EP1701612A2 - Chimeric murine model comprising human hepatocytes user for screening compounds - Google Patents
Chimeric murine model comprising human hepatocytes user for screening compoundsInfo
- Publication number
- EP1701612A2 EP1701612A2 EP05701083A EP05701083A EP1701612A2 EP 1701612 A2 EP1701612 A2 EP 1701612A2 EP 05701083 A EP05701083 A EP 05701083A EP 05701083 A EP05701083 A EP 05701083A EP 1701612 A2 EP1701612 A2 EP 1701612A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- mouse
- murine model
- anyone
- hepatocytes
- human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 210000003494 hepatocyte Anatomy 0.000 title claims abstract description 207
- 241000282414 Homo sapiens Species 0.000 title claims abstract description 137
- 238000010172 mouse model Methods 0.000 title claims abstract description 104
- 150000001875 compounds Chemical class 0.000 title claims abstract description 56
- 238000012216 screening Methods 0.000 title claims abstract description 17
- 244000052769 pathogen Species 0.000 claims abstract description 86
- 241001529936 Murinae Species 0.000 claims abstract description 63
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 59
- 208000015181 infectious disease Diseases 0.000 claims abstract description 57
- 108700019146 Transgenes Proteins 0.000 claims abstract description 27
- 230000009395 genetic defect Effects 0.000 claims abstract description 25
- 230000034994 death Effects 0.000 claims abstract description 20
- 230000003044 adaptive effect Effects 0.000 claims abstract description 19
- 206010061598 Immunodeficiency Diseases 0.000 claims abstract description 15
- 230000001627 detrimental effect Effects 0.000 claims abstract description 11
- 230000004060 metabolic process Effects 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 69
- 210000004185 liver Anatomy 0.000 claims description 69
- 238000000034 method Methods 0.000 claims description 66
- 108090000623 proteins and genes Proteins 0.000 claims description 39
- 210000002540 macrophage Anatomy 0.000 claims description 34
- 241000223960 Plasmodium falciparum Species 0.000 claims description 33
- 238000002513 implantation Methods 0.000 claims description 30
- 230000000694 effects Effects 0.000 claims description 23
- 230000001553 hepatotropic effect Effects 0.000 claims description 22
- 210000003743 erythrocyte Anatomy 0.000 claims description 20
- 102000004169 proteins and genes Human genes 0.000 claims description 20
- 210000005260 human cell Anatomy 0.000 claims description 16
- 238000001727 in vivo Methods 0.000 claims description 16
- 239000002676 xenobiotic agent Substances 0.000 claims description 14
- 238000011579 SCID mouse model Methods 0.000 claims description 13
- 239000002502 liposome Substances 0.000 claims description 13
- 210000000822 natural killer cell Anatomy 0.000 claims description 13
- 230000002034 xenobiotic effect Effects 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 11
- 238000000386 microscopy Methods 0.000 claims description 11
- 238000012360 testing method Methods 0.000 claims description 11
- 241000224016 Plasmodium Species 0.000 claims description 9
- 238000010186 staining Methods 0.000 claims description 8
- 241000124008 Mammalia Species 0.000 claims description 7
- 230000036983 biotransformation Effects 0.000 claims description 7
- 230000003993 interaction Effects 0.000 claims description 7
- 208000002491 severe combined immunodeficiency Diseases 0.000 claims description 7
- 238000011830 transgenic mouse model Methods 0.000 claims description 7
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 claims description 6
- 238000005259 measurement Methods 0.000 claims description 6
- 239000002207 metabolite Substances 0.000 claims description 6
- 206010028980 Neoplasm Diseases 0.000 claims description 5
- 230000001173 tumoral effect Effects 0.000 claims description 5
- 102100029591 V(D)J recombination-activating protein 2 Human genes 0.000 claims description 4
- 239000005557 antagonist Substances 0.000 claims description 4
- 230000001413 cellular effect Effects 0.000 claims description 4
- 206010012601 diabetes mellitus Diseases 0.000 claims description 4
- 238000010166 immunofluorescence Methods 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000012753 partial hepatectomy Methods 0.000 claims description 4
- 230000009467 reduction Effects 0.000 claims description 4
- 230000001225 therapeutic effect Effects 0.000 claims description 4
- 239000003440 toxic substance Substances 0.000 claims description 4
- 101001061851 Homo sapiens V(D)J recombination-activating protein 2 Proteins 0.000 claims description 3
- 210000001616 monocyte Anatomy 0.000 claims description 3
- 231100000331 toxic Toxicity 0.000 claims description 3
- 230000002588 toxic effect Effects 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 2
- 229920002521 macromolecule Polymers 0.000 claims description 2
- 239000011159 matrix material Substances 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims 4
- 231100000167 toxic agent Toxicity 0.000 claims 3
- 238000003757 reverse transcription PCR Methods 0.000 claims 2
- WMTIOGUVKBSEOW-UHFFFAOYSA-N ClC1(Cl)OP(=O)OP(=O)O1 Chemical compound ClC1(Cl)OP(=O)OP(=O)O1 WMTIOGUVKBSEOW-UHFFFAOYSA-N 0.000 claims 1
- 238000011282 treatment Methods 0.000 abstract description 20
- 241000699670 Mus sp. Species 0.000 description 47
- 241000699666 Mus <mouse, genus> Species 0.000 description 33
- 241001465754 Metazoa Species 0.000 description 31
- 108091006905 Human Serum Albumin Proteins 0.000 description 28
- 102000008100 Human Serum Albumin Human genes 0.000 description 28
- 239000003814 drug Substances 0.000 description 25
- 229940079593 drug Drugs 0.000 description 24
- 238000010240 RT-PCR analysis Methods 0.000 description 16
- 241000700721 Hepatitis B virus Species 0.000 description 13
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 13
- 244000045947 parasite Species 0.000 description 13
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 12
- 238000002347 injection Methods 0.000 description 12
- 239000007924 injection Substances 0.000 description 12
- 241000711549 Hepacivirus C Species 0.000 description 11
- 230000008901 benefit Effects 0.000 description 11
- 238000002054 transplantation Methods 0.000 description 11
- 102000015395 alpha 1-Antitrypsin Human genes 0.000 description 10
- 229940024142 alpha 1-antitrypsin Drugs 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 230000004083 survival effect Effects 0.000 description 10
- 102000009027 Albumins Human genes 0.000 description 9
- 108010088751 Albumins Proteins 0.000 description 9
- 101000823116 Homo sapiens Alpha-1-antitrypsin Proteins 0.000 description 9
- 230000012010 growth Effects 0.000 description 9
- 238000012317 liver biopsy Methods 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- 210000003046 sporozoite Anatomy 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 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 8
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 7
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 7
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 230000028327 secretion Effects 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- 108010050122 alpha 1-Antitrypsin Proteins 0.000 description 6
- 210000003719 b-lymphocyte Anatomy 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 108010057081 Merozoite Surface Protein 1 Proteins 0.000 description 5
- 102100039867 Outer mitochondrial transmembrane helix translocase Human genes 0.000 description 5
- 241000288906 Primates Species 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 238000010171 animal model Methods 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 201000004792 malaria Diseases 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- 230000002440 hepatic effect Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 210000005228 liver tissue Anatomy 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 230000010412 perfusion Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 229940002612 prodrug Drugs 0.000 description 4
- 239000000651 prodrug Substances 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 102100022712 Alpha-1-antitrypsin Human genes 0.000 description 3
- 102000029816 Collagenase Human genes 0.000 description 3
- 108060005980 Collagenase Proteins 0.000 description 3
- 102100029115 Fumarylacetoacetase Human genes 0.000 description 3
- 206010035500 Plasmodium falciparum infection Diseases 0.000 description 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 229960002424 collagenase Drugs 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 3
- 239000002547 new drug Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 238000003118 sandwich ELISA Methods 0.000 description 3
- 210000001563 schizont Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 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 2
- 108700028369 Alleles Proteins 0.000 description 2
- 101710081722 Antitrypsin Proteins 0.000 description 2
- 101710181333 Chaperone protein dnaK1 Proteins 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- 101100373503 Enterobacteria phage T4 y06Q gene Proteins 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 102100040352 Heat shock 70 kDa protein 1A Human genes 0.000 description 2
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 2
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 2
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 241000282579 Pan Species 0.000 description 2
- 240000009188 Phyllostachys vivax Species 0.000 description 2
- 239000013614 RNA sample Substances 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000001475 anti-trypsic effect Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001647 drug administration Methods 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 210000000973 gametocyte Anatomy 0.000 description 2
- 238000012239 gene modification Methods 0.000 description 2
- 230000005017 genetic modification Effects 0.000 description 2
- 235000013617 genetically modified food Nutrition 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 230000001744 histochemical effect Effects 0.000 description 2
- 102000051631 human SERPINA1 Human genes 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- 239000007943 implant Substances 0.000 description 2
- 238000010874 in vitro model Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000003278 mimic effect Effects 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000001558 permutation test Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000002331 protein detection Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000002629 repopulating effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000002459 sustained effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000010415 tropism Effects 0.000 description 2
- 239000002753 trypsin inhibitor Substances 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 1
- 241001414900 Anopheles stephensi Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 101710117490 Circumsporozoite protein Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 101100193633 Danio rerio rag2 gene Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 208000037262 Hepatitis delta Diseases 0.000 description 1
- 206010057212 Hepatitis viral infections Diseases 0.000 description 1
- 101001078133 Homo sapiens Integrin alpha-2 Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102100034349 Integrase Human genes 0.000 description 1
- 102100025305 Integrin alpha-2 Human genes 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 241000581650 Ivesia Species 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 101100225689 Mus musculus Enah gene Proteins 0.000 description 1
- 101100390735 Mus musculus Figla gene Proteins 0.000 description 1
- 101100193635 Mus musculus Rag2 gene Proteins 0.000 description 1
- 208000031662 Noncommunicable disease Diseases 0.000 description 1
- HCUVEUVIUAJXRB-UHFFFAOYSA-N OC1=C(C=C(CNC(CCCC=2SC=CC=2)=O)C=C1)OC Chemical compound OC1=C(C=C(CNC(CCCC=2SC=CC=2)=O)C=C1)OC HCUVEUVIUAJXRB-UHFFFAOYSA-N 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 241000580858 Simian-Human immunodeficiency virus Species 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 108010032099 V(D)J recombination activating protein 2 Proteins 0.000 description 1
- 206010058874 Viraemia Diseases 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000007321 biological mechanism Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008614 cellular interaction Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 210000003040 circulating cell Anatomy 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- ACSIXWWBWUQEHA-UHFFFAOYSA-N clodronic acid Chemical compound OP(O)(=O)C(Cl)(Cl)P(O)(O)=O ACSIXWWBWUQEHA-UHFFFAOYSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000005218 dilaceration Effects 0.000 description 1
- 210000001840 diploid cell Anatomy 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 239000002359 drug metabolite Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 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 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 108010022687 fumarylacetoacetase Proteins 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 208000035474 group of disease Diseases 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000005534 hematocrit Methods 0.000 description 1
- 210000005161 hepatic lobe Anatomy 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 208000029570 hepatitis D virus infection Diseases 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000334 hepatotoxic Toxicity 0.000 description 1
- 230000003082 hepatotoxic effect Effects 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000008076 immune mechanism Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000007233 immunological mechanism Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 108040006849 interleukin-2 receptor activity proteins Proteins 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000002350 laparotomy Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229940102859 methylene diphosphonate Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 101150061302 och1 gene Proteins 0.000 description 1
- 238000000399 optical microscopy Methods 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 210000004303 peritoneum Anatomy 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000007859 qualitative PCR Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003393 splenic effect Effects 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000022814 xenobiotic metabolic process Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6472—Cysteine endopeptidases (3.4.22)
- C12N9/6475—Interleukin 1-beta convertase-like enzymes (3.4.22.10; 3.4.22.36; 3.4.22.63)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
- C07K14/8125—Alpha-1-antitrypsin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
- C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Definitions
- the present invention provides a method of making a chimeric murine model comprising human hepatocytes.
- the invention also discloses a chimeric murine model.
- the invention also relates to applications in pathogen studies having recourse to said model, including screening compounds or assessing efficacy of compounds in the treatment of pathogen infections or detrimental effects resulting from said infection.
- the invention also concerns the use of said model to evaluate the interest of compounds in treatment of patients.
- the chimeric murine model comprising human hepatocytes can further be useful for the study of metabolism of said human hepatocytes, when said cells are submitted to contact with various agents including drug compounds or drug candidates.
- pathogens include microorganisms encompassing viruses, bacteria, fungi or parasites.
- Other pathogens can be substances inducing or favouring toxic or detrimental reactions to emerge or to spread in hosts, said substances including components derived from microorganisms or produced by the same or can be molecules having a different origin.
- Pathogen infections in humans, sometimes leading to premature death, have been controlled to some extent in industrialized countries in the last decades due to a better comprehension of pathogen life cycle and to the design and availability of new drugs including vaccines.
- known pathogens keep on infecting people in various regions, whereas in other situations, resistance strains to existing drugs have occurred or new pathogens emerge.
- cellular cultures do not represent a sufficient model to study the various interactions between the pathogen and the cell in a manner, which would mimic in vivo interactions.
- In vivo models often represent more relevant models than cultured cells; experiments generally are carried on mammals and particularly on mice, but also on primates. Mouse models have a lot of advantages such as being cost efficient, easy to reproduce and to manipulate.
- many pathogens cannot develop in such a host because of their restricted tropism.
- biological mechanisms in mice are different in many respects from those observed in human and results obtained in mice can sometimes hardly be transposed to human.
- experiments are performed on primates where the mechanisms of infection are more or less the same as in human, at least in higher primates such as chimpanzees .
- HBV hepatitis B virus
- HCV hepatitis C virus
- Plasmodium falciparum for malaria can be fought by different treatments: preventive vaccine or antiviral therapy for HBV and antiviral therapy for HCV and malaria.
- HBV hepatitis B virus
- HCV hepatitis C virus
- Plasmodium falciparum for malaria can be fought by different treatments: preventive vaccine or antiviral therapy for HBV and antiviral therapy for HCV and malaria.
- some patients do not respond to treatment and resistant strains of said pathogens are increasing both in prevalence and degree of resistance.
- a chimeric murine according to the invention which comprises functional human hepatocytes, allows the study of metabolic pathways following administration of compounds. Due to differences existing in metabolic pathways between human patients and animal models usually used for screening it appears that the effects of a compound on a human biological system can sometimes be ascertained in clinical trials only.
- the resulting model comprised three genetically disparate sources of tissues.
- the transplantation of ex vivo HBV-infected human liver fragment in such a mouse enabled HBV to replicate for a period of one month, and to generate viremia in the recipient mouse.
- This model enabled the infected transfected cells to maintain in the recipient and sustained the replication of the pathogen.
- Such a model also showed the survival of non-infected hepatocytes up to 1 month after transplantation, but not the growth of these latter.
- Another strategy was adopted by the team of Ohashi et al.
- NOD/SCID mice non obese diabetic/severe combined immunodeficiency mice were transplanted, in the kidney capsule, with hepatocytes mixed with Matrigel. The loss of the human transplanted hepatocytes was however observed and the hypothesis was made of the absence of an essential growth factor i.e., the hepatocyte growth factor (HGF).
- HGF hepatocyte growth factor
- mice having no functional T and B cells were crossed with Alb-uPA transgenic mice. These latter express a transgene, the urokinase-type plasminogen activator (uPA) under the control of the albumin promoter, leading to the death of transgene-carrying hepatocytes and resulting in a growth advantage for transplanted cells devoid of said gene.
- uPA urokinase-type plasminogen activator
- the effectiveness of human hepatocyte transplantation in these crossed mice was controlled by h ⁇ lAT signal measurement. The results showed that some recipient mice had an extinction of signal around 14 weeks after transplantation, whereas a second subset maintained a strong signal beyond 30 weeks.
- uPA transgenic mice were crossed with RAG- 2 mice (lacking mature T and B lymphocytes), and hemizygous uPA mice were transplanted with primary human hepatocytes.
- a successful transplantation and partial repopulation (highest degree estimated up to 15% of mouse liver) were obtained with hepatocytes from perfused donor liver specimen.
- the other experiments with hepatocytes from tissues surrounding tumours or from cell solution failed to produce successful transplantation.
- Injection of HBV-infectious human serum in uPA/RAG-2 mice resulted in human hepatocyte infection and in presence of viral envelope protein in transplanted mouse serum. Accordingly, the transplanted hepatocytes were permissive for HBV indicating that they are functional.
- the invention provides a method of making a chimeric murine model comprising: a. obtaining an immunocompromised murine host, which carries a transgene or a genetic defect responsible for murine hepatocyte death, b. implanting human hepatocytes therein, c. controlling non-adaptive defences, d. recovering a chimeric murine model harbouring settled human hepatocytes capable of maintaining, differentiating and growing. Alternatively in such process the above steps of implanting human hepatocytes and controlling non-adaptive defences can be inverted.
- the invention also provides a chimeric murine model which is an immunocompromised murine host harbouring at least one transgene or genetic defect responsible for murine hepatocyte death, which non-adaptive defences are controlled and which is implanted with human hepatocytes, said control of non-adaptive defences enable said hepatocytes to maintain, differentiate and grow.
- the chimeric murine model of the invention is also implanted with human red blood cells, in addition to human hepatocytes.
- the invention provides a method for studying a hepatotropic pathogen, in a chimeric murine model according to the invention comprising: a.
- the invention also relates to the use of a chimeric murine model of the invention for the testing of a compound for a potential therapeutic interest.
- Another aspect of the invention is a method for screening active compounds against the infection by a hepatotropic pathogen or against its detrimental effects in a chimeric murine model according to the invention comprising: a. infecting said chimeric murine model with a hepatotropic pathogen, in conditions enabling said pathogen to penetrate the settled human cells of the chimeric murine model; b.
- a further aspect of the invention provides a method for screening the in vivo metabolism of xenobiotic compounds, in a chimeric murine model of the invention comprising: a. administrating the xenobiotic compound to be tested to said chimeric murine model in conditions allowing the compound to interact with settled human hepatocytes, b. observing its biotransformation by said settled hepatocytes.
- the human hepatocytes to be implanted can be obtained from donor liver specimens, from partial hepatectomy, from tissues surrounding tumour or can be human hepatocytes isolated from another mammal , especially murine model.
- a particular murine host is an immunocompromised mouse homozygous for the SCID defect, lacking T and B cells, and homozygous or hemizygous for the Alb-uPA gene and enabling the implantation of human hepatocytes.
- Particular hepatotropic pathogens for life cycle studies or drug screening are Plasmodium strains (P. fa/ciparum or P. vivax), HBV or HCV.
- Fig 1 FACS profiles a) FACS profile for macrophage depletion in untreated and treated mice. In black, cells incubated without F4/80 antibody; in red, cells incubated with F4/80 antibody. b) FACS profile for NK depletion in untreated and treated mice.0 Fig 2: Immunohistological analysis of human hepatocytes in chimeric mouse livers.
- serial liver sections were immunostained with a monoclonal antibody against human hepatocytes (a) or with an anti-mouse macrophage5 monoclonal antibody Mac2 (b) Twelve weeks after transplantation in untreated animals, serial liver sections were immunostained with an anti-human ⁇ 1-antitrypsin antibody (c) or with an anti-mouse macrophage monoclonal antibody Mac2 (d). Untreated (e) and treated (f) chimeric mouse livers, one month after
- Fig 3 Proportion of mice positive for human albumin (HA) and ⁇ 1- 5 antitrypsin ( ⁇ 1AT) secretion in treated as compared to untreated implanted animals.
- the serum levels of HA (a) and human ⁇ 1AT (b) in uPA/SCID mice were determined by a standard sandwich ELISA, and the results expressed in % positive animals.
- Fig 5 The detection of HA transcripts in RNA samples from the chimeric livers of treated (uPA 131 , 144 and 146) and untreated (uPA 137, 138 and 149) animals 3 months after implantation. Human liver (Hu liver) and H 2 0 were used as positive and negative controls, respectively.
- Fig 6 Plasmodium falciparum liver stage development in chimeric human liver. Bar represents 50 ⁇ m in A and 10 ⁇ m in B. a) Liver sections stained with hematoxylin-eosin. The arrows show two developing P.
- Fig 7 RT-PCR using primers of LSA1 (a) and HSP70 (b) on liver biopsies from implanted mice (uPA1-6) and not implanted (uPA9) and on genomic DNA from blood stages of P. falciparum as positive control. RT-PCR using primers of LSA1 (a) and HSP70 (b) on liver biopsies from implanted mice (uPA1-6) and not implanted (uPA9) and on genomic DNA from blood stages of P. falciparum as positive control. RT-PCR using
- HA primers on liver biopsies results for human albumin from implanted mice (uPA1-6) and not implanted (uPA9) and on human liver (Hu liver) as positive control.
- the symbol “+” and “-” refer to test (RT-PCR) and control respectively.
- the present invention provides a method of making a chimeric murine model comprising: a. obtaining an immunocompromised murine host, which carries a transgene or a genetic defect responsible for murine hepatocyte death, b. implanting human hepatocytes therein, c. controlling non-adaptive defences, d. recovering a chimeric murine model harbouring settled human hepatocytes capable of maintaining, differentiating and growing.
- a murine host useful for the preparation of the chimeric murine model of the invention can be obtained by carrying out the following steps.
- an immunocompromised murine animal is crossed with a murine animal carrying a transgene or a genetic defect responsible for murine hepatocyte death and resulting in a survival disadvantage of the murine hepatocytes.
- a murine host homozygous for the immunocompromised phenotype, and carrying at least one copy of the transgene or the genetic defect responsible for murine hepatocyte death is obtained.
- a step is performed to control non-adaptive defences of the host, to recover in a last step a chimeric murine model enabling the settled human hepatocytes to maintain, differentiate and grow.
- An alternative in the method of generating a chimeric murine host of the invention encompasses the possibility to control non-adaptive defences before implanting human hepatocytes.
- "Chimeric" as used herein relates to a murine host, which comprises cells from a xenogenic origin i.e., originating from a different organism, in particular originating from a different animal species. In the invention, said cells encompass human hepatocytes.
- Said human hepatocytes can be provided to the murine host i.e., implanted by known appropriate methods such as grafting, injection... When implanted in the host, the human hepatocytes maintain, differentiate and grow.
- said implanted human hepatocytes are capable of substituting to the resident hepatocytes and/or of repopulating the model.
- “Host” as used herein is a murine animal exhibiting two important features enabling xenogenic cells to settle: - the capacity to tolerate the xenogenic human hepatic cells because of altered immunologic mechanisms resulting from genetic mutations, treatments or surgery to provide an "immunocompromised state"; - the transgene or genetic defect responsible for murine hepatocyte death leading to the survival advantage for the xenogenic human hepatocytes and facilitating their expansion or colonisation.
- the genetic defect responsible for murine hepatocyte death can be located in a genetic region naturally present in the host.
- the term genetic defect encompasses any genetic modification changing gene expression such as nucleotide substitution, deletion, insertion that alter the gene transcription, the mRNA stability, the protein translation or the protein stability or activity. These modifications can occur in coding or in non-coding sequences such as promoter, 5'UTR, introns or 3'UTR (untranslated region).
- This defect can be the result of a naturally occurring mutation or can be generated by techniques well known in the art, such as homologous recombination.
- the genetic defect can also result from the insertion of a genetic construct in mammal cells.
- the genetic construct such as a transgene, can be integrated in the host genome (genomic or mitochondria DNA) or can remain unintegrated, e.g., as a vector.
- the genetic construct can carry promoter and regulation elements and a coding sequence DNA, including cDNA, originally present or absent from the host genome.
- this genetic defect is harboured in at least one copy; it can be present in all cells or in some cell types only provided it is expressed in such a way that only resident hepatocytes of the murine host are altered, because of the different gene expression patterns.
- the Alb-uPA transgenic mouse carries urokinase gene controlled by an albumin promoter which targets urokinase overproduction to the liver resulting in a severe hypofibrinogenemic state and accelerated hepatocyte death.
- This deficiency causes the accumulation of a hepatotoxic compound leading to a continuous process of hepatocytes lyses. - a caspase inducible gene under an inducible promoter that can selectively destroy murine hepatocytes by activation of the promoter.
- "Implanting" as used herein is the process of incorporating xenogenic cells, e.g. human hepatocytes, into a recipient murine host. The implantation can take place in various locations, e.g., intrahepatic, intrasplenic, intraperitoneal or intraorbital and the implanted cells when settled in the chimeric murine model can circulate or to the contrary can remain at a determined location.
- implantation is realized with adult or foetal primary hepatocytes, bone marrow cells that can differentiate in hepatocytes or hepatocyte cell lines.
- implanted hepatocytes can also have previously undergone various treatments, including genetic modifications.
- hepatocytes can be prepared as described (Dandri M. et al. 2001 . Hepatology 33, 981-988) by collagenase treatment. They can also be kept under in vitro conditions in cultures (e.g., one to 3 weeks) before being implanted into the murine host.
- Dissociated hepatocyte suspension following collagenase treatment will be implanted in the liver, the spleen or the kidney capsule, and human liver slices of various thicknesses will be for example implanted in the muscle. Implantation can also occur in the peritoneal cavity where isolated human cells are attached to collagen and other matrix sponges.
- the chimeric murine model can be used to implant, human hepatocytes, and besides said human hepatocytes, enucleated cells such as red blood cells (RBC).
- RBC red blood cells
- the implanted hepatocytes are healthy cells, encompassing non-infected and non-tumoral hepatocytes.
- “Non-infected” refers to hepatocytes that have not undergone, previously to the implantation, interactions with the hepatotropic pathogen which effect on said cells might be tested later in the obtained chimeric murine model.
- Such hepatocytes are for example obtained from a liver of a patient who has been tested negative for said pathogen or whose background enables to support that he was free from infection with said pathogen for the period of concern (e.g. histological and/or biological signs).
- the current invention providing a chimeric murine model with human hepatocytes, the implanted hepatocytes should not be infected by Plasmodium strains and/or by HVB and/or by HCV.
- "Non-tumoral" refer to hepatocytes having controlled cellular proliferation and spread and having a stable karyotype, i.e. hepatocytes containing the same number of chromosomes after multiple divisions.
- the implanted human hepatocytes although they are non-infected, and non-tumoral cells can nevertheless carry a mutation which effect may be studied when these hepatocytes are brought in contact with a determined compound administered to the chimeric murine model.
- Non adaptive defences refer to cells involved in the non-specific immunity, such as macrophages, monocytes or NK cells, in contrast to specific immunity directed by T and B lymphocytes.
- Settled refers to human hepatocytes that are not lost after the implanting step, and that succeed in surviving and repopulating in the chimeric murine model.
- the capacity of implanted hepatocytes "to maintain” as used herein refers to the capacity for these hepatocytes to survive in the host.
- the capacity of hepatocytes "to differentiate” as used herein refers to hepatocytes having the capacity to reach, after the implanting step, characteristics as similar as possible to those in their original host, e.g., in humans.
- This capacity can be determined in terms of secreted molecules (such as h ⁇ lAT for the hepatocytes), expressed surface receptors, pathogen infection, cell size or any other appropriate methods.
- the presence of human cell type-specific molecules expressed by the human cells can be measured, on murine sera, by well known techniques such as ELISA (enzyme-linked immunosorbent assay), Western Blot, dot blot, immunoprecipitation, direct or indirect immunostaining on histological sections using specific antibodies of implanted cell markers.
- the cell type- specific molecule transcripts can be detected by RT-PCR (reverse transcriptase-polymerase chain reaction) or real-time RT-PCR by using specific primers of implanted cell markers.
- Specific receptors can be detected by various techniques such as FACS analysis
- pathogen infection is controlled by detection of settled cell stage specific proteins by techniques including light microscopy, immunohistological staining on biopsies, by ELISA, PCR or RT-PCR on sera and by Western Blot, PCR or RT-PCR on cellular extracts.
- the capacity of implanted hepatocytes "to grow" as used herein refers to the capacity of settled hepatocytes, not only to survive in the recipient but also to multiply in the obtained chimeric model. Growth can be measured by quantitative imaging and evaluating the percentage of cells expressing a specific cell type marker. Measurements can be made at different time points to follow the repopulation of the settled hepatocytes.
- a first advantage of the invention lies in the fact that the success of human hepatocyte implantation does not depend on the strict homozygosity of the transgene or genetic defect responsible for murine hepatocyte death, in the murine model of the invention as described in previous models. Consequently, the possibility to use murine host carrying only one copy of said transgene or genetic defect, such as hemizygous or heterozygous host for this trait, widens the scope of this model. Consequently, the number of crosses to obtain a murine host, in which an implanting step is possible, is reduced, leading to a faster and cheaper host generation.
- homozygous is defined as presenting with two copies of the same allele of a determined gene
- heterozygous is defined as having two different alleles (e.g., A and a) at the corresponding gene loci on homologous chromosomes for said gene
- hemizygous is defined as presenting with a gene present in only one copy, not two, in an otherwise diploid cell or individual.
- a second advantage of the chimeric murine model according to the invention is the possibility to implant non-infected, non-tumoral hepatocytes from various origins. The hepatocytes needed for the implanting step can come directly from donor liver, partial liver resection or tumour-surrounding tissues. The model is less sensitive than previous ones to cell conservation conditions.
- Non-adaptive defences of the host is one of the parameters enabling the implanted hepatocytes to settle, differentiate and grow in the chimeric murine model.
- the efficiency of the control of non-adaptive defences can be checked by various techniques, such as FACS analysis.
- the main actors of the non-adaptive defences are macrophages and NK cells for which a strong reduction or depletion is expected after immunomodulation treatment according to the invention.
- "Macrophage depletion" as used herein is the process of reducing in a large amount the circulating and tissue macrophages.
- a convenient range of remaining macrophages after treatment is 0% to 50%.
- a preferred range of remaining macrophages is 0% to 20%.
- Macrophage number can be reduced by administrating in the host, antagonists of macrophages, such as toxic substances, like CI 2 MDP, or antibodies altering macrophage development or function and finally killing them.
- the administration of antagonists is performed by well-known techniques, including the use of liposomes.
- NK depletion as used herein is the process of reducing NK cells after treatment.
- a convenient range of remaining NK after treatment is
- NK antagonists or substances altering their function and development is made by well-known techniques using vectors including liposomes.
- the macrophage depletion is realized by injecting liposomes containing CI 2 MDP according to the technique of Van Rooijen et al. (Van Rooijen N. 1989. J. Immunol. Methods 124, 1-6).
- the liposome size can range from 0.5 to 7 ⁇ m to be ingested by macrophages, resulting in their killing.
- Possible alternatives for reducing the macrophage population are either the administration of another toxic, the cis-platinium or the use of the irradiation.
- the NK cells depletion is preferably realized by injecting an anti-NK antibody, such as the anti-Tim ⁇ 1 mouse monoclonal antibody, binding to the IL-2R ⁇ chain.
- an anti-NK antibody such as the anti-Tim ⁇ 1 mouse monoclonal antibody
- a particular protocol of macrophage depletion is the injection of CI 2 MDP embedded in liposomes, at 4-day interval, starting two days after implantation.
- Such liposomes fully clear macrophages from peritoneum, liver, spleen and kidney.
- Monocytes from the bone marrow colonise the liver after the clearance of all Kupfer cells and transform into very active large macrophages and new Kupfer cells. These are, again, destroyed by the next injection of liposomes.
- anti-NK antibodies are injected at monthly interval, starting two days after implantation.
- the interval between injection depend on the half-life of the antibody directed to NK cells, and can be from 2 to 4 weeks in order to keep NK cells continuously low.
- Particular immunocompromised models used to generate a host are SCID mouse (severe combined immunodeficiency), SCID/Nod mouse (severe combined immunodeficiency/non obese diabetic) or mouse with altered lymphocyte lineages such as BXN (NIHIII or Beige Xid Nude),
- the mouse host is obtained by: a. crossing a homozygous SCID mouse with a homozygous
- Alb-uPA transgenic mouse b. making selective backcrosses; c. recovering a homozygous SCID, hemizygous Alb-uPA mouse host.
- the present invention provides also a chimeric murine model which is an immunocompromised murine host harbouring at least one transgene or genetic defect responsible for murine hepatocyte death, which non-adaptive defences are controlled and which is implanted with human hepatocytes, said control of non-adaptive defences enable said hepatocytes to maintain, differentiate and grow.
- An advantage of the chimeric murine model of the invention is not only the maintenance of the settled human hepatocytes, but also their growth and differentiation.
- the growth and differentiation of the settled human hepatocytes of the chimeric murine model of the invention can be checked by various features, such as the presence of cell surface receptors, the secretion of proteins specific of the implanted hepatocytes, the cell size or the receptivity to pathogens.
- the model enables the settled human hepatocytes to secrete specific proteins characterizing their differentiation and growth, for several months especially for at least 3 months.
- Specific proteins of the implanted hepatocytes, such as albumin can be used as markers to follow the differentiation state as well as the repopulation.
- the model is suitable for in vivo study of metabolism of administered compounds and especially drugs, in said settled hepatocytes.
- the murine host is a homozygous
- SCID hemizygous Alb-uPA mouse, comprising implanted human hepatocytes.
- settled cells are receptive to hepatotropic pathogens, such as HBV, HCV or Plasmodium strains, e.g. P. falciparum or P. vivax.
- Another particular chimeric murine model is a chimeric murine animal, comprising implanted human hepatocytes and human red blood cells (RBC).
- RBC red blood cells
- the chimeric murine model of the present invention allows the study of restricted-tropism pathogens, for which no permissive line exists or for which permissive models are not fully satisfactory.
- the chimeric murine model of the present invention contains at least settled hepatocytes that are differentiated and are able to sustain a pathogen infection for several weeks. Because of the duration of the settled hepatocyte survival, their repopulation and their high number in the chimeric murine model, pathogen life cycle can be intensively studied including when said infected hepatocytes are put in contact with candidate drug compounds.
- the invention also provides a method for studying a hepatotropic pathogen, in a chimeric murine model according to the invention comprising: a.
- the first step is the introduction in the chimeric murine model of a pathogen in conditions in which it can interact with settled hepatocytes and especially can penetrate them.
- the introduction of the pathogen can be achieved by various ways, including intravenous or intracutaneous injections.
- the infection of said settled hepatocytes is monitored by well known methods including, but not limited to, light microscopy, immunofluorescence antibody test (IFAT) using pathogen specific antibodies, PCR (qualitative or quantitative) or RT-PCR using primers for a pathogen specific gene, ELISA or immunoprecipitation.
- IFAT immunofluorescence antibody test
- PCR quantitative or quantitative
- RT-PCR primers for a pathogen specific gene
- ELISA immunoprecipitation
- the results obtained to identify the pathogen infection are compared to a control model infected by the same pathogen but without cell implantation.
- the chimeric murine model tested is a mouse generated from a homozygous SCID, hemizygous Alb-uPA mouse host, in which hepatocytes are implanted.
- Another particular chimeric murine model is a murine host according to the invention implanted with both human hepatocytes and human red blood cells (RBC).
- hepatotropic pathogens are HBV (hepatitis B virus), HCV (hepatitis C virus) or Plasmodium strains including P. falciparum and P vivax.
- the monitoring of the hepatocyte infection by P. falciparum can be performed by testing pathogen proteins, specific for hepatocytes and/or erythrocytes, such as the circumsporozoite protein (sporozoite), the
- the monitoring of the hepatocyte infection by HCV can be performed by measuring the presence or absence of the viral RNA sequence (qualitative PCR) or the viral load (quantitative PCR).
- the monitoring of the hepatocyte infection by HBV can be performed by quantifying a viral envelop protein HbsAg with the ELISA technique.
- the invention provides also the use of of a chimeric murine model of the invention for the testing of a compound for a potential therapeutic interest, towards the infection or its consequences.
- the chimeric murine model can also be useful for the screening of drugs capable of altering the life cycle of pathogens. Owing to the capacity of the chimeric murine model to sustain a pathogen infection for several weeks, the effects of a drug administration on the infection can be observed and its efficacy can be evaluated.
- the invention provides a method for screening active compounds against the infection by hepatotropic pathogen or against its detrimental effects in a chimeric murine model of the invention comprising: a. infecting said chimeric murine model with a hepatotropic pathogen, in conditions enabling said pathogen to penetrate the settled human cells of the chimeric murine model; b. administering the tested compound in conditions allowing its activity; c.
- a drug as defined herein is any substance presented for treating, curing or preventing disease in human beings or in animals, including prodrugs.
- the drug can be administered under any appropriate forms including systemic or local routes. Several drugs (at least two) can be administrated together, alternatively or with specific protocols to show a possible synergy, redundancy or antagonism.
- the drug can be administrated by a lot of well known routes, including taken by mouth (orally), given by injection into a vein (intravenously), into a muscle (intramuscularly), beneath the skin (subcutaneously) or placed under the tongue (sublingually), inserted in the rectum (rectally) or vagina (vaginally), instilled in the eye (by the ocular route); sprayed into the nose and absorbed through the nasal membranes (nasally); breathed into the lungs (by inhalation) or applied to the skin (cutaneously).
- the effects of the drug on the pathogen infection or on its detrimental effects can be monitored.
- Various techniques can be used to observe the effects of the administrated drug, including light microscopy, immunofluorescence antibody test (IFAT), RT-PCR, PCR (qualitative or quantitative), or ELISA.
- IFAT immunofluorescence antibody test
- RT-PCR RT-PCR
- PCR quantitative or quantitative
- ELISA ELISA
- the infection are followed at different time points and various factors can be calculated: drug half-life, minimal effective dosage for infection elimination or reduction, drug dosage based on age, on body weight or on body surface area, the most efficient place or route for drug administration, the combination therapy efficiency.
- Other factors that can be observed could be the biological activity of the drug metabolites produced by settled human hepatocytes as well as the toxicity on the various organs of the murine model of these human specific metabolites.
- the invention also provides a method for screening the in vivo metabolism of xenobiotic compounds, in a chimeric murine model of the invention comprising: a. administrating the xenobiotic compound to be tested to said chimeric murine model in conditions allowing the compound to interact with settled human hepatocytes, b. observing its biotransformation by said settled hepatocytes. This subset of human hepatocytes can be exploited to follow the biotransformation of a xenobiotic, after its injection into the chimeric murine model.
- "Xenobiotic” as used herein refers to a chemical substance (or more generally, a chemical mix) that is not a normal component of the organism in which it is exposed to.
- Xenobiotics include most drugs (others than those compounds which naturally occur in the organism) and prodrugs, as well as other foreign substances.
- the xenobiotic is administrated into the chimeric murine model according to all routes and all forms cited above for the drug.
- One condition in the administration is that the xenobiotic can interact with the settled hepatocytes.
- the measurement of the level of the metabolites (degradation products), including intermediates and final products enables to track the xenobiotic metabolism kinetics, including its half-life.
- the model enables also to observe the effects of the compound on the settled cells, to evaluate the doses at which the effects appear. Finally, it is possible to study the potential interactions between reactive metabolites and cellular macromolecules.
- This model is important for prodrugs, i.e., any compound that undergoes biotransformation prior to exhibiting its pharmacologic effects. Indeed, the human hepatocytes can produce metabolites, especially the active form of the drug, from the prodrugs, whereas the murine hepatocytes cannot do it.
- a particular murine model tested is a chimeric mouse model generated from a homozygous SCID, hemizygous Alb-uPA mouse host, in which hepatocytes are implanted. In this chimeric model, one could monitor the cytotoxic effects of the drug on the liver, e.g., by measuring the circulating hepatic transaminases and by analysing the liver histology with optical microscopy techniques.
- the method of the invention can further comprise either a step of removing the graft from said non-human animal or the sacrifice of the non-human animal model harbouring settled heterologous nucleated cells capable of maintaining, differentiating and growing. This sacrifice can be carried out in a non-infected or infected animal, before or after the injection of an active or xenobiotic compound to be tested.
- the invention also provides technical platforms comprising at least the chimeric murine model of the invention such as: - a technical platform, useful to identify new compounds useful to treat mammal infections provoked by a hepatotropic pathogen, characterized in that it comprises at least a chimeric murine model as defined above and appropriate means to detect or to observe the effects of said compounds on a pathogen-generated infection of said murine model.
- a technical platform useful for screening the in vivo metabolism of xenobiotic compounds characterized in that it comprises at least a chimeric murine model according to the invention and appropriate means to observe the biotransformation of said compounds by implanted human hepatocytes in said murine model.
- Example 1 generation of a chimeric mouse model Animals Homozygous Alb-uPA transgenic mice and homozygous SCID mice were purchased from Jackson Laboratories (Maine, USA) and IFFA-CREDO, respectively. Animal were housed and maintained under specific pathogen-free conditions. To generate Alb-uPA/SCID mice, we crossed Alb-uPA and SCID mouse lines, and through selective backcrossed bred trait to homozygosis. uPA transgenic mice were identified by PCR of mouse-tail DNA with the following primers: p1 :5'- ATTCTGGAGGACCGCTTATCTGT-3'; p2: 5'-
- Human hepatocytes Primary human hepatocytes were isolated as described elsewhere (Guguen-Guillouzo C. et al. 1986. Prog Liver Dis 8, 33-50) from the healthy liver tissue of surgical liver biopsies specimens (approx. 20-25 cm 3 ) obtained with informed consent from patients who underwent therapeutic partial hepatectomy for liver metastasis and benign hepatic tumor. Subjects with viral infections (HCV, HBV, HIV), cirrhosis and primary hepatic carcinoma were excluded. Resected liver lobes were cut at a distance of at least 3 cm from the metastasis. Human hepatocytes were isolated by a two-step perfusion technique.
- Hepatocyte viability obtained by this method depends on 3 important factors which are the temperature: 37°C, pH: 7.6 and perfusate flow rate: 16-20ml according to the rejection size.
- Perfusion began with a HEPES buffer, without calcium, at a temperature, a pH and a perfusate flow rate as indicated above.
- Hepatocytes were then isolated with a perfusion of 200 ml of HEPES buffer supplemented in Collagenase H 0.05% (Roche Molecular Biochemicals) and CaCI 2 5 mM, and separated from non-parenchymatous cells by Percoll fractionation, as previously described (Giannini C. et al. 2003. Hepatology 38, 114-122; Guguen- Guillouzo C. et al. 1993. Cytotechnology 11 Suppl, S3-5; Guguen-Guillouzo C. et al. 1982. Cell Biol Int Rep 6(6), 625-8). Viable cells were determined by trypan blue exclusion
- hepatocyte implantation For hepatocyte implantation, mouse hosts were anesthetized and a laparotomy realized. A number of 5-6 x 10 5 viable human hepatocytes were injected into the splenic parenchyma of 10-14 days old recipients, as previously described (Giannini C. et al. 2003. Hepatology 38, 114-122).
- Macrophage and NK depletion treatment Mice were injected intraperitonealy at 4-day interval starting 2 days after implantation with 50 ⁇ l of a solution at 50% hematocrit of encapsulated-dichloro-methylene-diphosphonate (CI 2 MDP) liposomes.
- CI 2 MDP encapsulated-dichloro-methylene-diphosphonate
- mice received at monthly interval starting at day 2 post-implantation 1 mg of purified anti-Tim- ⁇ 1 (anti-IL-2 R ⁇ chain) mouse monoclonal antibody.
- Example 2 macrophage and NK detection
- FACS analysis The depletion of macrophages and NK, following treatment, were investigated by FACS analysis.
- Facs buffer Becton Dickinson
- the features of the F4/80 are the following: rat anti-mouse F4/80 antigen, conjugated to Fluorescein Isothiocyanate Isomer I (FITC) from Serotec.
- FITC Fluorescein Isothiocyanate Isomer I
- spleens from treated and untreated mice were taken and splenocytes were obtained after dilaceration (Cell strainer, Becton Dickinson). Cell suspension is then washed twice by centrifugation at 1500 rpm in RPMI 1650 medium (Life Sciences). Cells are then incubated with DX5 and anti-CD3 (clone17A2) antibodies, respectively coupled to FITC and Phycoerythrin for 30 minutes at room temperature. Cells are then washed and resuspended in Facs buffer (Becton Dickinson).
- the used antibodies have the following features: - DX5 antibody: rat antibody, specific of CD49b/Pan-NK cells (Bect
- - 17A2 antibody a rat anti-mouse CD3 (T-cell receptor-associated CD3 complex), conjugated with FITC (Becton Dickinson).
- Example 3 serum human albumin and ⁇ 1 antitrypsin detection
- human albumin and human ⁇ 1 -antitrypsin (h ⁇ lAT) in uPA/SCID mouse sera were quantified by a standard sandwich ELISA.
- Mouse anti human albumin 0.1 ⁇ g/ml, clone HSA-9, Sigma, St Louis, MO
- goat anti-h ⁇ 1AT 11 ⁇ g/ml; Rockland, Gilbertsville, PA
- HRP- conjugated rabbit anti-human albumin (0.16 ⁇ g/ml; Sigma Chemical Co.) and HRP-conjugated rabbit anti-h ⁇ 1AT (2 ⁇ g/ml) were used as antigen- specific indicator antibodies.
- the chromogen and the substrate were used according to the manufacturer indications (Sigma Chemical Co.).
- Absorbance values (405 nm) were converted to concentrations in ⁇ g/ml by comparison with a standard curve performed by using serial dilutions of defined amounts of purified human albumin and h ⁇ lAT (Sigma Chemical Co.).
- the ELISA result was considered as positive for the detection of human proteins (HA, h ⁇ lAT) when the OD value was higher than the OD means + 2 fold the standard deviation of 14 non-implanted Alb-uPA/SCID mice.
- Qualitative comparisons were done using the chi 2 test and Fischer exact test. P values of less than 0.05 were considered as significant.
- Example 4 albumin transcript detection RT-PCR was used for human albumin transcript detection.
- Total RNAs were isolated from human-mouse chimeric hepatocytes, murine and human liver tissues (and human cells) using RNeasy Protect Midi kit (Qiagen S.A., France) according to the manufacturer indication. Samples were quantitated using a spectrophotometer, and equal amounts of RNA from all samples were subjected to cDNA synthesis using random primers.
- Human specific albumin primers, 5'-CATTAGCTGCTGATTTTGTTGAAAG- 3' and 5 -TGTGCAGCATTTTGTGACTCTG-3' were used to detect human albumin transcripts.
- PCR Conditions were 95°C for 5 min; 94°C for 30s, 60°C for 1 min, and 72°C for 1 min for 40 cycles, with a final extension at 72° C for 5 min. Twenty ⁇ l of final PCR product were analyzed by electrophoresis (2% agarose gel with Ethidium Bromide) and PCR product bands (523 bp) were visualized under UV trans-illumination.
- Example 5 immunohistoloqical staining alpha-1 antitrypsin protein detection
- Mouse liver biopsies fixed in 10% formalin were embedded in paraffin.
- 5 ⁇ m sections pre-treated with Dako Biotin Blocking System (Dako, Glostrup, Denmark) were immunostained with a monoclonal antibody against human hepatocytes (Clone OCH1 E5, 1 :20 dilution; Dako, Glostrup, Denmark), using the Dako ARK system.
- 5 ⁇ m sections were immunostained with rabbit anti-human ⁇ 1- antitrypsin antibodies (Dako, Glostrup, Denmark) diluted (1 :2000) for 1 hour at room temperature.
- Slides were developed using a secondary anti-rabbit antibody conjugated with horseradish peroxidase (EN-VISION-kit; Dako) following the manufacturer's instructions.
- EN-VISION-kit horseradish peroxidase
- Example 6 liver repopulation estimation Quantitative imaging was performed on the liver biopsies of three untreated and treated animals obtained at one and three months after implantation. For each animal three different sections were immunostained with rabbit anti-h ⁇ 1AT and counterstained with Meyer's hematoxylin as described above. Adobe PhotoShop 6.02 software was then use to select and count the pixels corresponding to the total liver section (Meyer's hematoxylin staining) or to human hepatocyte clusters (anti-h ⁇ 1AT reactivity). The percentage of liver repopulation by human hepatocytes was expressed as the cell surface stained for h ⁇ lAT divided by the total cells surface of the tissue section. Qualitative comparisons were done using Fischer exact test. P values of less than 0.05 were considered significant.
- Example 7 effective depletion of NK cells and macrophages FACS analysis demonstrated, using F4/80 marker, that macrophage depletion in mice was effective since two days after the first liposome injection, macrophages represent 0.5% of lymphocyte cells in treated mice versus 8% in untreated mice (Figla). In addition, a successful depletion of NK cells was reached, since after Tim ⁇ -1 injection, DX5 + CD3 " NK represent 0.5% of spleen lymphocyte cells in treated mice versus 8% in untreated mice (Fig 1b). These depletion results were confirmed by histochemical analysis of human/mouse chimeric liver sections from animals implanted 6 weeks earlier.
- Example 8 human albumin and ⁇ 1 antitrypsin secretion
- Figure 3a human albumin
- 3b human ⁇ 1AT
- the number of positive mice for human albumin and ⁇ 1 antitrypsin secretion was dramatically improved by the treatment.
- untreated animals the number of positive mice for HA and h ⁇ lAT secretion, decreased over time during the follow-up (from month 1 to month 3: 13.6 to 0%, and 45.5 to 21.7%, respectively).
- mice positive for HA and h ⁇ lAT secretion were markedly higher at one month and, above all, remained high over time during the follow-up. At three months, 60.7 ⁇ 1.6% and 86.3 ⁇ 5.8% of mice secreted HA and h ⁇ lAT respectively. This first results showed a clear correlation between the treatment and the human albumin and ⁇ 1 antitrypsin secretion.
- Example 9 histochemical h ⁇ lAT protein expression Immunohistochemical investigations at 3 months post- implantation in untreated animals, revealed that human hepatocyte clusters surrounded by macrophage became negative for h ⁇ lAT (Fig. 2c-d), suggesting that human hepatocytes were no longer functional, in agreement with the lack of detectable human serum albumin at that time
- mice Individual data about 12 mice (6 treated and 6 untreated) are given in Table 1. This experiment showed that the implanted cells not only survive in their host, but also can multiply and repopulate the chimeric liver.
- Example 11 HA transcript expression HA transcripts were detectable by RT-PCR in RNA samples from the chimeric livers of treated animals but not from untreated animals, 3 months after implantation (Figure 5). Taken together, these results indicate that innate immune mechanisms deeply influence the survival and function of implanted human hepatocytes in the chimeric mouse model. This confirms the highly differentiated status of the settled cells obtained, following treatment. To demonstrate unequivocally that the implanted human hepatocytes were indeed functional, we took advantage of the stringent requirement of P.falciparum liver stages for fully differentiated human hepatocytes.
- Example 12 P.falciparum infection challenges Plasmodium falciparum sporozoites (strain NF54) were obtained from infected Anopheles stephensi mosquitoes salivary glands on day 14-16 after membrane feeding on gametocytes from NF54 cultured gametocytes as previously described (Ponnudurai T. et al. 1986.
- falciparum liver forms were detected by light microscopy of hematoxylin-eosin stained sections. Non implanted mice, or implanted mice negative for human albumin served as controls.
- Example 13 detection of the hepatocyte infection by P. falciparum Immunohistological staining
- P. falciparum Immunohistological staining For protein detection, several antibodies against pathogen specific-stage proteins were used. - an anti-circumsporozoite protein mouse monoclonal antibody, Mab2A10, specific of the circumsporozoite (CS) protein, a molecule expressed in sporozoites and carried over in liver stages (Atkinson C.T. et al. 1989. Am J Trop Med Hyg 40, 131-140; , Suhrbier A. et al. 1988.
- LSA1-J (Guerin-Marchand C. et al. 1987. Nature 329, 164-167; Fidock D.A. et al. 1994. Nature 161 , 126). - an antibody specific of MSP3, an antigen expressed only in blood stages. Anti-MSP3 affinity purified antibodies are directed to peptide MSP3-b, peptide MSP3-C and peptide MSP3-d (Brahimi K. et al. 1993. J
- RNAs were extracted, as described above, from a) implanted and non implanted infected mouse liver tissue taken 5 days following sporozoites inoculation, b) from human non-infected liver tissue and c) from P. falciparum erythrocytic stages, and treated with RNase free DNase.
- Reverse transcription was carried out under a final volume of 20 ⁇ l using 4 ⁇ g of total RNA and 200 units of M-MLV Reverse Transcriptase (Invitrogen) in the presence of specific LSA1 and HSP70-1 primers (from either LSA1 or HSP70-1 P. falciparum genes).
- Nested RT-PCR was carried out using 2 ⁇ l of the RT reaction, in the presence of 125 ⁇ M dNTPs, 125 nM of primers (LSA1-N and HSP70-N), and 2 units of Taq polymerase
- Example 14 Plasmodium falciparum infection in hepatocytes
- P. falciparum liver forms were found to be unexpectedly abundant (Fig. 6a) and this high number apparently correlated with serum albumin levels (ie. the largest number of parasites was seen in mice having a serum albumin level of about 100 ⁇ g/ml). Morphological indications were confirmed by immunofluorescence labeling with parasite-specific reagents.
- liver form-specific antibodies were negative on liver sections from non-implanted though infected control mice (over 100 sections screened). The results obtained both by light microscopy and immunofluorescence staining, were further confirmed by RT-PCR, using primers specific for parasite or human genes. Human albumin transcripts were detected in 7 of 7 albumin secreting mice, in 3 of 7 implanted mice without detectable HA in the serum and not in 3 non implanted control mice
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Toxicology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP05701083A EP1701612A2 (en) | 2004-01-09 | 2005-01-07 | Chimeric murine model comprising human hepatocytes user for screening compounds |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP04290065A EP1552740A1 (en) | 2004-01-09 | 2004-01-09 | Chimeric murine model comprising human hepatocytes - use for screening compounds |
PCT/EP2005/000546 WO2005067709A2 (en) | 2004-01-09 | 2005-01-07 | Chimeric murine model comprising human hepatocytes user for screening compounds |
EP05701083A EP1701612A2 (en) | 2004-01-09 | 2005-01-07 | Chimeric murine model comprising human hepatocytes user for screening compounds |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1701612A2 true EP1701612A2 (en) | 2006-09-20 |
Family
ID=34586020
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP04290065A Withdrawn EP1552740A1 (en) | 2004-01-09 | 2004-01-09 | Chimeric murine model comprising human hepatocytes - use for screening compounds |
EP05701083A Withdrawn EP1701612A2 (en) | 2004-01-09 | 2005-01-07 | Chimeric murine model comprising human hepatocytes user for screening compounds |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP04290065A Withdrawn EP1552740A1 (en) | 2004-01-09 | 2004-01-09 | Chimeric murine model comprising human hepatocytes - use for screening compounds |
Country Status (2)
Country | Link |
---|---|
EP (2) | EP1552740A1 (en) |
WO (1) | WO2005067709A2 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006019162A1 (en) | 2004-08-20 | 2006-02-23 | Daiichi Pure Chemicals Co., Ltd. | Method of presuming the metabolism of drug in human liver and liver function |
CN101550425B (en) * | 2008-03-31 | 2011-03-30 | 北京大学 | Method for manufacturing adjustable liver injury animal model and special carriers thereof |
JP5841322B2 (en) | 2010-04-22 | 2016-01-13 | オレゴン ヘルス アンド サイエンス ユニバーシティ | Fumaryl acetoacetate hydrolase (FAH) deficient pig and use thereof |
EP2747551B1 (en) | 2011-08-26 | 2020-02-12 | Yecuris Corporation | Fumarylacetoacetate hydrolase (fah)-deficient and immunodeficient rats and uses thereof |
EP2644027A1 (en) | 2012-03-26 | 2013-10-02 | Institut Pasteur | Transgenic immunodefficient mouse expressing human SIRPalpha. |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6509514B1 (en) * | 2000-03-17 | 2003-01-21 | Kmt Hepatech, Inc. | Chimeric animal model susceptible to human hepatitis C virus infection |
-
2004
- 2004-01-09 EP EP04290065A patent/EP1552740A1/en not_active Withdrawn
-
2005
- 2005-01-07 EP EP05701083A patent/EP1701612A2/en not_active Withdrawn
- 2005-01-07 WO PCT/EP2005/000546 patent/WO2005067709A2/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO2005067709A3 * |
Also Published As
Publication number | Publication date |
---|---|
WO2005067709A3 (en) | 2005-11-24 |
EP1552740A1 (en) | 2005-07-13 |
WO2005067709A2 (en) | 2005-07-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6509514B1 (en) | Chimeric animal model susceptible to human hepatitis C virus infection | |
Baer et al. | Kupffer cells are obligatory for Plasmodium yoelii sporozoite infection of the liver | |
Brown et al. | A long‐term hepatitis B viremia model generated by transplanting nontumorigenic immortalized human hepatocytes in Rag‐2–deficient mice | |
JP5172670B2 (en) | Nonalcoholic steatohepatitis model animal and fatty liver model animal | |
CN102460162A (en) | Method of expanding human hepatocytes in vivo | |
AU2001242163A1 (en) | Chimeric animal model susceptible to human hepatitis C virus infection | |
US20030126626A1 (en) | Propagation of human hepatocytes in non-human animals | |
US7456018B2 (en) | Human hepatoma lines, methods for obtaining same and uses thereof | |
WO2005067709A2 (en) | Chimeric murine model comprising human hepatocytes user for screening compounds | |
JP2004538012A (en) | Proliferation of human hepatoblastoma cells in non-human animals | |
US6660905B1 (en) | Mice comprising engrafted functional human hepatocytes | |
WO2006023593A2 (en) | Malarial animal model having a chimeric human liver | |
US20070044165A1 (en) | Non-human mammal model comprising heterologous nucleated cells - use for screening compounds | |
EP3777528A1 (en) | Non-human vertebrate transplanted with human hepatocytes and method for producing same | |
JP5737821B2 (en) | Hemophilia B therapeutic agent and method for producing the same | |
Kneteman et al. | In vivo study of HCV in mice with chimeric human livers | |
Krishnan et al. | Prolonged engraftment of human hepatocytes in mice transgenic for the deleted form of human hepatocyte growth factor | |
Rebus | Approaches to the Development of In Vivo Propagation Systems for Hepatitis C Virus (HCV) | |
Okuse | than anti-HCV antibody-negative patients" In addi | |
AU2002329766A1 (en) | Propagation of human hepatoblastoma cell in non-human animals |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20060706 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: RENIA, LAURENT Inventor name: LUNEL, FRANCOISE Inventor name: KREMSDORF, DINA Inventor name: HEZ, STEPHANIE Inventor name: MOROSAN, SERBAN Inventor name: BRECHOT, CHRISTIAN Inventor name: DRUILHE, PIERRE |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: DRUILHE, PIERRE Inventor name: MOROSAN, SERBAN Inventor name: BRECHOT, CHRISTIAN Inventor name: KREMSDORF, DINA Inventor name: RENIA, LAURENT Inventor name: HEZ-DEROUBAIX, STEPHANIE Inventor name: LUNEL, FRANCOISE |
|
DAX | Request for extension of the european patent (deleted) | ||
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1096822 Country of ref document: HK |
|
17Q | First examination report despatched |
Effective date: 20110928 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20120209 |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: WD Ref document number: 1096822 Country of ref document: HK |