were routinely cultured in DMEM containing glutaMAX and 4.5 g/L glucose (ThermoFisher; catalogue #61965026) supplemented with 10% FBS, and maintained in a 37°C in a 5% CO2 humidified atmosphere.
- Cells were authenticated using short tandem repeat (STR) profiling (Eurofms Genomics).
- Human astrocytes (T0281, expressing hTERT), mouse astrocytes (T0289, expressing the SV40 large T antigen), human hepatocytes (T0063, expressing HPV E6ZE7, hTERT and MycT58A) and human bronchial epithelial cells (T0753, expressing hTERT and Cdk4) used in migration assays were immortalized cells from Applied Biological Materials Inc. (ABM).
- ABS Applied Biological Materials Inc.
- Astrocytes were routinely cultured in DMEM with glutaMAX and 4.5 g/L glucose (ThermoFisher; catalogue #61965026) supplemented with 10% FBS and 5% astrocyte growth supplement (Sanbio; catalogue #1852; for human astrocytes only).
- Hepatocytes were maintained in PriGrow IX medium (ABM; catalogue #TM019) supplemented with 10% FBS, then progressively transferred to the same medium as cancer cells.
- Bronchial cells were maintained in Prigrow X medium (ABM; catalogue #TM0753), then progressively transferred to bronchial epithelial cell growth medium (BEGM; Lonza; catalogue #CC-3170).
- luciferase and GFP expression cells were infected with lentiviruses carrying the luciferase and GFP sequences along with puromycin resistance gene (Amsbio; catalogue #LPV020). Briefly, 70-80% confluent cells in a 24-wells plate were transduced with 2 pL per well of the lentivirus solution in ImL medium with 10 pL/mL polybrene. Cells were selected by a 48-72 h incubation with 1 pg/mL puromycin (InvivoGen), and FACS-sorted for GFP expression on a Becton Dickinson FACSArialll system.
- Amsbio catalogue #LPV020
- 70-80% confluent cells in a 24-wells plate were transduced with 2 pL per well of the lentivirus solution in ImL medium with 10 pL/mL polybrene. Cells were selected by a 48-72 h incubation with 1 pg/mL puromycin (Inviv
- COX7B gene silencing was performed using a CRISPR-Cas9 strategy following Zhang’s laboratory protocol with pSpCas9(BB)-2A-Puro (pX459; Addgene; catalogue #62988, puromycin selection) or pU6-(BbsI)_CBh-Cas9-T2A-mCherry (Addgene; catalogue #64324, red fluorescence selec-tion) plasmids. These plasmids contain both Cas9 and gRNA expression cassettes, with BbsI restriction sites for the insertion of gRNA sequences.
- Prevalidated guide RNA (gRNA) sequences were chosen in the GenScript genome-wide database for a non-overlapping duo of gRNAs: 5’- AGCGCACTAAATCGTCTCCA-3’ (SEQ ID NO: 15) and 5’- GAGTTACCCCAAAGGAATGG-3’ (SEQ ID NO: 16). Sticky ends were created for insertion in the vector plasmids, with CACCG at the 5’ of the gRNA sense sequence, and AAAC at the 5’ and C at the 3’ of the gRNA antisense sequence.
- gRNA oligonucleotides (Eurogentec) were annealed into double-stranded DNA with 1 L of stock solutions containing 100 pM of each sense and anti-sense oligonucleotides in 2 L of 5X T4 ligase buffer (ThermoFisher; catalogue #46300018), 0.5 L of T4 PNK (BIOKE; catalogue #M0201) and 5.5 L of DNase/RNase-free distilled water. Incubation times were 37°C for 30 min, followed by 95°C for 5 min, and then de-creasing 5°C/min until reaching 25°C.
- the Golden Gate DNA Assembly protocol was then used for inserting 1 L of annealed gRNA at 1 pM into 100 ng of vector plasmid, in a solution containing 5 L of 10X Fast Digest buffer (ThermoFisher; catalogue #B64), 0.5 L of ATP 0.1 M (ThermoFisher; catalogue #R1441), 0.5 L of BSA 10 mg/mL (Promega; catalogue #R396D), 1 L of restriction enzyme Bpil (ThermoFisher; catalogue #FD1014) and 2 L of T4 ligase 5U/pL (ThermoFisher; catalogue #EL0014) in a total volume of 50 L completed with water.
- 10X Fast Digest buffer ThermoFisher; catalogue #B64
- 0.5 L of ATP 0.1 M ThermoFisher; catalogue #R1441
- BSA 10 mg/mL Promega
- restriction enzyme Bpil ThermoFisher; catalogue #FD1014
- the mixture was incubated for 20 cycles at 37°C for 5 min and 20°C for 5 min, followed by 80°C for 20 min.
- Five microliters of the resulting solution were used for TOP 10 bacteria (ThermoFisher; catalogue #0404003) transformation using prewarmed LB agar plates (ThermoFisher; catalogue #22700-025) containing 100 pg/mL of ampicillin following manufacturer’s instructions.
- Single colonies were inoculated in LB broth (ThermoFisher; catalogue #12780-052) with 50 pg/mL ampicillin, and incubated at 37°C overnight.
- Plasmid DNA was then collected with the Pure Yield Plasmid Miniprep System (Promega; catalogue #A1223), and its concentration obtained using a NanoDrop device (ThermoFisher). Sequences were verified by Sanger Sequencing (Genewiz, Leipzig, Germany). Cells at 70-80% confluence were transfected with the Lipofectamine LTX/Plus transfection kit (ThermoFisher; catalogue #15338100) or with the jetOPTIMUS kit (Westburg; catalogue #117-01).
- the first kit was used with 0.25 pg of each gRNA plasmid, for a total of 0.5 pg of DNA in 100 pL of OptiMEM, containing 0.5 pL of Plus and 2.25 pL of lipofectamine LTX, with incubation times of 15 and 30 min, respec-tively.
- the second kit was used with 0.5 pg of each gRNA plasmid, for a total of 1 pg of DNA, with 1 pL of reagent in 200 pL of buffer for each 6-wells plate well.
- the human untagged COX7B cDNA ORF Clone in expression vector pCMV3 (Bio-Connect; catalogue #HG20762-UT) was used to overexpress COX7b in MDA-MB- 231 cancer cells.
- Cells at 70-80% confluence were transfected with 10 pg of the plasmid and Lipofectamine 3000 (ThermoFisher; catalogue #L3000001) in a 10 cm-dish, allowed to recover the next day in fresh DMEM containing glutaMAX and 4.5 g/L glucose (ThermoFisher; catalogue #61965026) supplemented with 10% FBS, and selected with hygro- mycin (400 pg/ml)-containing medium for 10 days. Medium was renewed every 3-4 days. Colonies individually picked, expanded, and tested by western blotting for the expression of COX7b.
- mice On day 0, 6-weeks-old female NMRI nude mice (Janvier) received image-guided intraventricular injections of 100,000 cells, using a Vevo 2100 imaging system (FUJIFILM VisualSonics) equipped with a 30 MHz transducer. Briefly, mice were anesthetized (80 mg/Kg ketamine and 8 mg/Kg xylazine), secured on the animal platform in supine position, and their thorax was shaved. Two-dimensional (2D) parasternal long- axis ultrasound images of the left ventricle were acquired, in order to ascertain the optimal point in the apex of the heart for the intraventricular injection.
- FUJIFILM VisualSonics Vevo 2100 imaging system
- a microinjector system with a 26G hypodermic needle was used to perform a precise echocardiography-guided intraventricular injection of 100,000 cancer cells constitutively expressing luciferase and GFP. Following injection, the blood flow within the left ventricle was closely observed (and images were recorded), confirming that the cells were successfully injected intraventricularly. All mice were followed-up for a few minutes with echography to ascertain that the injection did not lead to any injury and were closely monitored until recovery. Metastasis development was monitored using a Xenogen IVIS 50 bioluminescence imaging system (PerkinElmer), and quantified with the Living Image software (PerkinElmer).
- mice were injected i.p with 0.15 mg/g bodyweight of luciferin (PerkinElmer) and anesthetized using isoflurane after a 10-min incubation time. Chemiluminescence was detected with 1-12 seconds acquisition time. Mice were sacrificed after 4 weeks by cervical dislocation under terminal anesthesia, and organ chemiluminescence was acquired ex vivo before fixation in 4% paraformaldehyde (PF A).
- PF A paraformaldehyde
- Transwell inserts (Coming; catalogue #353097) were used to measure cell migration and invasion capacities. FBS 1% (general migration/invasion) or cells (astrocytes, hepatocytes, bronchial cells) seeded in the lower chamber were used as chemoattractants. Invasion was assessed by coating the inserts with 250 pg/mL of Matrigel (Corning; catalogue #356231) for 2 h at 37°C, and migration without Matrigel coating.
- Fifty thousand cells were seeded in the upper chamber of each transwell in 500 L of serum -free culture medium, while the lower chamber contained FBS 1% (general migration/invasion) or confluent nonmalignant astrocytes, hepatocytes, or bronchial cells that were used as chemoattractants. Tested cells were allowed to migrate/invade for 24 h in a 37°C and 5% CO2 humidified atmosphere. At the end of the assay, cells were fixed with 4% PF A for 10 min and PBS, rinsed 3 times with PBS, and immobile cells (upper compartment of the insert) were wiped away.
- Oxygen consumption rates were measured on a Seahorse XF96 bioenergetics analyzer using the XF Cell Mito Stress Test Kit and the Fuel Flex test kit following manufacturer's instructions (Agilent Technologies).
- Glucose and lactate concentrations were measured using an enzymatic CMA600 analyzer (Aurora Borealis), according to manufacturer’s instructions, in the supernatant of 150,000 (for 24 h assays) and 250,000 (for 48 h assays) cells were seeded in exactly 1 mL of culture medium. Wells containing medium only were used as controls for the calculation of glucose consumption and lactate production.
- the ATP content of 10.000 cells per well was measured using the Cell titer Gio assay of Promega (catalogue #G7570). All metabolic measurements were normalized to total protein content determined after overnight incubation with 0.5 M NaOH using the Bio-Rad protein assay (catalogue #5000006) on a SpectraMax i3 spectrophotometer equipped with a MiniMax imaging cytometer.
- mtDNA Mitochondrial DNA
- mtDNA Mitochondrial DNA
- RT-qPCR RT-qPCR as previously described.
- Total DNA was isolated with a QIAmp DNA kit (Qiagen, Antwerp, Belgium).
- the 12S-rRNAA mitochondrial gene forward primer: 5'-GTA CCC ACG TAA AGA CGT TAG G-3' (SEQ ID NO: 12); reverse primer: 3'-TAC TGC TAA ATC CAC CTT CG-5' (SEQ ID NO: 13); labeled probe: 5'-CCC ATG AGG TGG CAA GAA AT-3' FAM (SEQ ID NO: 14) in parallel with nuclear gene RNAseP (RNAseP VIC- labeled probe; ThermoFisher Scientific; catalogue #4401631) were then analyzed by RT- qPCR (50 ng of sample and 1 pL of each primer pair [10pM]), with TaqMan universal master mix II with UNG (Ther
- cDNAs were diluted 1 : 10 in DNase/RNase- free distilled water (Thermo Fisher), and 2 L were used with 5 L of 2X Takyon qPCR Master Mix, 0.2 L of each primer (10 M) and completed to 10 L with water for RT- qPCR analysis (ViiA 7417 Real-Time instrument, ThermoFisher).
- Brains were collected, cut along the separation between right and left hemispheres, and embedded in paraffin. Sections (5 pm-thick) were performed from the center of each hemisphere to produce 10 slides for each sample. For each hemisphere, 3 slides from the beginning, the middle and the end were used for immunostaining, and the process was repeated up to 3 times in order to analyze slices representative of the whole brain.
- OS Overall Survival curves were generated on Kaplan-Meier plotter (kmplot.com) with the auto select best cutoff for the 202110 Affy ID (C0X7B) on the RNA-seq mRNA dataset (breast cancer and renal clear cell carcinoma in pan-cancer) and on gene chip mRNA data sets for lung cancers.
- Sources for the databases include GEO, EGA and TCGA.
- the objective of the set of experiments presented herein was to identify metabolic protein(s) responsible for the brain tropism of human metastatic breast cancer.
- MDA-MB-231 triple-negative breast cancer (TNBC) cells were used, as well as two independently derived brain-seeking variant cell lines 231-BR and 231-BR-2 that were generated by serial cycles of in vivo selection in mice.
- the selection protocol involved intracardiac cancer cell injection in the left ventricle of female nude mice, surgical isolation, expansion of metastatic cancer cells retrieved from the brain, and intracardiac injection of these cells in next animals for several rounds until metastatic dissemination became restricted to the brain.
- STR profiling confirmed that all variants were genomically similar to parental MDA-MB-231 cells (data not shown).
- STR short tandem repeat
- cells were infected with lentiviruses to constitutively express luciferase and green fluorescent protein (GFP).
- the assay involves sequential inhibition of glucose-fueled (using 2 pM mitochondrial pyruvate carrier inhibitor UK5099), glutamine-fueled (using 3 pM glutaminase 1 inhibitor BPTES) and lipid-fueled (using 4 pM carnitine palmitoyl-transf erase 1A inhibitor Etomoxir) OXPHOS.
- ALDH1A3 (Genbank ID 220, on chromosome 15) encoding aldehyde dehydrogenase 1 family member A3, NDUFB8 (Genbank ID 4714, on chromosome 10) encoding NADH:ubiquinone oxidoreductase subunit B8, and PGM5 (Genbank ID 5239, on chromosome 9) encoding phosphoglucomutase 5.
- Table 1 Metabolic genes differentially expressed in 231-BR versus other tissuespecific variants of parental MDA-MB-231 human breast cancer cells
- Cytochrome c Oxidase Subunit 7b in Mitochondrial Complex IV is a Candidate Protein Supporting the Brain Tropism of Human Breast Cancer Cells
- ALDH9A1 encodes a cytosolic aldehyde hydrogenase that catalyzes the oxidation of y-aminobutyraldehyde and aminoaldehydes derived from polyamines. It is involved in carnitine biosynthesis, which facilitates the transport of fatty acids across the inner mitochondrial membrane for P-oxidation and, potentially, in a marginal pathway for the biosynthesis of neurotransmitter y-aminobutyric acid (GABA).
- GABA neurotransmitter y-aminobutyric acid
- FH encodes fumarate hydratase, the 7 th enzyme of the TCA cycle that catalyzes the hydration of fumarate to L-malate.
- FH can cause various diseases, including hereditary and sporadic forms of cancer.
- FH mRNA and protein expression was increased in 231-BR but not in 231-BR-2 cells ( Figure 3C-3D), thus disqualifying this enzyme as a shared metabolic sensor for brain tropism.
- NDUFB8 encodes an accessory subunit of NADH ubiquinone oxidoreductase, a large protein complex known as ETC Complex I at the inner mitochondrial membrane. The subunit is bound to NADH dehydrogenase 5 (ND5) in the proton-pumping module of Complex I.
- RT-qPCR showed significantly reduced NDUFB8 mRNA expression in 231-BR compared to MDA-MB-231 cells ( Figure 3E). However, it was significantly increased in 231-BR-2 cells, and changes in protein expression did not match changes in mRNA expression (Figure 3E-F). Overall, this disqualified NDUFB8 as a metabolic sensor for brain-selective metastasis.
- COX7B encodes subunit 7B of cytochrome c oxidase (COX), a large protein complex known as ETC Complex IV that catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen at the inner mitochondrial membrane.
- COX7B is a short 80 amino acid protein that stabilizes the complex and modulates COX activity.
- COX7B mRNA expression was increased in 231-BR but decreased in 231-BR-2 cells ( Figure 3G). However, the expression of the corresponding protein was increased in both variants ( Figure 3H). Considering that among the 4 candidates proteins, only COX7B protein expression showed a similar change in both brain-seeking variants, COX7B was retained for further investigation.
- COX7B expression drives human breast cancer cell migration towards astrocytes
- Transwell migration assays were used to establish a causal link between COX7B expression and MDA-MB-231 brain chemoattraction, mimicked by cell migration towards immortalized human and mouse astrocytes. Chemoattraction at other important metastatic sites was mimicked by immortalized human hepatocytes (T0063) and human bronchial epithelial cells (T0763) All four cell lines were nonmalignant.
- COX7B expression promotes the oxidative phenotype of human metastatic breast cancer cells
- COX7B overexpression in wild-type MDA-MB-231 cells induced the opposite effect, z'.e., an oxidative switch characterized by a raise in all basal OCR, maximal OCR and OCR associated to ATP production (Figure 5I-5L).
- COX7B is an OXPHOS inducer. They further established a positive correlation between the oxidative activities of human metastatic breast cancer cells and their preferential migration towards astrocytes.
- COX7B expression is responsible for the brain tropism of metastatic human breast cancer cells in mice
- Figure 6D-6I shows the number of metastases per mouse and the metastatic surfaces. Analyses revealed a strong decrease in the number of metastases per mouse and in the metastasis-positive tumor area per slice in mice injected with 231-BR and 23 l-BR-2 cells lacking COX7B compared to wild-type 231-BR and 23 l-BR-2 cells ( Figure 6D-6G). The opposite effects were seen in mice that received parental MDA- MB-231 cells overexpressing COX7B compared to wild-type MDA-MB-231 cells ( Figure 6H-6I). Collectively, these in vivo data established a cause-effect relationship between COX7B expression and the brain tropism of human TNBC in mice.
- Example 1 shows that high COX7B expression is responsible for brain-specific metastasis of human breast cancer cells MDA-MB-231, therefore it was reasoned that an experimentally induced overexpression of COX7B could serve to target cells to the brain in the context of cell therapy in neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Eluntington’s disease, amyotrophic lateral sclerosis and multiple sclerosis, and brain trauma, including ischemic stroke and accidental and surgical traumas, and lymphomas of the central nervous system.
- neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Eluntington’s disease, amyotrophic lateral sclerosis and multiple sclerosis
- brain trauma including ischemic stroke and accidental and surgical traumas, and lymphomas of the central nervous system.
- Cell therapy is indeed a promising strategy that can be used for neuroprotection (z'.e., to delay the progression of neurodegenerative diseases) and/or brain repair (see, e.g., Steffanoni, S., et al., Diagnosis and Treatment Using Autologous Stem-Cell Transplantation in Primary Central Nervous System Lymphoma: A Systematic Review. Cancers (Basel), 2023. 15(2)).
- a main research area for brain repair and regeneration therapy is the use of stem cells, among which mesenchymal stem cells and neural stem cells are multipotent cells capable of self-renewal and able to differentiate into various central nervous system neuronal and glial cell types.
- Eluman mesenchymal stem cells can be isolated from various sources of the body, such as abdominal fat, bone marrow, and umbilical cord blood.
- human neural stem cells originate from direct isolation from brain tissue and in vitro expansion, from the differentiation of pluripotent stem cells (such as human embryonic stem cells [hESCs] and induced pluripotent stem cells [iPSCs]) or from the transdifferentiation of somatic cells in culture.
- pluripotent stem cells such as human embryonic stem cells [hESCs] and induced pluripotent stem cells [iPSCs]
- iPSCs induced pluripotent stem cells
- mesenchymal stem cells are used to suppress inflammation and to take advantage of their secretome that can provide neuroprotection and promote regeneration
- neural stem cells produce neurotrophic factors, decrease neuroinflammation, enhance neuronal plasticity through integrating into host neuronal circuits, and replace damaged cells (Uchida, N., et al., Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A, 2000. 97(26): p. 14720-5).
- Injecting stem cells in the brain faces two major issues. The first issue is the transplantation procedure itself, which raises safety, ethical and efficacy issues.
- injected cells agglomerate and form clusters at the site of injection in the brain parenchyma, and the grafting efficiency/survival rate of the injected cells is estimated to be less than 5%.
- Stem cells can also be delivered by lumbar puncture injection into the cerebrospinal fluid. Currently, this procedure is more efficient than intravenous administration, notably because more cells are found in the injured site.
- stem cell therapy optimizing the delivery of stem cells to the brain is thus an important task in translational research.
- ReNcell VM human neural progenitor cells as an example, it is demonstrated that overexpressing COX7B in stem cells promotes their homing to the brain parenchyma after systemic delivery in the blood stream.
- ReNcell VM human neural progenitor cells were routinely grown in flasks coated with 1% of Matrigel (Corning, #356231) in proliferation medium DMEM F12 containing L-glutamine (Gibco, #11320-074), supplemented with 20 ng/mL hEGF (Sigma-Aldrich, #E9644-.2MG), 20 ng/mL hFGF (Sigma-Aldrich, #GF003AF-100UG), 2 pg/mL heparin (StemCell, #07980), 2% B27 supplement (ThermoFisher, #17504044) and 1% antibiotic-antimycotic (ThermoFisher, #15240062), in a humidified atmosphere with 5% CO2 at 37°C.
- DMEM F12 containing L-glutamine Gibco, #11320-074
- 20 ng/mL hEGF Sigma-Aldrich, #E9644-.2MG
- Expression vector pCMV3 encoding the human COX7B ORF cDNA sequence and possess ing an hygromycin selection cassette (Sinobio, #HG20762-UT) was used to overexpress COX7B in ReNcell VM cells. Briefly, cells at 30-60% confluence in a 10 cm-dish were transfected with 10 pg of the plasmid using the Lipofectamine Stem Transfection Reagent (ThermoFisher, #STEM00001). They were allowed to recover for 24h after transfection. Cells were then selected with 10 pg/mL hygromycin (InvivoGen, #ant-hg-l) for 14 days. Medium was renewed every 3-4 days. Colonies (clones) were then individually picked, expanded, and tested by western blotting for the expression of COX7B protein.
- luciferase and GFP expression cells were infected with lentiviruses carrying the luciferase and GFP sequences along with a neomycin resistance gene (Amsbio, #LVP403). Briefly, 70-80% confluent cells in a 96-wells plate were transduced with 20 viral particles. The next day, the cells were selected by incubation with 400 pg/mL neomycin (InvivoGen, #ant-gn-l) during 10 days. After treatment, the cells were FACS-sorted for GFP expression on a Becton Dickinson FACSAria III system (Erembodegem, Belgium).
- ReNcell VM human neural progenitor cell line (ReN) was used, which is an immortalized cell line that can be maintained for more than 45 passages in culture and can differentiate into neurons and glial cells with simple growth-factor deprivation. This cell line has already been used by others to study brain engraftment after intrathecal injection in rats (see, e.g., Hovakimyan, M., et al., Survival of transplanted human neural stem cell line (ReNcell VM) into the rat brain with and without immunosuppression. Ann Anat, 2012. 194(5): p. 429-35.)
- ReNcell VM cells were first genetically modified to express high levels of the COX7B protein.
- the pCMV3 plasmid carrying the human untagged COX7B open reading frame cDNA sequence as well as an hygromycin resistance sequence were used.
- Selection after transfection allowed to retrieve 14 ReNcell VM cell clones that expressed higher levels of the COX7B protein compared to the wild-type parental cells ( Figure 8).
- these 14 clones were pooled together to create the ReNcell VM COX7B OE stem cell line.
- ReNcell VM and ReNcell VM COX7B OE cells were modified to constitutively express luciferase and the green fluorescent protein (GFP).
- GFP green fluorescent protein
- a lentivirus carrying the luciferase and GFP sequences along with a neomycin resistance cassette was used.
- ReNcell VM cells with constitutive luciferase and constitutive GFP expression were obtained on the one hand, and ReNcell VM COX7B OE cells with constitutive luciferase and constitutive GFP expression were obtained on the other hand.
- the two cell lines were used to compare their homing in damaged mouse brain.
- NMRI nude mice treated with LPS (Ipg/gbw) were used, because this strain is compatible with the engraftment of human cells and because LPS was previously shown to induce neuroinflammation resulting in brain damage, which is now widely used as a model of brain damage and neurodegeneration (Trepanier, M.O., et al., Increased brain docosahexaenoic acid has no effect on the resolution of neuroinflammation following intracerebroventricular lipopolysaccharide injection. Neurochem Int, 2018. 118: p. 115- 126; Bodea, L.G., et al., Neurodegeneration by activation of the microglial complementphagosome pathway. J Neurosci, 2014. 34(25): p. 8546-56).
- mice received a bolus injection of either 100.000 of ReNcell VM cells or 100.000 ReNcell VM COX7B OE cells in the systemic blood circulation. All mice were sacrificed by cervical dislocation under terminal anesthesia 3 days after stem cell injection, and brains were collected and embedded in paraffin. For each mouse, a series of sections of the two brain hemispheres were immunostained for GFP expression, allowing to detect the injected human stem cells with full certitude. The results presented in Figure 9A-9H unequivocally show that COX7B overexpression improved the homing of human neural stem cells to the damaged brain of immunocompromised mice. Within the brain parenchyma, ReNcell VM COX7B OE cells were widely distributed in the cortex along areas that also stained positively for brain cell bodies, which are known to host a dense brain vasculature.
- Example 1 demonstrates that COX7B overexpression allows the tropism to target cells not only to mouse astrocytes but also to human astrocytes.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Developmental Biology & Embryology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Psychiatry (AREA)
- Hematology (AREA)
- Ophthalmology & Optometry (AREA)
- Hospice & Palliative Care (AREA)
- Pain & Pain Management (AREA)
- Psychology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
La présente invention concerne une population de cellules surexprimant la protéine de la sous-unité VIIb du cytochrome c oxydase (COX7B), pour une utilisation dans le traitement et/ou la prévention d'une maladie ou d'un trouble du cerveau chez un sujet qui en a besoin, ainsi que des compositions pharmaceutiques ou des trousses les comprenant. La présente invention concerne en outre une méthode pour induire le tropisme cérébral des cellules, de préférence des cellules souches, en utilisant la surexpression du COX7B.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP22191920 | 2022-08-24 | ||
EP22191920.2 | 2022-08-24 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2024042158A1 true WO2024042158A1 (fr) | 2024-02-29 |
Family
ID=83508845
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2023/073225 WO2024042158A1 (fr) | 2022-08-24 | 2023-08-24 | Cellules souches à tropisme cérébral |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2024042158A1 (fr) |
-
2023
- 2023-08-24 WO PCT/EP2023/073225 patent/WO2024042158A1/fr unknown
Non-Patent Citations (9)
Title |
---|
ALESSIA INDRIERI ET AL: "Synthetic long non-coding RNAs [SINEUPs] rescue defective gene expression in vivo", SCIENTIFIC REPORTS, vol. 6, no. 1, 6 June 2016 (2016-06-06), XP055675489, DOI: 10.1038/srep27315 * |
BLACKMAN MARINE C N M ET AL: "Mitochondrial Protein Cox7b Is a Metabolic Sensor Driving Brain-Specific Metastasis of Human Breast Cancer Cells.", CANCERS 08 SEP 2022, vol. 14, no. 18, 8 September 2022 (2022-09-08), XP002808453, ISSN: 2072-6694 * |
BODEA, L.G. ET AL.: "Neurodegeneration by activation of the microglial complement-phagosome pathway", J NEUROSCI, vol. 34, no. 25, 2014, pages 8546 - 56 |
DE GIOIA, R ET AL.: "Neural Stem Cell Transplantation for Neurodegenerative Diseases", INT J MOL SCI, vol. 21, no. 9, 2020 |
HOVAKIMYAN, M ET AL.: "Survival of transplanted human neural stem cell line (ReNcell VM) into the rat brain with and without immunosuppression", ANN ANAT, vol. 194, no. 5, 2012, pages 429 - 35 |
TANAKA NOBUYUKI ET AL: "Single-cell RNA-seq analysis reveals the platinum resistance gene COX7B and the surrogate marker CD63", vol. 7, no. 12, 26 October 2018 (2018-10-26), GB, pages 6193 - 6204, XP093013730, ISSN: 2045-7634, Retrieved from the Internet <URL:https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fcam4.1828> DOI: 10.1002/cam4.1828 * |
TREPANIER, M.O. ET AL.: "Increased brain docosahexaenoic acid has no effect on the resolution of neuroinflammation following intracerebroventricular lipopolysaccharide injection", NEUROCHEM INT, vol. 118, 2018, pages 115 - 126 |
UCHIDA, N ET AL.: "Direct isolation of human central nervous system stem cells", PROC NATL ACAD SCI USA, vol. 97, no. 26, 2000, pages 14720 - 5, XP002336911, DOI: 10.1073/pnas.97.26.14720 |
VAN HEE, V.F. ET AL.: "Lactate does not activate NF-kappaB in oxidative tumor cells", FRONT PHARMACOL, vol. 6, 2015, pages 228 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11576872B2 (en) | Compositions for facilitating membrane fusion and uses thereof | |
US20210187018A1 (en) | Cytobiologics and therapeutic uses thereof | |
RU2766120C2 (ru) | Соединения, композиции, способы и наборы, связанные с удлинением теломер | |
CN112888788A (zh) | 用于治疗线粒体疾病或病症和异质性的方法和组合物 | |
JP2021513849A (ja) | 膜タンパク質送達のための組成物および方法 | |
JP2017513520A (ja) | メトトレキサートによる選択と組み合わせたSleeping Beautyトランスポゾンによる遺伝子改変T細胞の製造 | |
US20230022146A1 (en) | Compositions and methods for editing beta-globin for treatment of hemaglobinopathies | |
JP7403461B2 (ja) | ピルビン酸キナーゼ欠損症を治療するためのpklr送達用レンチウイルスベクター | |
JP2016513974A (ja) | 造血幹細胞系列を初期化するための組成物および方法 | |
WO2014022423A2 (fr) | Cellules hla-g modifiées et procédés associés | |
US20210213062A1 (en) | Drug-Resistant Immune Cells and Methods of Use Thereof | |
KR20220119063A (ko) | 치료용 조작 세포 | |
CN112601812A (zh) | 基因疗法 | |
Gunasekaran et al. | Comparative efficacy and mechanism of action of cardiac progenitor cells after cardiac injury | |
CN109641063B (zh) | 用于增强pklr的基因表达的组合物和方法 | |
WO2024042158A1 (fr) | Cellules souches à tropisme cérébral | |
Lou et al. | Neural progenitor cells expressing herpes simplex virus-thymidine kinase for ablation have differential chemosensitivity to brivudine and ganciclovir | |
US20230192784A1 (en) | KLF Induced Cardiomyogenesis | |
WO2020171222A1 (fr) | Procédé d'intégration d'un gène étranger long dans une région sûre d'une cellule souche pluripotente humaine et lui permettant de fonctionner normalement dans cette dernière | |
EP1363995B1 (fr) | Utilisation d'une composition comprenant de la n-acétyl-cystéine pour le conditionnement de cellules souches | |
WO2023176709A1 (fr) | Procédé de traitement d'un patient ayant subi un processus de pré-transplantation impliqué dans une transplantation de cellules souches hématopoïétiques, et composition destinée à être utilisée dans ledit procédé | |
US20210252072A1 (en) | Engineered hematopoietic stem cells for the treatment of acute myeloid leukemia | |
JP2023519744A (ja) | ミトコンドリア増強療法 | |
KR20210092149A (ko) | 저산소 환경 하에서 높은 적응력을 가지는 세포 및 이의 용도 | |
JP2015522273A (ja) | 造血幹細胞増幅プロトコル及び移植療法のためのチロシンキナーゼレシピエントアゴニストト分子の使用、及びチロシンキナーゼレシピエントアゴニストのキット |
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: 23761849 Country of ref document: EP Kind code of ref document: A1 |