WO2001032841A2 - Method of in vitro t cell differentiation of cd34+ progenitor cells - Google Patents
Method of in vitro t cell differentiation of cd34+ progenitor cells Download PDFInfo
- Publication number
- WO2001032841A2 WO2001032841A2 PCT/US2000/029655 US0029655W WO0132841A2 WO 2001032841 A2 WO2001032841 A2 WO 2001032841A2 US 0029655 W US0029655 W US 0029655W WO 0132841 A2 WO0132841 A2 WO 0132841A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cell
- cells
- cγtokine
- hematopoietic
- factor
- Prior art date
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 187
- 238000000034 method Methods 0.000 title claims abstract description 154
- 238000000338 in vitro Methods 0.000 title claims description 19
- 230000024245 cell differentiation Effects 0.000 title description 14
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 219
- 210000001165 lymph node Anatomy 0.000 claims abstract description 101
- 230000003394 haemopoietic effect Effects 0.000 claims abstract description 84
- 102000004127 Cytokines Human genes 0.000 claims abstract description 23
- 108090000695 Cytokines Proteins 0.000 claims abstract description 23
- 241000288906 Primates Species 0.000 claims abstract description 20
- 208000029462 Immunodeficiency disease Diseases 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 322
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 101
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 claims description 101
- 102000004140 Oncostatin M Human genes 0.000 claims description 62
- 108090000630 Oncostatin M Proteins 0.000 claims description 62
- 239000003795 chemical substances by application Substances 0.000 claims description 41
- 102000036693 Thrombopoietin Human genes 0.000 claims description 38
- 108010041111 Thrombopoietin Proteins 0.000 claims description 38
- 241000725303 Human immunodeficiency virus Species 0.000 claims description 33
- 108010002350 Interleukin-2 Proteins 0.000 claims description 31
- 108090001005 Interleukin-6 Proteins 0.000 claims description 30
- 108010002586 Interleukin-7 Proteins 0.000 claims description 29
- 230000004069 differentiation Effects 0.000 claims description 24
- 241000700605 Viruses Species 0.000 claims description 22
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 21
- 239000013598 vector Substances 0.000 claims description 18
- 238000012258 culturing Methods 0.000 claims description 16
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims description 16
- 102000004058 Leukemia inhibitory factor Human genes 0.000 claims description 15
- 108090000581 Leukemia inhibitory factor Proteins 0.000 claims description 15
- 230000012010 growth Effects 0.000 claims description 15
- 208000032839 leukemia Diseases 0.000 claims description 15
- 208000026278 immune system disease Diseases 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 10
- 239000013604 expression vector Substances 0.000 claims description 9
- 230000001177 retroviral effect Effects 0.000 claims description 9
- 210000005260 human cell Anatomy 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 239000013603 viral vector Substances 0.000 claims description 6
- 206010061598 Immunodeficiency Diseases 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 230000007813 immunodeficiency Effects 0.000 claims description 4
- 241001430294 unidentified retrovirus Species 0.000 claims description 3
- 241000713772 Human immunodeficiency virus 1 Species 0.000 claims description 2
- 208000033065 inborn errors of immunity Diseases 0.000 claims 1
- 208000028529 primary immunodeficiency disease Diseases 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 44
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 55
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 55
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 48
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 47
- 102000015696 Interleukins Human genes 0.000 description 44
- 108010063738 Interleukins Proteins 0.000 description 44
- 230000000638 stimulation Effects 0.000 description 41
- 230000014509 gene expression Effects 0.000 description 32
- 150000007523 nucleic acids Chemical class 0.000 description 28
- 108090000623 proteins and genes Proteins 0.000 description 25
- 230000004044 response Effects 0.000 description 20
- 239000000203 mixture Substances 0.000 description 19
- 239000000523 sample Substances 0.000 description 18
- 210000001519 tissue Anatomy 0.000 description 17
- 239000012634 fragment Substances 0.000 description 15
- 210000004698 lymphocyte Anatomy 0.000 description 15
- 239000003550 marker Substances 0.000 description 14
- 238000003752 polymerase chain reaction Methods 0.000 description 14
- 108091028043 Nucleic acid sequence Proteins 0.000 description 13
- 108091008874 T cell receptors Proteins 0.000 description 12
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 12
- 239000000427 antigen Substances 0.000 description 12
- 208000035475 disorder Diseases 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 238000003556 assay Methods 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 238000011282 treatment Methods 0.000 description 11
- 210000000987 immune system Anatomy 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 150000001875 compounds Chemical class 0.000 description 9
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 9
- 229920002477 rna polymer Polymers 0.000 description 9
- 230000000692 anti-sense effect Effects 0.000 description 8
- 210000001185 bone marrow Anatomy 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 210000003743 erythrocyte Anatomy 0.000 description 8
- 239000011886 peripheral blood Substances 0.000 description 8
- 210000005259 peripheral blood Anatomy 0.000 description 8
- -1 CD8 Proteins 0.000 description 7
- 230000000961 alloantigen Effects 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- CBVCZFGXHXORBI-PXQQMZJSSA-N indinavir Chemical compound C([C@H](N(CC1)C[C@@H](O)C[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H]2C3=CC=CC=C3C[C@H]2O)C(=O)NC(C)(C)C)N1CC1=CC=CN=C1 CBVCZFGXHXORBI-PXQQMZJSSA-N 0.000 description 7
- 239000010410 layer Substances 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 108020004707 nucleic acids Proteins 0.000 description 7
- 102000039446 nucleic acids Human genes 0.000 description 7
- 108020003175 receptors Proteins 0.000 description 7
- 102000005962 receptors Human genes 0.000 description 7
- 230000008685 targeting Effects 0.000 description 7
- 239000006285 cell suspension Substances 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 229960001936 indinavir Drugs 0.000 description 6
- 238000002955 isolation Methods 0.000 description 6
- 239000002502 liposome Substances 0.000 description 6
- 210000005087 mononuclear cell Anatomy 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 239000013641 positive control Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 208000030507 AIDS Diseases 0.000 description 5
- 241000713666 Lentivirus Species 0.000 description 5
- 241000124008 Mammalia Species 0.000 description 5
- 101710149951 Protein Tat Proteins 0.000 description 5
- 241000191967 Staphylococcus aureus Species 0.000 description 5
- 238000012264 coculture process Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 230000001605 fetal effect Effects 0.000 description 5
- 238000012239 gene modification Methods 0.000 description 5
- 230000005017 genetic modification Effects 0.000 description 5
- 235000013617 genetically modified food Nutrition 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 210000002861 immature t-cell Anatomy 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 238000002054 transplantation Methods 0.000 description 5
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 4
- 101710205625 Capsid protein p24 Proteins 0.000 description 4
- 241000282693 Cercopithecidae Species 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 4
- 102100034343 Integrase Human genes 0.000 description 4
- 102000003729 Neprilysin Human genes 0.000 description 4
- 108090000028 Neprilysin Proteins 0.000 description 4
- 101710177166 Phosphoprotein Proteins 0.000 description 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 4
- 101710149279 Small delta antigen Proteins 0.000 description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 4
- 102100022563 Tubulin polymerization-promoting protein Human genes 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 230000000735 allogeneic effect Effects 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 4
- 239000002775 capsule Substances 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- 230000006801 homologous recombination Effects 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 210000003563 lymphoid tissue Anatomy 0.000 description 4
- 210000002540 macrophage Anatomy 0.000 description 4
- 239000003226 mitogen Substances 0.000 description 4
- 230000007193 modulation by symbiont of host erythrocyte aggregation Effects 0.000 description 4
- 238000004806 packaging method and process Methods 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000002356 single layer Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 229960000814 tetanus toxoid Drugs 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 230000002992 thymic effect Effects 0.000 description 4
- 210000001541 thymus gland Anatomy 0.000 description 4
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 101710146739 Enterotoxin Proteins 0.000 description 3
- 208000031886 HIV Infections Diseases 0.000 description 3
- 208000037357 HIV infectious disease Diseases 0.000 description 3
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 3
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 description 3
- 210000000612 antigen-presenting cell Anatomy 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000001143 conditioned effect Effects 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000000147 enterotoxin Substances 0.000 description 3
- 231100000655 enterotoxin Toxicity 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000000527 lymphocytic effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 210000001778 pluripotent stem cell Anatomy 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- QWENRTYMTSOGBR-UHFFFAOYSA-N 1H-1,2,3-Triazole Chemical compound C=1C=NNN=1 QWENRTYMTSOGBR-UHFFFAOYSA-N 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
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 description 2
- 208000029483 Acquired immunodeficiency Diseases 0.000 description 2
- 102100035248 Alpha-(1,3)-fucosyltransferase 4 Human genes 0.000 description 2
- 108091093088 Amplicon Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000016911 Deoxyribonucleases Human genes 0.000 description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 description 2
- 208000000398 DiGeorge Syndrome Diseases 0.000 description 2
- 102400001368 Epidermal growth factor Human genes 0.000 description 2
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 101000867232 Escherichia coli Heat-stable enterotoxin II Proteins 0.000 description 2
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 2
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 description 2
- 101001022185 Homo sapiens Alpha-(1,3)-fucosyltransferase 4 Proteins 0.000 description 2
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 2
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 2
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 2
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 2
- 241000282560 Macaca mulatta Species 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- 241000713311 Simian immunodeficiency virus Species 0.000 description 2
- 101150052863 THY1 gene Proteins 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000036436 anti-hiv Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 210000002798 bone marrow cell Anatomy 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 230000011712 cell development Effects 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000001246 colloidal dispersion Methods 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 229940116977 epidermal growth factor Drugs 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 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 2
- 125000000524 functional group Chemical group 0.000 description 2
- 208000016361 genetic disease Diseases 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 210000000777 hematopoietic system Anatomy 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000001365 lymphatic vessel Anatomy 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 239000000693 micelle Substances 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000008707 rearrangement Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004043 responsiveness Effects 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 150000003431 steroids Chemical class 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 210000002536 stromal cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 239000000724 thymus hormone Substances 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 2
- 230000029812 viral genome replication Effects 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- LIFNDDBLJFPEAN-BPSSIEEOSA-N (2s)-4-amino-2-[[(2s)-2-[[2-[[2-[[(2s)-5-amino-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-2-[[(2s)-5-oxopyrrolidine-2-carbonyl]amino]propanoyl]amino]hexanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]acetyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@@H]1CCC(=O)N1 LIFNDDBLJFPEAN-BPSSIEEOSA-N 0.000 description 1
- IQFYYKKMVGJFEH-OFKYTIFKSA-N 1-[(2r,4s,5r)-4-hydroxy-5-(tritiooxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound C1[C@H](O)[C@@H](CO[3H])O[C@H]1N1C(=O)NC(=O)C(C)=C1 IQFYYKKMVGJFEH-OFKYTIFKSA-N 0.000 description 1
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 102100022749 Aminopeptidase N Human genes 0.000 description 1
- 241000282708 Aotus <primate> Species 0.000 description 1
- 241000282709 Aotus trivirgatus Species 0.000 description 1
- 241000282672 Ateles sp. Species 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 description 1
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 description 1
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 102000013925 CD34 antigen Human genes 0.000 description 1
- 108050003733 CD34 antigen Proteins 0.000 description 1
- 108010041397 CD4 Antigens Proteins 0.000 description 1
- 102100032912 CD44 antigen Human genes 0.000 description 1
- 108010032795 CD8 receptor Proteins 0.000 description 1
- 241000288950 Callithrix jacchus Species 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000282668 Cebus Species 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 108010003422 Circulating Thymic Factor Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 1
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 102100026234 Cytokine receptor common subunit gamma Human genes 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 102100021260 Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Human genes 0.000 description 1
- 208000015872 Gaucher disease Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010027044 HIV Core Protein p24 Proteins 0.000 description 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 241000175212 Herpesvirales Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000757160 Homo sapiens Aminopeptidase N Proteins 0.000 description 1
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 1
- 101001055227 Homo sapiens Cytokine receptor common subunit gamma Proteins 0.000 description 1
- 101000908391 Homo sapiens Dipeptidyl peptidase 4 Proteins 0.000 description 1
- 101000894906 Homo sapiens Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Proteins 0.000 description 1
- 101000994365 Homo sapiens Integrin alpha-6 Proteins 0.000 description 1
- 101001046677 Homo sapiens Integrin alpha-V Proteins 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 101001018097 Homo sapiens L-selectin Proteins 0.000 description 1
- 101001063392 Homo sapiens Lymphocyte function-associated antigen 3 Proteins 0.000 description 1
- 101001008874 Homo sapiens Mast/stem cell growth factor receptor Kit Proteins 0.000 description 1
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 101000934346 Homo sapiens T-cell surface antigen CD2 Proteins 0.000 description 1
- 101000980827 Homo sapiens T-cell surface glycoprotein CD1a Proteins 0.000 description 1
- 101000934341 Homo sapiens T-cell surface glycoprotein CD5 Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 101100321817 Human parvovirus B19 (strain HV) 7.5K gene Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102100023915 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102100032816 Integrin alpha-6 Human genes 0.000 description 1
- 102100022337 Integrin alpha-V Human genes 0.000 description 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102100027268 Interferon-stimulated gene 20 kDa protein Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- 108090000177 Interleukin-11 Proteins 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 108010002335 Interleukin-9 Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 102100033467 L-selectin Human genes 0.000 description 1
- 102100023981 Lamina-associated polypeptide 2, isoform alpha Human genes 0.000 description 1
- 101710163560 Lamina-associated polypeptide 2, isoform alpha Proteins 0.000 description 1
- 101710189385 Lamina-associated polypeptide 2, isoforms beta/gamma Proteins 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 102100030984 Lymphocyte function-associated antigen 3 Human genes 0.000 description 1
- 102000004083 Lymphotoxin-alpha Human genes 0.000 description 1
- 108090000542 Lymphotoxin-alpha Proteins 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 102100027754 Mast/stem cell growth factor receptor Kit Human genes 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010050619 Monokines Proteins 0.000 description 1
- 102000013967 Monokines Human genes 0.000 description 1
- 206010048723 Multiple-drug resistance Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000714177 Murine leukemia virus Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 208000001388 Opportunistic Infections Diseases 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 241000282516 Papio anubis Species 0.000 description 1
- 241001504519 Papio ursinus Species 0.000 description 1
- 241000237988 Patellidae Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 241000288961 Saguinus imperator Species 0.000 description 1
- 241000282695 Saimiri Species 0.000 description 1
- 241000282676 Sapajus apella Species 0.000 description 1
- 241000699772 Sciurus Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 108700042075 T-Cell Receptor Genes Proteins 0.000 description 1
- 102100024219 T-cell surface glycoprotein CD1a Human genes 0.000 description 1
- 102100025244 T-cell surface glycoprotein CD5 Human genes 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 101150003725 TK gene Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 239000000898 Thymopoietin Substances 0.000 description 1
- 108010046075 Thymosin Proteins 0.000 description 1
- 102000007501 Thymosin Human genes 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000007503 antigenic stimulation Effects 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 239000000823 artificial membrane Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- MXWJVTOOROXGIU-UHFFFAOYSA-N atrazine Chemical compound CCNC1=NC(Cl)=NC(NC(C)C)=N1 MXWJVTOOROXGIU-UHFFFAOYSA-N 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 239000012578 cell culture reagent Substances 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 229930183167 cerebroside Natural products 0.000 description 1
- 150000001784 cerebrosides Chemical class 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 229940047120 colony stimulating factors Drugs 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 208000024190 congenital T-cell immunodeficiency Diseases 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 229940088900 crixivan Drugs 0.000 description 1
- 230000002338 cryopreservative effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000005860 defense response to virus Effects 0.000 description 1
- 230000005742 definitive hemopoiesis Effects 0.000 description 1
- 230000005786 degenerative changes Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 210000000267 erythroid cell Anatomy 0.000 description 1
- 230000000925 erythroid effect Effects 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000012595 freezing medium Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 150000002270 gangliosides Chemical class 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 210000001280 germinal center Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000004013 groin Anatomy 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 230000002607 hemopoietic effect Effects 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 239000008241 heterogeneous mixture Substances 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000037451 immune surveillance Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229940102223 injectable solution Drugs 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- RCRODHONKLSMIF-UHFFFAOYSA-N isosuberenol Natural products O1C(=O)C=CC2=C1C=C(OC)C(CC(O)C(C)=C)=C2 RCRODHONKLSMIF-UHFFFAOYSA-N 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 208000021601 lentivirus infection Diseases 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- 125000003473 lipid group Chemical group 0.000 description 1
- 238000002690 local anesthesia Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 210000004925 microvascular endothelial cell Anatomy 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 239000002088 nanocapsule Substances 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 239000002831 pharmacologic agent Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000009696 proliferative response Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 108700004030 rev Genes Proteins 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 150000003408 sphingolipids Chemical class 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 231100000617 superantigen Toxicity 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 108700004027 tat Genes Proteins 0.000 description 1
- 101150098170 tat gene Proteins 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000017613 viral reproduction Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 210000001325 yolk sac Anatomy 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/204—IL-6
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/464838—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/145—Thrombopoietin [TPO]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2306—Interleukin-6 (IL-6)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2307—Interleukin-7 (IL-7)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/237—Oncostatin M [OSM]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Definitions
- the present invention relates generally to the field of T cell differentiation, and more specifically, to systems useful for inducing the growth and differentiation of T cells.
- the hematopoietic stem cell is the progenitor for all of the ieukocytic and erythrocytic blood cells, including the lymphocytic and myelomonocytic lineages, as well as other types of cells such as osteoclasts. These cells provide an enormous range of functions and are believed to be the only cells that are self-regenerating and maintain their pluripotent potential during the life of the host.
- hematopoietic stem cells originate in coordinate waves within the extra-embryonic yolk sac and the fetal aortagonadomesonephros (AGM). Later, during definitive hematopoiesis, pluripotent stem cells populate the fetal liver and the bone marrow.
- Pluripotent hematopoietic stem cells migrate from these regions to the thymus and serve as the precursors for T lymphocytes. Postnatally, pluripotent stem cells account for approximately 1 % of the low density nucleated bone marrow cells; these provide a renewable source of stem cells to regenerate the hematopoietic system during adult life. The differentiation of hematopoietic cells is dependent on a variety of factors, including the stromal microenviro ⁇ ment and differentiation factors.
- T cells The differentiation of T cells occurs through a series of discrete developmental stages characterized by specific T cell markers.
- the pluripotent progenitor cell has been shown to express high levels of the CD34 marker on the cell surface. As the pluripotent cells develop and commit to either the iymphoid, mo ⁇ omyeloid, or erythroid cells, the level of CD34 expression decreases.
- the pluripotent hematopoietic CD34 * T cell progenitor cell does not express the T cell markers CD3, CD4, and CD8.
- Differentiating CD34 * cells progress from Stage 0 (the undifferentiated state), to Stages 1, 2, 3, and 4, as shown in Figure 1. In Stage 0, cells are double negative for CD4 and CD8 and appear in the lower left quadrant.
- CD34+ progenitor cells (CD34 * CD3 CD4 CD8 ) subsequently express lymphocyte specific makers and lose the expression of CD34 as they differentiate (CD34 CD3 * CD4 * CD8 * )(Stages 1, 2, and 3). Developing T cells become double positive for the expression of CD4 and CD8 and express variable levels of CD3 on the surface of the cell.
- CD3 * CD4 * CD8 CD3 * CD4 CD8 +
- CD3 * CD4 CD8 + CD3 * CD4 CD8 +
- mu ⁇ ne thymic explant studies (Plum, J., et al., Blood 84:1587-1593, 1994) and simian thy ic monolayer cultures (Rosenzweig, M., et al., Blood 87( 10): 4040-4048, 1996) have been employed.
- Human thymic explant studies (U.S. Patent 5,677,139) have also been utilized. All of the techniques require the use of allogeneic thymic stromal cells in order to culture or differentiate adult stem cells. T cells that develop in a xenogeneic environment may not demonstrate appropriate host MHC restriction.
- the present invention relates to the generation of a population of T cells derived from a population of hematopoietic T cell progenitor cells.
- one embodiment of the invention is a method for in vitro T cell production, comprising the steps of culturing a lymph node cell in vitro with a primate hematopoietic T cell progenitor cell.
- one embodiment of the invention is a method of treating a subject having an immune disorder, comprising the steps of culturing a lymph node cell in vitro with a primate hematopoietic T cell progenitor cell to produce a descendent cell, and administering a therapeutically effective amount of the progenitor cell or the descendent of the progenitor cell to the subject.
- Another therapeutic embodiment of the invention is a method of treating an immune disorder in a subject, comprising administering a therapeutically effective amount of a c ⁇ tokine to a lymph node in the subject, wherein the c ⁇ tokine comprises at least IL-2 and IL-6.
- Another embodiment of the invention is a method for modifying a hematopoietic stem cell to produce a descendent cell containing a nucleotide sequence of interest.
- the method comprises the steps of culturing a lymph node cell in vitro with a primate hematopoietic T cell progenitor cell to produce a descendent cell, contacting the primate hematopoietic T cell progenitor cell and the lymph node cells with an expression vector comprising a nucleotide sequence of interest, wherein the contacting is for a period of time sufficient for the virus to enter the hematopoietic T cell progenitor cell.
- the invention comprises the steps of culturing a lymph node cell in vitro with a primate hematopoietic T cell progenitor cell to produce a descendent cell, contacting the primate hematopoietic T cell progenitor cell and the lymph node cells with an expression vector comprising a nucleotide sequence
- one embodiment contemplated by the invention is a method for testing the effect of an agent on a hematopoietic cell, comprising the steps of co- culturing a lymph node cell in vitro with a primate hematopoietic T cell progenitor cell, contacting the hematopoietic T cell progenitor cell with an agent, and comparing the differentiation or growth of the hematopoietic T cell progenitor cell with the differentiation or growth of a control cell not contacted with the agent.
- Still another embodiment of the present invention is a kit comprising a container containing a cr ⁇ opreserved lymph node cell and instructions for culture of the lymph node cell with a hematopoietic T cell progenitor cell.
- FIGURE 1 shows a graphic representation of the stages of T cell progenitor cell development as such cells would appear during FACS analysis.
- FIGURE 2 shows FACS graphs A through 0 that depict a number of results concerning various growth conditions used in the methods described herein.
- T cell surface markers CD3, CD4, and CD8, and other markers are followed in this series of graphs.
- a number of fluorescent probes are used in these experiments. For example, CY (CY5), FITC, PE, QR, and TCR-ab FITC are all used.
- FIGURE 3 is a three dimensional FACS representation of the expression of CD3, CD4 and CD8 in a lymph node cocultured with CD34 + progenitor cells obtained from a patient with end stage HIV utilizing the methods described herein.
- FIGURE 4 is a graphic representation of various populations of cells and their response to mitogen stimulation. Mitogens used were (PHA) and the response to mitogen is represented as the Stimulation Index, which is calculated by quantitating the amount of tritiated thymidine incorporated by the assayed cells.
- the Stimulation Index is the quotient of the total number of counts divided by the number of counts assayed from an unstimulated control.
- the abbreviation LNMC means lymph node mononuclear cells.
- FIGURE 5 is a graphic representation of various populations of cells obtained from an end stage HIV + patient utilizing the methods described herein and testing responses to alio-antigen stimulation.
- the lymphocyte response to stimulation by allo-antigen is MHC-restricted but it is not antigen specific at the T cell receptor (TCR).
- FIGURE 6 is a graphical representation of various populations of cells and their response to antigen stimulation when the antigens were SE A and SE-B.
- a method for in vitro T cell production wherein a lymph node tissue fragment is cultured in vitro with a primate hematopoietic CD34 + CD3 progenitor cell.
- "Hematopoietic T cell progenitor cells” are those cells capable of differentiating into mature T cells. T cell progenitor cells include stem cells.
- a “stem cell” is a pluripotent cell that is capable of self-renewal and differentiation into all myeloid and lymphoid cell lineages, including T cells.
- a pluripotent stem cell gives rise to progeny of all defined hemato-l ⁇ mphoid lineages; and limited numbers of these cells are capable of fully reconstituting an immuno-compromised host in all blood cell types and their progenitors, including the pluripotent hematopoietic stem cell by cell renewal.
- the T cell progenitor cells which are employed may be fresh, frozen, or have been subject to a treatment prior to culture. They may be fetal, neonate, adult, and can be isolated from sources including fetal liver, bone marrow, umbilical cord blood, or peripheral blood.
- T cell progenitor cells include peripheral blood stem cells obtained from the peripheral blood of subjects who have been treated with che otherapeutic agents and/or cytokines (e.g., G-CSF or GM-CSF) to increase hematopoietic T cell progenitor cells circulating in the peripheral blood.
- cytokines e.g., G-CSF or GM-CSF
- the hematopoietic T cell progenitor cells are derived from a mammal.
- the T cell progenitor cells are derived from a primate.
- the primate can be a human or a non-human primate.
- Non-human primates include, but are not limited to Aotus triviruius (owl monkey), Ateletes geoffroy (spider monkey), Cebus Albifroms (white and brown capuchin monkey), Cercopitecus aethiops (African green monkey), Calitnx jacch ⁇ s (common marmoset monkey), Macaca mulatta (rhesus monkey), Macaca fasciculans (crab eater (cynomolgus) monkey), Pan traogtodytes (chimpanzee), Papio anubis (baboon), Sagumus fuscicollis (tamarin monkey), Salmiri sciurus
- the T cell progenitor cells can be isolated from a normal subject or a subject with a disorder of the immune system.
- Immune disorders include "immunodeficiency disorders," wherein a general deficiency of T cells exists or a deficiency of specific populations of T cells exists.
- An example of an acquired immunodeficiency disorder is AIDS, an example of a congenital T cell immunodeficiency is DiGeorge's Syndrome (Complete Athymia), or an acquired immunodeficiency secondary to chemotherapy.
- Immune disorders include "immunoproliferative" disorders, wherein an increase in the overall numbers of lymphocytes or a specific population of T cells exists.
- An example of an immunoproliferative disorder is a I ⁇ mphoma.
- a subject's own cells can be removed, expanded and/or differentiated, and optionally genetically altered.
- the subject is infected with an immunodeficiency lentivirus.
- lentivirus is used in its conventional sense to describe a genus of viruses containing reverse transc ⁇ ptase.
- the lentiviruses include the "immunodeficiency viruses” which include human immunodeficiency virus (HIV) type 1 and type 2 (HIV-1 and HIV- 2) and simian immunodeficiency virus (SIV).
- Hematopoietic T cell progenitor cells at various stages of differentiation may be used, although it is preferable to use progenitor cells in very early stages of differentiation.
- CD34 + progenitor cells can be used.
- Hematopoietic stem cells are cells that are both self-renewing and pluripotent. These cells constitute approximately 0.01 % of low density nucleated bone marrow cells Pluripotent hematopoietic stem cells express a high level of CD34 antigen. These cells eventually develop and commit to the erythroid, monomyeloid, or lymphoid cells, including T cells.
- CD34 + T cell progenitor cells do not express the T cell markers CD3, CD4, and CD8 on the cell surface During differentiation into mature T cells, the cells pass through an intermediate state where the cells express variable levels of CD3, CD4 and CD8 (termed CD3 t CD4 + CD8 + , or double positive cells for CD4 + CD8 + ).
- CD3 + CD4 + CD8 + double positive hematopoietic T cell progenitor cells are immature T cells at an intermediate stage of differentiation which have lost expression of CD34. These cells subsequently mature into cells that are CD34 CD3 4 (bright), and further express only one of the CD4 or CD8 cell surface markers. Thus, mature T cells are either positive for CD4 or CD8 which is expressed with the CD3 receptor as CD3 + CD4 + or CD3 + CD8 ⁇
- the manner in which the T cell progenitor cells are separated from other cells of the hematopoietic or other lineage is not critical to this invention.
- the stem cells may be separated as described in U.S. Patent No. 5,061,620 or U.S. Patent 5,799,944.
- a "substantially enriched" hematopoietic T cell progenitor cell population refers to a substantially homogeneous population of hematopoietic T cell progenitor cells that are substantially free from other cells with which they are naturally associated. As described in U.S. Patent Nos.
- a substantially enriched population of stem cells may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with the stem cells.
- the population of hematopoietic T cell progenitor cells will comprise less than about 10% lineage committed cells, more usually less than about 5% lineage committed cells.
- the hematopoietic T cell progenitor cells are characterized by both the presence of markers associated with specific epitopes identified by antibodies and the absence of certain markers as identified by the lack of binding of specific antibodies. At such time as a specific marker is identified for hematopoietic T cell progenitor cells, binding of an antibody to such a marker may provide the desired composition.
- a large proportion of the differentiated or lineage committed cells may be removed initially by using a relatively crude separation technique, where the major cell populations of the hematopoietic system, such as the lymphocytic and myelomonocytic lineages, are removed, as well as minor populations, such as megakaryoc ⁇ tic, mast cells, eosinophils and basophils. Usually, at least about 70 to 90 percent of the hematopoietic cells will be removed.
- a prior separation may be employed to remove erythrocytes, by employing ficoll-hypaque (Sigma, St. Louis, MO) separation. The gross separation may be achieved using magnetic beads, cytotoxic agents, affinity chromatography, panning, or the like.
- Antibodies which find use in these methods include antibodies to CD34, CD38, or other markers, which allow for selection of stem cells and removal of most mature cells.
- markers will include CD2, CD3, CD7, CD8, CD10, CD14, CD15, CD16, CD19, CD20 and glycopho ⁇ n where combinations comprising at least CD3, CD8, CD10, CD19 and CD20, normally including at least CD10 and CD19 or at least CD2, CD14, CD15,
- CD 16 CD 19 and glycopho ⁇ n are employed. These combinations of markers used to isolate hematopoietic T cell progenitor cells may vary as other markers become available.
- the hematopoietic cell composition substantially depleted of dedicated cells may then be further positively selected using a marker for Thy-1 (CD90), whereby a substantially homogeneous stem cell population is achieved.
- a progenitor cell population is a population which is CD34 + Thy 1 ⁇ which approximates the substantially homogeneous stem cell composition
- the CD34 + molecule is a single chain type I transmembrane glycoprotein serving as a differentiation stage specific receptor which is associated with hematopoietic T cell progenitor cells, stromal cell precursors, and microvascular endothelial cells.
- the CD34 + stem cell population is composed of a heterogeneous mixture of cell types, the major fraction represent committed progenitor cells and a minor fraction of CD34 + stem cells remain capable of generating hematopoietic T cell progenitor cells in long term culture.
- bone marrow is used as the source for the CD34 + cells.
- CD34 + cells are purified from peripheral blood ononuclear cells (PBMC).
- T cells Coculture of hematopoietic T cell progenitor cells with lymph node cells results in an expansion in the number of cells and in the differentiation of cells into hematopoietic cells at all stages including mature T cells that are either CD3 * CD4 + CD8 or CD3 + CD4CD8 ⁇
- T cells mature in the thymus, and then once mature, they emigrate out of the thymus to populate secondary lymphoid tissue, consisting of the lymph nodes (LNs) and lymphatic vessels.
- LNs lymph nodes
- lymphatic vessels In vivo, LNs are normally found in locations such as the neck, axilla, abdomen, and groin, and are responsible for immune surveillance.
- a LN is an aggregation of lymphoid tissue (stroma) surrounded by a fibrous capsule that is found along the course of lymphatic vessels.
- the outer cortex contains populations of B lymphocytes within germinal center follicles, whereas the paracortical cords contain transiting T lymphocytes and antigen presenting cells (APCs).
- APCs antigen presenting cells
- LN material comprises whole lymph nodes (LN), lymph node fragments (LNF), and lymph node cells.
- LN material is obtained from a subject using standard surgical techniques well known in the art.
- the subject can be any mammal, preferably a primate, and more preferably a human.
- the LN material is used in an organ culture, such that the entire LN or a LNF is placed into a culture.
- the LN material is placed directly into a liquid media on a substrate
- a suitable substrate is laminin
- any substrate can be used which plays a role in progenitor cell adhesion (e.g., via the ,/ 7 integnns) or migration during early T cell differentiation.
- Lymphoid cells can be derived from the disaggregation of a piece of lymph node.
- lymphoid cells can be derived from a LNF from which the lymph cells have migrated during tissue culture. Lymphoid cells migrating from LN material often form a monolayer of cells surrounding the isolated LN material. This phenomenon is known as "skirting". Lymphoid cells may include, but are not limited to, all cell types present in the lymph node which are not lymphocytes or lymphocytic precursors or progenitors
- the lymph node cells are used to stimulate the T cell progenitor cells by cocultunng the lymph node cells together with the T cell progenitor cells. In general, direct contact may be used for efficient stimulation.
- the lymph node cells may be obtained from the lymph nodes of a mammal at any time after the organ has developed to a stage at which it can support the differentiation of cells.
- the lymph node cells can also be grown as a monolayer.
- a monolayer is an nonconfluent layer of lymph node cells, a confluent layer of lymph node cells having a thickness of a single cells, or a confluent layer of lymph node cells where the cells are stacked on one another to a thickness of two or more cells.
- the monolayer is formed of cells disaggregated from their native tissue structure, which are then plated on a vessel and allowed to attach to a surface.
- a lymph node cell line can be established and used to differentiate T cell progenitor cells.
- the cell line can be human, or derived from another mammalian species.
- the LN material may be cryopreserved for storage and shipment to remote locations, such as for use in kits.
- Cells may be cryopreserved by suspending the cells in a cryopreservation medium and freezing the cell suspension.
- the cell suspension is generally frozen gradually and then stored in liquid nitrogen or at an equivalent temperature in a medium containing serum and a cryopreservative such as dimethyl sulfoxide (DMSO).
- DMSO dimethyl sulfoxide
- CD34 + cells into T cells of various stages of maturity including immature T cells that are (double positive) CD3"CD4 + CD8 + or single positive mature T cells that are either CD3 * CD4 + CD8 or CD3 * CD4CD8 ⁇
- Coculture media are commercially available and can be used during the coculture process.
- suitable media include Dulbecco's Modified Eagle Medium (dMEM), Iscove s medium, and the like.
- Coculture media is frequently supplemented with serum, usually fetal bovine serum, generally at a concentration of from about 1-15%, preferably about 5%, vitamins, non-essential ammo acids, and other cell culture reagents.
- serum usually fetal bovine serum
- serum usually fetal bovine serum
- vitamins non-essential ammo acids
- other cell culture reagents for example, appropriate antibiotics to prevent bacterial growth and other additives, such as p ⁇ ruvate (0.1 5 mM), glutamine (0.5 5 mM), 2 mercaptoethanol (1-10 x 10 6 M) may also be included.
- the pH of the media ranges from about 6.0 to 8.0, but is preferably 7.4 after 5% C0 2 equilibration.
- the osmolant ⁇ of the media ranges from 200 to 1000 mOsm, and in one embodiment is about 290-300 mOsm.
- the temperature of the media ranges from 30 to 40°C, and in one embodiment is 37°C.
- tissue culture plates or flasks can also be employed.
- the cells may be cultured in the presence of a mitogemc agent.
- a mitogemc agent is an agent capable of supporting the expansion of a population of hematopoietic T cell progenitor cell descendants when incubated or cultured with these cells. These agents include, but are not limited to lecti ⁇ s, such as concavalin A and phytohemagglutimn.
- a cytokme is included during the coculture process.
- the term "cytokine" is used as a generic name for a diverse group of soluble proteins, peptides, and polypeptides, which act as humoral regulators at nano to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues.
- Cytokines comprise interleukins, initially thought to be produced exclusively by leukocytes; I ⁇ mphokines, initially thought to be produced exclusively by lymphocytes; monokines, initially thought to be produced exclusively by monocytes; interferons, initially thought to be involved in antiviral responses; colony stimulating factors, initially thought to support the growth of cells in semisolid media; and chemokines, thought to be involved in chemotaxis; and a variety of other proteins. Cytokines also comprise thymic hormones. Thymic hormones are postulated to promote T cell maturation.
- thymosin thymopoietin
- thymulin thymic humoral factor.
- cytokines In vivo, the expression of most cytokines is strictly regulated; these factors are usually produced only by activated cells in response to an induction signal.
- cytokines examples include: tumor necrosis factor, transforming growth factor ⁇ , lymphotoxin, migration inhibition factor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL- 13, IL-14, IL-15, various FLT receptor ligands such as fit 3, granulocyte-macrophage colony stimulating factor (GM-
- IFN- ⁇ IFN- ⁇
- M-CSF macrophage CSF
- a combination of cytokines is included during the coculture process into combinations or cocktail formulations.
- the cocktails include: (1) IL-2; (2) IL-2 and IL-6 and oncostatin M (OSM); (3) IL-2, IL-6, IL-7, OSM, and stem cell factor (SCF); (4) IL-2, IL-6, IL-7, OSM, SCF, fit 3; (5) IL-2, IL-6, IL-7, OSM, SCF, fit 3, and thrombopoietin (TPO); (6) IL-2, IL-6, IL-7, SCF, fit 3, and TPO, and leukemia inhibitory factor
- the factors which are employed may be naturally occurring or synthetic, e.g., prepared recombinantly, and may be human or of other species, e.g., non-human primate or murine.
- a human cytokine is used.
- the amount of the cytokine factors will generally be in the range from about 0.1 ng/ml to 1000 ngfml.
- a range from about 1 ng/ml to 100 ngfml, and preferably from about 5 ng/ml to 50 ng/ml is used with the methods described herein.
- FACS fluorescence-activated cell sorter
- a population of cells is incubated with one or more probes that are each labeled with a different fluorescent tag.
- the different fluorescent tags each emitted a unique fluorescent signal that is used to differentiate the binding of one probe to a specific target on a cell.
- a marker for T cells generally, CD4, a marker for the T helper cell, and CD8, a maker for the T cytolytic cell, it is possible to monitor the differentiation of CD34 + cells into T cells. It is also possible to assay various cytokines for their effectiveness as potential T cell differentiating cytokines.
- lymph node ononuclear cells LNMC
- peripheral blood mononuclear cells LNMC
- PBMC stem cells
- CD34 + cells stem cells
- CD1A CD3, CD4, CD5, CD8,
- CMFDA labeled (CD34 * cells) are also analyzed by flow cytometr ⁇ .
- Various immunological, histochemical, and molecular biological techniques may also be used to assay for maturing T cells
- PCR polymerase chain reaction
- This PCR assay uses a reverse transc ⁇ ptase polymerase chain reaction (RT)
- PCR reverse transcriptase kinase
- TCR T cell receptor
- T cell receptor rearrangement excision circles composed of genetic material from the T cell receptor are generated in maturing T cells as the genetic components of the T cell receptor are rearranged into a mature form.
- the number of TREC molecules is highest in newly generated T cells that have recently undergone T cell receptor gene rearrangement The number of
- TREC molecules in a cell decreases with each cell division T cells that have few TREC molecules are likely to have undergone multiple cell divisions and may represent residual mature T cells within the LN material used in the coculture process as these cells are older than the population of T cells differentiated de novo from the progenitor cell population.
- T cells that contain relatively higher numbers of TREC are more likely to have differentiated from CD34 * progenitor cells
- an assay that can determine the number of TREC in a cell can be used to determine the age of a T cell population.
- the TREC assay exploits PCR technology to amplify TREC sequences to determine the number of TREC in a target cell populations.
- a general TREC protocol is described in Douek DC, et al., Nature 396(67121:690-5 (1998).
- a retroviral vector capable of integration into a host cell genome is introduced to the population of CD34 + cells before coculture with the LN material. Methods of nucleic acid introduction into a target cell are discussed herein.
- the retroviral vector contains one or more markers which can be used to discriminate between CD3 + cells that differentiated from a CD34 + cell introduced during the coculture procedure, and any CD3 * cells that might have been present in the LN material when it was taken from the host.
- the act of evaluating the cocultured population of T cells for the presence of previously matured CD3 + cells can occur by screening for a marker or unique sequence contained within the retroviral vector using standard techniques well known in the art.
- lymphocyte proliferation assay LPA
- neoantigens can be introduced to the developing T cells during culture, the neoantigen can be used in the LPA to then examine the ability of the differentiated T cells to respond to novel, synthetic antigens.
- recombination activation gene analysis is contemplated for use with the methods described herein.
- the hematopoietic T cell progenitor cells or descendants thereof may be genetically modified by contacting the cells with a nucleic acid sequence capable of genetically modifying the hematopoietic T cell progenitor cells or descendants thereof.
- the resulting cells may then be grown under conditions as described for unmodified hematopoietic T cell progenitor cells, whereby the modified hematopoietic T cell progenitor cells may be expanded and differentiated and used for a variety of purposes.
- Genetic modification of a hematopoietic T cell progenitor cell includes all transient and stable changes of the cellular genetic material that are created by the exogenous addition of a nucleic acid sequence Techniques for the modifying the progenitor cells by introducing nucleic acid sequences into
- CD34 + stem cells are known to one of ordinary skill in the art (Dick et al., Blood 78:624-634, 1991 ; Rosenzweig, M., et al., Blood 90:48224831 , 1997; Rosenzweig, M., et al., J Med Primatol. 25:192-200, 1997; Fletcher et al., Blood 76:1098-1103, 1990).
- a polynucleotide or "a nucleic acid sequence” refers to a polymeric form of nucleotides at least 10 bases in length.
- isolated nucleic acid sequence is meant a polynucleotide that is not immediately contiguous with both of the coding sequence with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
- the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule [e.g., a cDNA) independent of other sequences.
- the nucleotides of use with the method of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
- the term includes single and double stranded forms of DNA.
- the nucleic acid sequence encodes a pol ⁇ peptide of interest.
- the nucleic acid can also encode a mutagen, antisense, ribozyme, or a dominant negative form of a polypeptide of interest.
- Nucleic acid sequences which encode a polypeptide of interest, or functional fragment thereof, can be operatively linked to expression control sequences.
- “Operativel ⁇ linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
- An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences.
- expression control sequences refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operativel ⁇ linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
- expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon [i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of the mRNA, and stop codons.
- control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
- Expression control sequences can include a promoter.
- promoter minimal sequence sufficient to direct transcription. Also included in the definition are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell- type specific, tissue-specific, or inducible b ⁇ external signals or agents; such elements ma ⁇ be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters, are included in the invention (see e.g., Bitter et al, Methods in Enzymology 153:516-544, 1987).
- Promoters derived from the genome of mammalian cells [e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) ma ⁇ be used. Promoters produced b ⁇ recombinant DNA or s ⁇ nthetic techniques ma ⁇ also be used to provide for transcription of the nucleic acid sequences of the invention.
- the nucleic acid sequences emplo ⁇ ed can include a marker, which allows for selection of cells into which the
- DNA has been integrated, and against cells which have not integrated the DNA construct.
- markers exist, particularly antibiotic resistance markers, such as resistance to G418, h ⁇ grom ⁇ cin, and the like. Less convenientl ⁇ , negative selection ma ⁇ be used, where the marker is the HSV tk gene, which will make the cells sensitive to agents, such as ac ⁇ clovir and ga ⁇ c ⁇ clovir.
- multiple drug resistance gene(s), e.g., pgp ⁇ , ma ⁇ be introduced to protect the cells against cytotoxic drugs.
- nucleic acid sequences which provide resistance to viral infection or viral replication can be introduced. In one nonlimiting example, nucleic acid sequences which provide resistance to an immunodeficiency lentivirus infection such as HIV can be introduced.
- nucleic acid sequences include antisense and riboz ⁇ me sequences.
- the transcription of riboz ⁇ mes has been shown to protect against viral infection, or viral replication of an immunodeficienc ⁇ lentivirus (Rosenzweig, M. et a/., Blood 90:4822-4831, 1997).
- a number of nucleic acid sequences are contemplated for use with the present invention.
- a suitable nucleic acid sequence for introduction into a CD34' cell is a sequence whose expression or presence within the host CD34 * cell results in an increase in the viabilit ⁇ of the host cell.
- an antisense sequence that is complementar ⁇ to the tat/rev Exon I is introduced into a cell infected with the HIV virus, the product of this nucleic acid sequence produces an short segment of antisense nucleic acid that interferes with the expression of both the tat and rev genes.
- the interference generated b ⁇ the antisense sequences protects the host cell and other cells b ⁇ limiting viral reproduction.
- the nucleic acid sequences contemplated here can be prepared in a variety of conventional ways. Numerous vectors are now available which provide for the desired features, such as long terminal repeats, marker genes, and restriction sites.
- the vector can be introduced into an appropriate plasmid and manipulated by restriction, insertion of the desired gene, with appropriate transcriptional and translational initiation and termination regions, and then the plasmid is introduced into an appropriate packaging host. At each of the manipulations, the plasmid can be grown in an appropriate prokar ⁇ otic host, and then anal ⁇ zed to ensure that the desired construct has been obtained. The construct can then be subject to further manipulation. When completed, the plasmid or excised virus can be introduced into the packaging host for packaging and isolation of virus particles for use in the genetic modification.
- Man ⁇ vectors such as viral vectors, episomal plasmid vectors, stabl ⁇ integrating plasmid vectors, and artificial chromosome vectors can be utilized with the methods of the invention.
- Viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpesviral vectors and pox viral vectors.
- a retroviral vector is emplo ⁇ ed for the introduction of the DNA construct into the stem cell host.
- An example of a suitable retroviral vector is a lentivirus-derived vector.
- Vectors ma ⁇ be designed to integrate into a specific location in the genome b ⁇ insertion or homologous recombination, ma ⁇ integrate randomly or ma ⁇ remain in the nucleus as a stable episomal nucleic acid.
- retroviral vectors ma ⁇ be emplo ⁇ ed for genetic modification. Combinations of retroviruses and an appropriate packaging line, where the capsid proteins will be functional for infecting cells of a primate, such as a human, can be utilized.
- Various amphotropic virus-producing cell lines are known, such as PA12 (Miller et al., Mol.
- the cells and virus will be incubated for at least 24 hours in the culture medium.
- the cells are then allowed to grow in the culture medium for at least one week, and may be allowed to grow for two weeks or more, before analysis.
- Other delivery techniques are well known in the art can be used to deliver a nucleic acid sequence capable of geneticall ⁇ modif ⁇ ing the hematopoietic T cell progenitor cells or descendants thereof. These techniques include, but are not limited to, a colloidal dispersion s ⁇ stem.
- Colloidal dispersion s ⁇ stems include macromolecule complexes, nanocapsules, microspheres, beads, and hpid-based s ⁇ stems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
- the preferred colloidal s ⁇ stem of this invention is a hposome.
- Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. It has been shown that large particularlyamellar vesicles (LUV), which range in size from 0.2-4.0 m can encapsulate a substantial percentage of an aqueous buffer containing large macromoiecules.
- RNA, DNA and intact vinons can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al., Trends Bioche . Sci., 6:77, 1981).
- liposomes In addition to mammalian cells, liposomes have been used for deliver ⁇ of pol ⁇ nucleotides in plant, ⁇ east and bacterial cells.
- a hposome In order for a hposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell c ⁇ toplasm at high efficiency; and (4) accurate and effective expression of genetic information (Manmno et al., Biotechmques, 6:682, 1988).
- the composition of the hposome is usually a combination of phosphoiipids, particularly high phase-transition temperature phosphoiipids, usually in combination with steroids, especially cholesterol. Other phosphoiipids or other lipids may also be used.
- the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in hposome production include phosphatid ⁇ l compounds, such as phosphatidylgl ⁇ cerol, phosphatid ⁇ icholme, phosphatid ⁇ lse ⁇ ne, phosphatid ⁇ lethanolamine, sphingolipids, cerebrosides, and gangliosides.
- diacylphosphatidyl gl ⁇ cerols where the lip id moiety contains from 14 18 carbon atoms, particularly from 16 18 carbon atoms, and is saturated.
- Illustrative phosphoiipids include egg phosphatid ⁇ lcholine, dipalmito ⁇ lphosphatid ⁇ lcholme and distearoylphosphatid ⁇ lcholine.
- the targeting of liposomes can be classified based on anatomical and mechanistic factors Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle specific Mechanistic targeting can be distinguished based upon whether it is passive or active.
- Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo endothelial system (RES) in organs which contain sinusoidal capillaries.
- Active targeting involves alteration of the hposome by coupling the hposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the hposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization
- lipid groups can be incorporated into the lipid bila ⁇ er of the hposome in order to maintain the targeting ligand in stable association with the hposomal biia ⁇ er.
- Various linking groups can be used for joining the lipid chains to the targeting ligand.
- Also provided here is a method of treating a subject having an immune disorder, b ⁇ culturing a I ⁇ mph node cell in vitro with a primate hematopoietic T cell progenitor cell to produce a descendent cell, and then administering a therapeutically effective amount of the T cell progenitor cell or a descendent thereof to the subject.
- subject is meant an ⁇ mammal, preferabl ⁇ a primate, more preferably a human.
- a therapeutically effective dose of hematopoietic T cell progenitor cells refers to that amount of hematopoietic T cell progenitor cells or descendants thereof that is of sufficient quantity to alleviate a s ⁇ mptom of the disease or to ameliorate the immune disorder.
- “Ameliorate” refers to lessening or lowering the disease's or disorder's detrimental effect in the patient receiving the therap ⁇ .
- the treatment can increase the number of T cells.
- Immune disorders include "immunodeficienc ⁇ disorders" and " immunoproliferative" disorders, described above.
- An example of an immunodeficienc ⁇ disorder is AIDS, and an example of an immunoproliferative disorder is a I ⁇ mphoma.
- a patient's own T cell progenitor cells can be removed, expanded and/or differentiated, and optionally genetically altered The cells can then be remtroduced into the patient.
- transplantation treatments described can be used in conjunction with standard viral or anti HIV treatments.
- transplantation as described above would be accompanied by on-going antiviral treatments and more specifically anti HIV 1 treatments.
- the hematopoietic T cell progenitor cells ma ⁇ be administered in an ⁇ physiologically acceptable medium, normally intravascularly, although the ⁇ ma ⁇ also be introduced into bone or other convenient site, where the cells ma ⁇ find an appropriate site for further differentiation or for activation.
- the cells can be introduced into a I ⁇ mph node or the spleen.
- Usuall ⁇ at least 1 x10 5 cells will be administered, preferabl ⁇ 1 x10 6 or more.
- Transplantation or implantation is typically by simple injection through a hypodermic needle having a bore diameter sufficient to permit passage of a suspension of cells therethrough without damaging the cells
- the cells may be introduced by injection through a hypodermic needle, catheter, or the like.
- factors may also be included, such as the cytokines IL 2, IL 6, leukemia inhibitory factor (LIF), IL 7, stem cell factor (SCF), fit 3, oncostatin M (OSM), and thrombopoietin (TPO), and others.
- nucleic acid sequences into the progenitor cells or descendants thereof can be used for a wide va ⁇ et ⁇ of purposes, such as gene therap ⁇ , introduction of novel capabilities into the hematopoietic T cell progenitor cells, virus resistance, enhancement of maturation to a particular subset, or the like.
- genes specific for hematopoietic cells, including combined immunodeficienc ⁇ and leukemia. These diseases ma ⁇ be treated b ⁇ homologous recombination, where at least one cop ⁇ of the defective gene ma ⁇ be modified to the wild-type or a functioning gene. It may be necessary to correct onl ⁇ one cop ⁇ to sufficiently provide for therapeutic treatment of such disorders.
- a particular polymorphic region of a polymorphic protein such as a T-cell receptor or major histocompatibility complex antigen which is involved with susceptibility to a particular disease, e.g., an autoimmune disease
- the particular exon ma ⁇ be "knocked out" b ⁇ homologous recombination. In this manner hematopoietic cells are provided which will not be susceptible or responsive to the disease process.
- the therapy involves removal of bone marrow or another source of hematopoietic T cell progenitor cells from a human host, isolating the hematopoietic T cell progenitor cells from the source and expanding and differentiating the cells.
- the host ma ⁇ be left intact, or ma ⁇ be treated to substantially or complete ablate native hematopoietic capability.
- the hematopoietic T cell progenitor cells ma ⁇ be modified so as to provide for hematopoietic T cell progenitor cells having the desired genetic modification.
- the modified hematopoietic T cell progenitor cells or descendants thereof are then restored to the host to provide for the new capability. If necessary, the process may be repeated to ensure the substantial absence of the original hematopoietic T cell progenitor cells and the substantial population of the modified hematopoietic T cell progenitor cells and descendants thereof.
- lymph node tissue can be conditioned in vivo to induce the proliferation or differentiation of progenitor cells b ⁇ the administration of c ⁇ tokines.
- the cocktails include: (1) IL 2; (2) IL
- compositions described above are preferabl ⁇ prepared and administered in dose units
- compositions ma ⁇ be accomplished by any means known to the skilled artisan.
- Solid dose units are tablets, capsules and suppositories.
- different doses are necessar ⁇ Under certain circumstances, however, higher or lower doses ma ⁇ be appropriate.
- the administration of the dose can be carried out both b ⁇ single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
- the cytokine compositions may be administered locally or s ⁇ stemicall ⁇ .
- the compositions can be administered b ⁇ injection.
- the compositions according to the invention are preferabl ⁇ administered intravenousl ⁇ .
- other routes of administration are within the scope of the invention.
- the compositions can be administered topically, intravenousl ⁇ , orally or parenterally or as implants, but even rectal use is possible in principle.
- Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customaril ⁇ used as described above.
- the compositions are suitable for use in a variet ⁇ of drug deliver ⁇ s ⁇ stems.
- B ⁇ "therapeutically effective dose” is meant the quantity of a compound according to the invention necessary to prevent, to cure or at least partially arrest the symptoms of the disease and its complications. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the patient. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the composition, and animal models ma ⁇ be used to determine effective dosages for treatment of particular disorders. Various considerations are described, e.g., in Gilman et al. (eds.) (1990) Goodman and Gilma ⁇ 's: The
- a method for testing the effect of an agent on cells is provided.
- agent as used herein describes an ⁇ molecule, e.g., a protein, peptide, pharmaceutical or biological agent with the capabilitiesit ⁇ of altering growth or differentiation of hematopoietic T cell progenitor cells.
- the agent can be an ⁇ thing known to be or suspected of being capable of affecting the growth and development of T cells. These agents include s ⁇ nthetic chemical agents, biochemical agents, cells, extracts, and homogenates.
- the test agent ma ⁇ also be a combinatorial librar ⁇ for screening a plurality of compounds.
- Compounds identified can be further evaluated, detected, cloned, sequenced, and the like, either in solution of after binding to a solid support, b ⁇ an ⁇ method usually applied to the detection of a specific DNA sequence, such as PCR, oligo er restriction (Saiki et al., Bio/Technology 3:1008- 1012, 1985), allele-specific oligonucleotide (ASO) probe anal ⁇ sis (Conner et al., Proc. Natl. Acad. Sci. USA 80:278, 1983), oligonucleotide ligation assa ⁇ s (OLAs) (Landegren et al., Science 241:1077, 1988), and the like.
- PCR oligo er restriction
- ASO allele-specific oligonucleotide
- OVAs oligonucleotide ligation assa ⁇ s
- Candidate agents for use with the present method encompass numerous chemical classes.
- the ⁇ can be organic molecules, preferabl ⁇ small organic agents having a molecular weight of more than 50 and less than about 2,500 Daltons.
- Candidate agents comprise functional groups necessary for structural interaction with proteins, particularl ⁇ h ⁇ drogen bonding, and t ⁇ picall ⁇ include at least an amine, carbon ⁇ l, h ⁇ drox ⁇ l or carbox ⁇ l group, preferabl ⁇ at least two of the functional chemical groups.
- the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or pol ⁇ aromatic structures substituted with one or more of the above functional groups.
- Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, p ⁇ rimidines, derivatives, structural analogs or combinations thereof.
- Candidate agents are obtained from a wide variety of sources including libraries of s ⁇ nthetic or natural agents. For example, numerous means are available for random and directed s ⁇ nthesis of a wide variety of organic agents and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural agents in the form of bacterial, fungal, plant and animal extracts are available or readil ⁇ produced. Additionally, natural or synthetically produced libraries and agents are readily modified through conventional chemical, ph ⁇ sical and biochemical means, and ma ⁇ be used to produce combinatorial libraries. Known pharmacological agents ma ⁇ be subjected to directed or random chemical modifications, such as ac ⁇ lation, alk ⁇ lation, esterification, amidification, etc. to produce structural analogs.
- the coculture of the I ⁇ mph node cells and hematopoietic T cell progenitor cells can be carried out in the presence of the agent.
- “Incubating” includes conditions which allow contact between the test compound and the hematopoietic T cell progenitor cells and descendants thereof.
- Contacting includes in solution and solid phase.
- the growth and/or differentiation of the progenitor cells, or descendants thereof, contacted with the agent is then compared to the growth or differentiation of control progenitor cells or descendants thereof as a determination of the effect of the agent.
- the control cells can be a control culture subject to otherwise identical conditions, but without the agent. Other suitable controls can readil ⁇ be determined b ⁇ one of skill in the art.
- control cells can be incubated with a vehicle, or can be incubated with a different dose of the agent, or can be incubated with an agent of known effect and activit ⁇ .
- a time course, wherein the cells are exposed to an agent for varying amounts of time, can also be performed.
- a stud ⁇ wherein cells are exposed to different concentrations of an agent, can be performed.
- kits comprise a carrier means containing one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
- the kit includes a cr ⁇ opreserved sample of I ⁇ mph node or a cr ⁇ opreserved sample of a suspension of I ⁇ mph node cells, together with instructions for co-culturing such cells with hematopoietic T cell progenitor cells.
- the instructions ma ⁇ be specific to primate cells, such as human cells.
- the kit ma ⁇ also contain a container comprising one or more c ⁇ tokines, or a combination of cytokines.
- a c ⁇ tokine combination comprises at least IL-2 and IL-6.
- the c ⁇ tokine combination ma ⁇ further comprise leukemia inhibitor ⁇ factor (LIF), stem cell factor (SCF), fit 3, thrombopoietin (TPO), oncostatin M (OSM), or IL-7.
- LIF leukemia inhibitor ⁇ factor
- SCF stem cell factor
- TPO thrombopoietin
- OSM oncostatin M
- IL-7 IL-7
- Bone marrow is one of the richest source of CD34 * cells (1-3% in normal adult individuals).
- U.S. Patent No. 5,766,944 describes a method for purification of CD34 * cells from bone marrow. Briefly, bone marrow aspirates were obtained from a human source using standard techniques well known in the art, and diluted 1:2 with sterile PBS or normal saline and mixed gentl ⁇ . Depletion of red blood cell/high densit ⁇ cells was accomplished by the addition of an equal volume of 3% gelatin that was mixed gently and allowed to settle b ⁇ gravit ⁇ sedimentation. Sedimentation time ma ⁇ var ⁇ with different donors and the qualit ⁇ of the marrow aspirate.
- the nuclear cells were harvested b ⁇ pipetting the plasma la ⁇ er into 50 ml tubes. Cells were washed with NS or PBS and centrifuged at 1350 rpm. The RBC depleted fraction was resuspended in 35 ml of complete media (such as Dulbecco's modified
- DMEM Eagle's medium
- RPMI-10 fetal calf serum
- the ficoll-h ⁇ paque gradient was then centrifuged at 1350 rpm (400 g) at room temperature (22°C) for 35 minutes. Then, the interface mononuclear cells were harvested with a pipette.
- the mononuclear cells were then diluted with an equal volume of PBS, spun at 1000 rpm for 10 minutes, and the supernatant was aspirated and discarded. The cells were resuspended in PBS +2% FCS, spun and aspirated two more times. The mononuclear marrow cells were resuspended in 5 ml RPMI and counted. The mononuclear marrow cell concentration was then adjusted to 10 x 10 6 cells/ ml with RPMI-5% HI-ADS FCS (note-maximum of 70 x 10 6 cells/tube).
- a 0.14M 2-aminoeth ⁇ lisothiouronium bromide (AET) solution was prepared and mixed 4:1 with sedimented red blood cells (SRBC) and incubated at 37°C for twent ⁇ minutes.
- the AET treated SRBC's (AET-SRBC) was then washed four times with PBS.
- a 1% AET-SRBC solution was prepared b ⁇ diluting 0.5 ml of packed AET-SRBC with 50 ml PBS.
- An equal volume of the 1 % AET-SRBC solution was then added to the mononuclear marrow cell suspension and the mixture was incubated at 37°C for 5 minutes.
- the cell mixture was then centrifuged at 600 rpm for 5 minutes then incubated at 4°C for 60 minutes. Alternately, a 2% AET-SRBC solution mixed with a cell suspension of 2.5 x 10 6 cells/ml was used to deplete CD3 + T cells. A sample of the rosetting cells was checked under the microscope as rapid rosetting with large agglutination to entrap non-T cells, the percent rosetting should not exceed that of a t perennial sample.
- the cell suspension was gentl ⁇ underlaid with a volume of ficoll-h ⁇ paque which was 2 times the cell suspension volume.
- the ficoll-h ⁇ paque gradient was centrifuged at 600 rpm for 10 minutes and then at 1350 rpm for 25 minutes.
- the T cells were located in the pellet and the non-T cells (B cells, macrophages/dendritic cells, mixed stem cell population including CD34 * cells) were located at the interface.
- the interface mononuclear cells was harvested with a pipette.
- the cells were diluted with an equal volume of PBS, centrifuged at 1000 rpm for 10 minutes, aspirated and the supernatant was discarded.
- the wash step was repeated.
- the cells were then resuspended in complete RPMI (without serum) and the cell concentration was adjusted to 2 x 10 6 cells/ml. Non-CD34 cells were then depleted b ⁇ immunomagnetic selection.
- the cells were resuspended in 2-3 ml complete media and a cell count was performed.
- the CD34 + cell recover ⁇ was calculated. An aliquot was reserved for Colon ⁇ Forming Unit Assa ⁇ s/phenot ⁇ ping in a separate tube with complete media.
- the CD34 * cells t ⁇ picall ⁇ display sheared epitopes and/or down regulation of the receptor induced b ⁇ the binding of the capture antibody.
- the CD34 + receptor recycles within 24-36 hours after binding to the capture antibod ⁇ in the selection process. The aliquot reserved for phenot ⁇ ping should therefore be rested prior to testing. There may be considerable variability in detection of the CD34 receptor among different mAb ⁇ -CD34 used for FACS anal ⁇ sis.
- the epitope which has provided an even performance is the HPCA-2 (8G12 clone).
- the CD34 + cells were now read ⁇ to be used. If the CD34 + cells are to be co-cultivated with I ⁇ mphoid cells, the absolute minimal stem cell concentration should be about 10,000 cells per fragment/monola ⁇ er. The cells will undergo a rapid expansion during the initial phase of the coculture reflecting the expansion and terminal differentiation of the pre-com itted progenitors in the CD34 * population. At set intervals of the coculture, prior to functional testing or phenot ⁇ ping these "non-mononuclear" cells were removed after cell harvesting b ⁇ low densit ⁇ cell centrifugation with ficoll-h ⁇ paque.
- peripheral blood mononuclear cells were obtained b ⁇ ficoll-h ⁇ paque (F/H) centrifugation as described above.
- PBMC peripheral blood mononuclear cells
- F/H ficoll-h ⁇ paque
- PBMC peripheral blood mononuclear cells
- sRBC standard AET- sheep red blood cell
- SRBC-CD3 * cells were depleted of sRBC b ⁇ treatment with ACK RBC I ⁇ sing buffer and cultured on laminin coated plates in media containing one of seven c ⁇ tokine cocktails (#1-7) with or without INDINAVIR (CRIXIVAN) (7 vM) (Merck, Whitehouse
- the cocktails were: (1) IL-2; (2) IL-2, IL-6, and OSM; (3) IL-2, IL 6, IL-7, OSM, and SCF; (4) IL-2, IL-6, IL-7, OSM, SCF, and fit 3; (5) IL-2, IL-6, IL-7, OSM, SCF, fit 3, and TPO; (6) IL-2, IL 6, IL-7, SCF, fit 3, TPO, and LIF; (7) IL 2, IL 6, and LIF.
- T and NK cell-depleted population was further depleted of monocyte/macrophages with 20 30 minutes of plastic adherence.
- the non-adherent population was then selected for CD34 + cells using immunomagnetic anti-CD34 Mab and/or ant ⁇ -AC133 Mab coupled beads.
- An aliquot of CD34/AC133 + cells was analyzed by flow c ⁇ tometr ⁇ for pu ⁇ t ⁇ and the population was reselected if more than 1 % CD3 + cells were present.
- CD34/AC133 * cells were added to culture wells, as well as cultured alone in 96 RB laminin coated wells at 5,000 cells/well in various c ⁇ tokine containing media.
- CD34/AC133 + cells were tagged with the fluorescent marker 5-chlorometh ⁇ lfluoresce ⁇ n diacetate (CMFDA)(Molecular Probes, Eugene, OR) as per manufacturer instructions prior to adding to lymph node fragment containing wells.
- CMFDA 5-chlorometh ⁇ lfluoresce ⁇ n diacetate
- NIH/NIAID protocols Protocol No. #92-1 0125 from both HIV umnfected and HlV-infected individuals under local anesthesia.
- One to two (1 2) mm sections were cut from the LN for pathologic and HIV RNA in situ analyses prior to removal of the fibrous capsule, fat and scar tissue from the remainder of the tissue. The tissue was then cut into consecutively smaller pieces allowing a portion of the I ⁇ mph node mononuclear cells to be harvested to serve as controls, to migrate out into the media over time.
- LNFs (1 2 mm) were then placed (1 6 LNFs/well) on laminin (30 ⁇ g/ml; Gibco/ ⁇ fe Science Products) coated wells (24 or 12 well plates) in 75 200 ⁇ L of media, covered with sterile glass cover slips and incubated at 37°C for 1 hr.
- laminin (30 ⁇ g/ml; Gibco/ ⁇ fe Science Products) coated wells (24 or 12 well plates) in 75 200 ⁇ L of media, covered with sterile glass cover slips and incubated at 37°C for 1 hr.
- C ⁇ tokine containing media in some cases ⁇ INDINAVIR,
- Example 4 Culture Method Cell populations were maintained in a base media [1 :1 vol/vol Iscoves modified essential media: HAMS F12 media plus 100 U/ml penicillm/streptom ⁇ cin, 1 mM glutamine, 1 mM hepes buffer, 1 % sodium pyruvate (all from Gibco/Life Science Products), 1 % BIT (Stem Cell Technologies, Vancouver, Canada), 0.5% MEM vitamins (Sigma), 0.5% non essential ammo acids (Sigma), 0.5% linoleic acid (Sigma) and 5% fetal bovine serum containing one or more of the following c ⁇ tokines (Peprotech, Rock ⁇ Hill, NJ) with or without the addition of 7 ⁇ M INDINAVIR (Merck): a) Interleukin (IL) 2 at 20 U/ml, b) IL 6 at 50 ng/ml, c) IL-7 at 10 ng/ml, d) Oncostatin M (OSM) at 10
- LNFs #1 (a) only; #2 (a+b+d); #3 (a-e); #4 (a-f); #5 (a-g); #6 (a-c plus e-h) and #7 (a + b+h).
- the LN material was placed in the bottom of the upper chamber of a transwell plate containing
- the I ⁇ mphoid culture media was completely aspirated from the upper and lower chambers of the transwell and coculture media was added to the capacit ⁇ of the outer well and to the halfwa ⁇ point of the inner well.
- the stem cells were then seeded to the upper well of the transwells containing the I ⁇ mphoid fragments at a concentration of between 10,000 and 100,000 CD34 + CD38 stem cells per fragment (depending on the lineage status).
- the I ⁇ mphoid fragments which are being cocultured with the purified stem cells were checked periodically under the microscope for CD34 " cell expansion (to precommitted lines) and for assessing whether a portion of the cells were tracking to the I ⁇ mphoid fragments.
- CD34 * stem cells were added to LN material containing wells (10, 000-70, 000/well) and monitored by a variet ⁇ of parameters for indicators of I ⁇ mphopoiesis.
- CD34 * stem cells were added to LNF containing wells several times over approximately a 1 month period.
- RNA deox ⁇ ribonucleic acid
- TRIAZOL ribonucleic acid
- the RNA used in the RT-PCR method was isolated from a sample of cells taken from the coculture using TRIAZOL (Life Technologies, Rockville, MD). The cells were pelleted b ⁇ centrifugation and resuspended in 1 ml of TRIAZOL. To the suspension was added 200 ⁇ l of chloroform.
- the ⁇ ield of the above-described method was a nucleic acid pellet derived from the cultured cells.
- the next step of the assa ⁇ method was to quantitate the amount of RNA present in the pellet and to determine the purit ⁇ of the ⁇ ield using ultraviolet spectroscop ⁇ using an absorbance ratio of A260/280. This ratio ⁇ ields information on the nucleic acid to protein absorbance present in the sample.
- the nucleic acid sample was treated with 3U DNase for 30 minutes at 37°C to digest an ⁇ deox ⁇ ribonucleic acid that was present in the sample.
- RNA was purified.
- a complementar ⁇ DNA strand was s ⁇ nthesized from the isolated RNA.
- the cDNA was constructed using the reverse transcriptase protocol from the SUPERSCRIPT KIT (Life Technologies, Rockville, MD). According to this protocol, in a 20 ⁇ L reaction volume was placed: the purified RNA and 100 Units of
- the solution was brought to a final concentration of the following compounds: 50 mM Tris-HCI, 75 mM KCI, 3 mM MgCI 2 , 2 mM DTT, and 500 ⁇ M of the four deox ⁇ ribonucleosides (dNTPs).
- dNTPs deox ⁇ ribonucleosides
- the resulting product was used as a template for the pol ⁇ merase chain reaction.
- the primers used in the reaction were: Forward Primer Sequence: 5'-GGCACACCCTTTCCTTCTCTG-3'
- the PCR conditions were as follows: an initial heating step at 94°C for 2 minutes followed b ⁇ 56°C for 2 minutes.
- the thermoc ⁇ cler in which the PCR samples were inserted were processed for 35 c ⁇ cles.
- the c ⁇ cles consisted of the following steps: first, a round of heating for 30 seconds at 94°C, then a 30 second heating round at 56°C, and then a heating round for 1 minute at 72°C.
- PCR amplicons were run on a 2% agarose gel to examine the extent of pT alpha amplification.
- the amplicon from pT alpha was approximatel ⁇ 620 base pairs and compared in intensit ⁇ with controls subjected to the same PCR conditions.
- the controls were RNA from fetal th ⁇ ic tissue, PBMC, CD4 + T cells, and CD34 + stem cells, and an immature T cell line (MOLT-4) known to express pT alpha.
- MOLT-4 immature T cell line
- a T cell Receptor Excision Circle Assay for use with the method described herein includes the following steps and materials.
- TREC standards were prepared from a plasmid containing an insert that corresponded to the signal joint (sj) region of TCR delta. Standards were prepared at 10 fold dilutions that ranged from down 10 2 to 10 7 .
- Cells to be assa ⁇ ed in the TREC s ⁇ stem were isolated from the coculture, pelleted, and frozen. Frozen cell pellets were dissolved in protemase K in a 10mM Tris-HCI pH 8.0 solution, at a concentration of 100,000 cells per 10 ⁇ l. Cell I ⁇ sates were incubated for 2 hours at 56°C to digest protein. The protemase K was then inactivated b ⁇ heating the cell lysate sample at 94° C for 15 minutes. Samples were then stored at -20°C until assayed.
- PCR assa ⁇ 5 ⁇ l of cell I ⁇ sate (50,000 cells) was added to 20 ⁇ l of master PCR mix.
- the master mix consisted of 2.5 ⁇ l of 10X PCR Buffer; 1.75 ⁇ l 50mM MgCI 2 ; 0.5 ⁇ l 10mM dNTP; I ⁇ l 12.5 ⁇ M 5' sj primer; 1 ⁇ l 12.5 ⁇ M 3' sj primer; 1 ⁇ l 3.75 ⁇ M probe; 0.25 ⁇ l 5U/ ⁇ l Taq; 0.125 ⁇ l of BD636 reference; and, 11.875 ⁇ l of water for a total volume of 20 ⁇ l.
- a chronically infected HIV * patient with degenerative changes in lymph node tissue was examined for the ability to assist in the generation of mature T I ⁇ mphoc ⁇ tes from T I ⁇ mphoc ⁇ te precursor cells.
- Assessment of HIV viral load was performed b ⁇ ELISA, in which the p24 antigen was measured using the commercially available HIV 1 p24 Antigen Assa ⁇ (Coulter Corporation, Miami, FL).
- Subject R021898 had CD4 count of ⁇ 50, a high viral load, and was insensitive to INDINAVIR.
- An HIV umnfected individual was used as a control.
- the procedure used to generate the mature T lymphocytes was as follows. LNF fragments were isolated and cocultured with CD34' T cell progenitor cells as described above. Additionally, several different controls were generated for use in the analysis. Cellular growth of the LN material alone, the CD34 ' cells alone, and the coculture of I ⁇ mph node material plus CD34+ progenitor cells were monitored for viability using standard cell culture techniques, such as thymidine blue d ⁇ e anal ⁇ sis.
- IL-2 (1) IL-2, (2) IL 2, IL 6, and OSM; (3) IL 2, IL 6, IL 7, OSM, and SCF; (4) IL 2, IL 6, IL-7, OSM, SCF, and fit 3; (5) IL 2, IL 6, IL 7, OSM, SCF, fit 3, and TPO; (6) IL 2, IL 6, IL 7, SCF, fit 3, TPO, and LIF; (7) IL 2, IL 6, and LIF.
- LPAs I ⁇ mphoproliferative assa ⁇ s
- FIGURE 2 shows a number of FACS results obtained from the cells of Subject R021898 that were grown using the culture methods described herein.
- FIGURE 2 graphs A and B show the presence of CD3 + CD8 * (56.5%) and CD3 + CD4 + (9.1 %), respectively which were obtained from PBMCs on day #1.
- FIGURE 2 graphs C and D examined cells taken from the isolated LN material which showed CD3 + CD8 + (0.5%) and CD3 + CD4 + (0.1 %), respectively. These results show that although there were CD3 * CD8 + and CD3 + CD4 * single positive cells present within the subject's immune system, these ceils were substantially removed from the isolated LN material.
- FIGURE 2 graph E examined the expression of CD4 + vs. CD8 + cells from the LN material on day 14, just prior to the addition of the CD34 + cells, in the presence of T cell cocktail No. 1.
- the cocktail used here was a control to detect the presence or absence of contaminating T cells in the isolated LN material.
- those cells that displayed both the CD4 and CD8 markers appeared in the upper right quadrant of the graph (Stage 1, 2, and 3 of developing double positive T cells). This quadrant in graph E shows only a population of 2.2% cells with these markers.
- Graph F shows FACS results looking at the expression of CD3 and CD4 markers. This graph shows a reading of 8.9% for cells displaying both markers (single positive mature CD3 + CD4+ T cells).
- FIGURE 2 graphs G and H show results from the culture that was grown in T cell differentiating cocktail
- CD4 + CD8 + double positive cells CD4 + CD8 + double positive cells. Also, substantial populations of CD4 * or CD8 + cells (mature T cells) were also present.
- the displa ⁇ ed results of graph K are t ⁇ pical of developing th ⁇ moc ⁇ te population obtained from a health ⁇ subject. Thus, the results discussed show that the methods described herein are capable of producing a viable population of T cells expressing both CD4 and CD8 markers.
- the results shown in FIGURE 3 demonstrate the complex expression of CD3, CD4 and CD8 receptors which occur during T cell differentiation. The level of expression of CD8 increased along the ⁇ -axis, the level of expression of
- CD4 increased along the x-axis and the level of expression of CD3 increased along the z-axis (oriented toward the viewer).
- the 3 D cube on the left represents CD34 + progenitor cells cocultured with LN stroma in c ⁇ tokine combination #1 , there were onl ⁇ stage 4 single positive mature T cells present, single positive CD3 * CD8 * T cells are represented along the rear left plane and single positive CD3 * CD4 * T cells are represented along the forward plane.
- stages 1 , 2, and 3 There were transitional stage 4 single positive mature T cells present along the base of the double positive population along with mature single positive CD3 + CD8 + T cells which are represented along the rear left plane and mature single positive CD3 + CD4 + T cells which are represented along the forward plane.
- the results shown in FIGURE 4 are the Stimulation Indexes of various samples stimulated with PHA.
- PHA is a mitogen which stimulates cellular replication in I ⁇ mphoc ⁇ tes
- the Stimulation Index is an adjusted presentation of the number of radioactive counts found in a particular cell population over a control. Specifically, the Stimulation Index is the number of counts produced in an experimental well divided by the number of counts in a corresponding negative control well. Thus the Stimulation Index represents the number of counts in a particular well expressed in the number of fold above or below the unstimulated control Typically, a three fold increase in counts from an experimental well is required for the incorporation of thymidme to be considered relevant.
- the first column represents I ⁇ mphoc ⁇ tes purified from an HIV negative donor.
- the remaining columns show results from various samples taken from subject R021898.
- the second column shows the Stimulation Index results from LN material taken from da ⁇ 1 , which indicate there was onl ⁇ limited incorporation of the label.
- the remaining columns indicate the Stimulation Index measurements taken on da ⁇ 80 for cocultured cells with T cell differentiating cocktails Nos 1 through 6.
- the third column (LN d80#1 ), cells cultured in the presence of cocktail No.
- the results shown in the sixth column showed the highest stimulation response for all of the culture conditions. These cells produced a Stimulation Index reading of (25) when stimulated with PHA.
- the results from the seventh (LN d80#5) and eighth (LN d80#6) columns were less pronounced but still well above the LNMC d#1 levels of approximatel ⁇ (3).
- the seventh column had measurements of (10) for PHA stimulation.
- the eighth column also had a (10) for PHA stimulation.
- the results shown in FIGURE 4 support a number of observations.
- the I ⁇ mphoc ⁇ te proliferative response of cells obtained from the peripheral blood of the test subject was reduced (data not shown), indicating a need for I ⁇ mphoc ⁇ te replenishment.
- the results indicate that the LN material isolated from the subject had little I ⁇ mphoc ⁇ te proliferative capabilities, indicating little contaminating I ⁇ mphoc ⁇ te material, even though the LN material was not completei ⁇ T I ⁇ mphoc ⁇ te depleted. This result is supported b ⁇ the results in the column labeled LN d80#1.
- CD34 * cells cocultured in the presence of these c ⁇ tokines were not expected to differentiate into mature T cells.
- FIGURE 5 shows a study similar to that shown in FIGURE 4, however the I ⁇ mphoc ⁇ tes were stimulated b ⁇ allo-antigens.
- L ⁇ mphoc ⁇ te response to stimulation b ⁇ allo-antigen is MHC restricted but is not antigen specific at the TCR receptor.
- Cocktails No. 3, 4, 5, and 6, showed increasing levels of responsiveness to allo-antigen compared to
- FIGURE 6 shows a study similar to that shown in FIGURE 4, however, the antigens used in FIGURE 6 are thought to stimulate different populations of T cells.
- Staphylococcus aureus Enterotoxin A (SE-A) stimulates V chain families 1, 3, 10, 11, and 17, while Staphylococcus aureus Enterotoxin B (SE-B) stimulates V chain families 3, 7, 8, and 17. Accordingly, the results of this experiment permit one to assess which V chain families were represented in the mature T cells including those differentiated in culture.
- Cells from this culture responded well to SE-A but showed almost no response to SE-B stimulation (V ⁇ 7, 8 cells were not present or not responding in the tested culture).
- a pattern of skewed responsiveness is noted during advanced HIV infection.
- the Stimulation Index from cells in column 4 (LNd80#2) showed a response to both SE-A and SE B stimulation, although this response was well below that of the positive control.
- the results shown in columns 5 and 6 (LNd80#3 and LNd80#4, respectively) showed antigen induced stimulation response comparable to those of the positive control. Cocktail No.
- T cell differentiation c ⁇ tokine cocktails 2-6 all produced cells that either differentiated into new T cells capable of responding to superantigens, as cocktail No. 1 would onl ⁇ rescue existing T cells and potentially restore their abilit ⁇ to respond to antigenic stimulation.
- a human subject with an immune s ⁇ stem compromised by infection with HIV is treated with the methods described above.
- Autologous I ⁇ mphoid tissue is isolated according to the methods described above.
- CD34 * ceils that are free of the HIV virus are isolated and purified according to the teachings of the present method.
- the isolated CD34 * cells are cocultured with the autologous I ⁇ mphoid tissue in the presence of a c ⁇ tokine cocktail containing IL 2, IL-6, IL 7, OSM, SCF, and fit 3.
- the cells are cocultured as described above. Samples of the coculture are taken on da ⁇ s 1 (prior to the addition of CD34+ progenitor cells), 14, 45, and 60 to determine the extent to which T cell differentiation has occurred using the assa ⁇ methods described herein.
- the mature T ceils are administered to the subject so as to suppl ⁇ the subject with viable, HIV-free T cells.
- the administration of the cocultured T cells occurs subsequent to an aggressive antiviral chemotherapeutic regime to protect the cocultured HIV-free T cells from infection.
- a subject with an immune system damaged by chemotherap ⁇ is treated with the methods of the present invention to provide the subject with a viable population of mature T cells.
- a population of mature T cells is generated from CD34 * stem cells and I ⁇ mphoid tissue as described above.
- Mature T cells are isolated from the coculture and administered to the subject.
- a course of hemopoietic differentiation stimulating compounds such as EPO are also administered at efficacious concentrations to stimulate the generation of er ⁇ throid cells.
- Example 11 Reconstitution of a Human Immune System Damaged by Radiotherapy
- CD34 * cells are acquired from a donor shown to be compatible with the host patient's immune s ⁇ stem.
- the exogenous stem cells are differentiated according to the methods described above. The result is a patient with a reconstituted immune system.
- Example 12 Generating CD34 * Progenitor Cells with an Antisense Tat/Rev HIV Seguence
- CD34 * stem cells are isolated from the patient as described above.
- an expression vector containing an inverted segment of the HIV genome corresponds to a fragment of Exon I of the tat gene with the sequence 5'- CCAGGAAGUCAGCCUAAAA-3' (SEQ ID NO. 3).
- an antisense to the tr gene sequence 5'- CAGACUCAUCAAGCUUCUC-3' (SEQ ID NO. 4) ma ⁇ be used.
- a nucleic acid sequence encoding a riboz ⁇ me corresponding to SEQ ID NO. 3 can be used.
- An example of such a riboz ⁇ me is 5'-
- GGUCCUUCAAAGCAGGAGUGCCUGAGUAGUCUCGGAUUUU-3' SEQ ID NO. 5
- Another embodiment of such a riboz ⁇ me is 5'-GUCUGAGUAAAGCAGGAGUGCCUGAGUAGUCUUCGAAGAG-3' (SEQ ID NO. 6).
- the riboz ⁇ mes discussed above are disclosed in U.S. Patent No. 5,695,938.
- the expression vector is introduced using standard molecular biolog ⁇ techniques well known in the art.
- the expression vector also contains a drug selection marker, such as the tetrac ⁇ cline resistance marker, so that cells containing a functional cop ⁇ of the expression vector ma ⁇ be selected for using tetrac ⁇ cline selection.
- the cells are subjected to tetracycline selection. Accordingl ⁇ , an effective amount of tetrac ⁇ cline is added to the culture media and the cells are grown for an additional 24 hour period. Those cells that survive the selection are then introduced to conditioned I ⁇ mph node cells as discussed above. Mature T cells produced from the process are then administered to the HIV infected patient.
- Mature CD4 * T cells are vulnerable to HIV infection.
- the mature CD4 * cells generated in this Example are prevented from producing virus because the ⁇ contain the expression vector with the tat antisense sequence. This sequence prevents the expression of this essential viral protein. Accordingl ⁇ , the mature CD4 + cells administered to the HIV patient are protected from HIV infection.
- a cocktail of T cell differentiating c ⁇ tokines is administrated to I ⁇ mphoid tissue in the subject to be treated.
- the cocktail of T cell differentiating c ⁇ tokines contains: IL 2, IL 6, IL 7, OSM, SCF, and fit 3.
- the cocktail is administered b ⁇ injection according to basic surgical techniques well known in the art. Specifically, a catheter is introduced through the skin and positioned in a I ⁇ mph node located under the right arm of the subject. Following insertion of the catheter, the cocktail is administered.
- the components of the c ⁇ tokine cocktail function to convert the tissue of the I ⁇ mph node into a microenvironment in which migrating CD34 + stem cell differentiate into mature T cells.
- Example 14 Transplantation of Secondary Lymphoid Tissue
- a patient suffering from complete DiGeorges Syndrome Congenital Athymia
- T cell differentiating c ⁇ tokines a I ⁇ mph node from the subject to be treated
- the lymph node as an intact sample, is cultured for one week in the presence of a T cell differentiating cocktail of cytokines consisting of IL 2, IL 6, IL 7, OSM, SCF, and fit 3 Following this culture period the lymph node is replaced in the bod ⁇ of the subject.
- the subject receives regular injections of the T cell differentiating cocktail of cytokines.
- the conditioned lymph node is replaced in the bod ⁇ of the subject to provide a microenvironment in which CD34 + cells can differentiate into mature T cells.
- SEQ ID NO. 1 Primer Sequence: 5'-GGCACACCCTTTCCTTCTCTG-3'
- SEQ ID NO. 2 Primer Sequence: 5'-GCAGGTCCTGGCTGTAGAAGC-3'
- SEQ ID NO. 3 tat/rev txon I of HIV 5'-CCAGGAAGUCAGCCUAAAA-3'
- SEQ ID NO. 4 tr gene sequence fragment 5'-CAGACUCAUCAAGCUUCUC-3'
- SEQ ID NO. 5 HIV ribozyme sequence
- SEQ ID NO. 6 HIV riboz ⁇ me sequence 5'-GUCUGAGUAAAGCAGGAGUGCCUGAGUAGUCUUCGAAGAG-3'
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU17531/01A AU1753101A (en) | 1999-10-29 | 2000-10-27 | Method of in vitro T cell differentiation of CD34+ progenitor cells |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16257499P | 1999-10-29 | 1999-10-29 | |
US60/162,574 | 1999-10-29 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2001032841A2 true WO2001032841A2 (en) | 2001-05-10 |
WO2001032841A3 WO2001032841A3 (en) | 2002-02-07 |
WO2001032841A9 WO2001032841A9 (en) | 2002-07-04 |
Family
ID=22586221
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/029655 WO2001032841A2 (en) | 1999-10-29 | 2000-10-27 | Method of in vitro t cell differentiation of cd34+ progenitor cells |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU1753101A (en) |
WO (1) | WO2001032841A2 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005063969A2 (en) * | 2003-12-24 | 2005-07-14 | Assistance Publique, Hopitaux De Paris | Methods for the identification and preparation of regulator/suppressor t lymphocytes, compositions and uses thereof |
WO2011068962A1 (en) * | 2009-12-03 | 2011-06-09 | The University Of Utah Research Foundation | Methods for generating t lymphocytes from hematopoietic stem cells |
US8772028B2 (en) | 2008-11-07 | 2014-07-08 | Sunnybrook Health Sciences Centre | CD34+CD7+CD5+CD1a-human progenitor T-cells produced in vitro and methods of using |
CN111718899A (en) * | 2020-06-19 | 2020-09-29 | 珠海贝索细胞科学技术有限公司 | Culture method for in vitro induced NK (natural killer) cells after resuscitation of cryopreserved human PBMCs (peripheral blood mononuclear cells) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993018137A1 (en) * | 1992-03-04 | 1993-09-16 | Systemix, Inc. | Culturing of hematopoietic stem cells and their genetic engineering |
DE4240635A1 (en) * | 1992-12-03 | 1994-06-09 | Kanz Lothar Dr | Compsn. for ex vivo propagation of haematopoietic precursor cells - contains interleukin(s) 1,3 and 6, erythropoietin and stem-cell growth factor, generating cells of high clonogenic potential |
WO1996033265A1 (en) * | 1995-04-21 | 1996-10-24 | President And Fellows Of Harvard College | In vitro differentiation of cd34+ progenitor cells into t lymphocytes |
WO1997030590A1 (en) * | 1996-02-21 | 1997-08-28 | Cira Technologies, Inc. | Cellular immunotherapy |
WO1999007831A1 (en) * | 1997-08-07 | 1999-02-18 | Dompe' S.P.A. | Method for the ex-vivo expansion of hematopoietic stem cells |
WO1999043788A1 (en) * | 1998-02-27 | 1999-09-02 | Anticancer, Inc. | In vitro model for viral infection and immune response |
WO2000027999A2 (en) * | 1998-11-12 | 2000-05-18 | Cell Science Therapeutics, Inc. | Lymphoid tissue-specific cell production from hematopoietic progenitor cells in three-dimensional devices |
-
2000
- 2000-10-27 AU AU17531/01A patent/AU1753101A/en not_active Abandoned
- 2000-10-27 WO PCT/US2000/029655 patent/WO2001032841A2/en active Application Filing
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993018137A1 (en) * | 1992-03-04 | 1993-09-16 | Systemix, Inc. | Culturing of hematopoietic stem cells and their genetic engineering |
DE4240635A1 (en) * | 1992-12-03 | 1994-06-09 | Kanz Lothar Dr | Compsn. for ex vivo propagation of haematopoietic precursor cells - contains interleukin(s) 1,3 and 6, erythropoietin and stem-cell growth factor, generating cells of high clonogenic potential |
WO1996033265A1 (en) * | 1995-04-21 | 1996-10-24 | President And Fellows Of Harvard College | In vitro differentiation of cd34+ progenitor cells into t lymphocytes |
WO1997030590A1 (en) * | 1996-02-21 | 1997-08-28 | Cira Technologies, Inc. | Cellular immunotherapy |
WO1999007831A1 (en) * | 1997-08-07 | 1999-02-18 | Dompe' S.P.A. | Method for the ex-vivo expansion of hematopoietic stem cells |
WO1999043788A1 (en) * | 1998-02-27 | 1999-09-02 | Anticancer, Inc. | In vitro model for viral infection and immune response |
WO2000027999A2 (en) * | 1998-11-12 | 2000-05-18 | Cell Science Therapeutics, Inc. | Lymphoid tissue-specific cell production from hematopoietic progenitor cells in three-dimensional devices |
Non-Patent Citations (1)
Title |
---|
PAWELEC GRAHAM ET AL: "Finite lifespans of T cell clones derived from CD34+ human haematopoietic stem cells in vitro." EXPERIMENTAL GERONTOLOGY, vol. 34, no. 1, January 1999 (1999-01), pages 69-77, XP000926432 ISSN: 0531-5565 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005063969A2 (en) * | 2003-12-24 | 2005-07-14 | Assistance Publique, Hopitaux De Paris | Methods for the identification and preparation of regulator/suppressor t lymphocytes, compositions and uses thereof |
WO2005063969A3 (en) * | 2003-12-24 | 2005-10-13 | Assist Publ Hopitaux De Paris | Methods for the identification and preparation of regulator/suppressor t lymphocytes, compositions and uses thereof |
US8772028B2 (en) | 2008-11-07 | 2014-07-08 | Sunnybrook Health Sciences Centre | CD34+CD7+CD5+CD1a-human progenitor T-cells produced in vitro and methods of using |
US9533009B2 (en) | 2008-11-07 | 2017-01-03 | Sunnybrook Health Sciences Centre | Producing human CD34+CD7+CD5+CD1a− progenitor T cells and method of treatment |
US10266805B2 (en) | 2008-11-07 | 2019-04-23 | Sunnybrook Health Sciences Centre | Human progenitor T-cells into NK cells |
WO2011068962A1 (en) * | 2009-12-03 | 2011-06-09 | The University Of Utah Research Foundation | Methods for generating t lymphocytes from hematopoietic stem cells |
US8871510B2 (en) | 2009-12-03 | 2014-10-28 | University Of Utah Research Foundation | Methods for generating T lymphocytes from hematopoietic stem cells |
CN111718899A (en) * | 2020-06-19 | 2020-09-29 | 珠海贝索细胞科学技术有限公司 | Culture method for in vitro induced NK (natural killer) cells after resuscitation of cryopreserved human PBMCs (peripheral blood mononuclear cells) |
CN111718899B (en) * | 2020-06-19 | 2021-11-23 | 珠海贝索细胞科学技术有限公司 | Culture method for in vitro induced NK (natural killer) cells after resuscitation of cryopreserved human PBMCs (peripheral blood mononuclear cells) |
Also Published As
Publication number | Publication date |
---|---|
WO2001032841A3 (en) | 2002-02-07 |
WO2001032841A9 (en) | 2002-07-04 |
AU1753101A (en) | 2001-05-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Gattinoni et al. | Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells | |
EP1135463B1 (en) | Lymphoid tissue-specific cell production from hematopoietic progenitor cells in three-dimensional devices | |
US7919316B2 (en) | Hematopoietic stem cell identification and isolation | |
EP2177601A1 (en) | Antigen-presenting cell populations and their use as reagents for enhancing or reducing immune tolerance | |
US7763251B2 (en) | Kits to assess the risk of tumor progression | |
JP2018531022A (en) | Methods for generating modified human primary blood dendritic cell lines | |
JP2018531022A6 (en) | Methods for generating modified human primary blood dendritic cell lines | |
US20070196335A1 (en) | Immune Modulation By Regulating Expression Of The "Minor" Gene In Immune Dendritic Cells | |
Glienke et al. | GMP-compliant manufacturing of TRUCKs: CAR T cells targeting GD2 and releasing inducible IL-18 | |
US11708559B2 (en) | Engineered red blood cells having rare antigen phenotypes | |
US5837507A (en) | Hox-induced enhancement of in vivo and in vitro proliferative capacity and gene therapeutic methods | |
US7494807B2 (en) | Mammalian megakaryocyte progenitor cell | |
WO2001032841A2 (en) | Method of in vitro t cell differentiation of cd34+ progenitor cells | |
Mumprecht et al. | Chronic myelogenous leukemia maintains specific CD8+ T cells through IL‐7 signaling | |
Sportoletti et al. | Interleukin-7–engineered mesenchymal cells: in vitro effects on naïve T-cell population | |
WO2003089592A2 (en) | Enhancement of hematopoietic stem cell survival | |
US20230149523A1 (en) | Treatment of autoimmunity and transplant rejection through establishment and/or promotion of tolerogenic processes by fibroblast-mediated reprogramming of antigen presenting cells | |
Peault | THE ORIGIN OF HUMAN HEMATOPOIETIC STEM CELLS |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ CZ DE DE DK DK DM DZ EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): AE AG AL AM AT AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ CZ DE DE DK DK DM DZ EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
AK | Designated states |
Kind code of ref document: C2 Designated state(s): AE AG AL AM AT AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ CZ DE DE DK DK DM DZ EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: C2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
COP | Corrected version of pamphlet |
Free format text: PAGES 1/6-6/6, DRAWINGS, REPLACED BY NEW PAGES 1/6-6/6; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: JP |