ZA200502111B - Ex-vivo expansion of hematopoietic stem cell popu lations in mononuclear cell cultures. - Google Patents
Ex-vivo expansion of hematopoietic stem cell popu lations in mononuclear cell cultures. Download PDFInfo
- Publication number
- ZA200502111B ZA200502111B ZA200502111A ZA200502111A ZA200502111B ZA 200502111 B ZA200502111 B ZA 200502111B ZA 200502111 A ZA200502111 A ZA 200502111A ZA 200502111 A ZA200502111 A ZA 200502111A ZA 200502111 B ZA200502111 B ZA 200502111B
- Authority
- ZA
- South Africa
- Prior art keywords
- cells
- hematopoietic
- mononuclear cells
- nicotinamide
- vivo
- Prior art date
Links
- 210000003958 hematopoietic stem cell Anatomy 0.000 title claims description 322
- 210000005087 mononuclear cell Anatomy 0.000 title claims description 251
- 238000004113 cell culture Methods 0.000 title description 12
- 210000004027 cell Anatomy 0.000 claims description 350
- 230000003394 haemopoietic effect Effects 0.000 claims description 240
- 238000000034 method Methods 0.000 claims description 170
- 210000000130 stem cell Anatomy 0.000 claims description 169
- 230000004069 differentiation Effects 0.000 claims description 104
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical class NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 98
- 239000003795 chemical substances by application Substances 0.000 claims description 91
- 230000014509 gene expression Effects 0.000 claims description 91
- 108090000623 proteins and genes Proteins 0.000 claims description 81
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 claims description 80
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 claims description 80
- 239000010949 copper Substances 0.000 claims description 74
- 239000002738 chelating agent Substances 0.000 claims description 72
- 229910052802 copper Inorganic materials 0.000 claims description 71
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 70
- 230000000694 effects Effects 0.000 claims description 70
- 230000002401 inhibitory effect Effects 0.000 claims description 69
- 150000005480 nicotinamides Chemical class 0.000 claims description 60
- 230000004663 cell proliferation Effects 0.000 claims description 54
- 238000011124 ex vivo culture Methods 0.000 claims description 53
- 235000005152 nicotinamide Nutrition 0.000 claims description 50
- 102000039446 nucleic acids Human genes 0.000 claims description 50
- 108020004707 nucleic acids Proteins 0.000 claims description 50
- 150000007523 nucleic acids Chemical class 0.000 claims description 50
- 229960003966 nicotinamide Drugs 0.000 claims description 49
- 239000011570 nicotinamide Substances 0.000 claims description 49
- 102000004127 Cytokines Human genes 0.000 claims description 42
- 108090000695 Cytokines Proteins 0.000 claims description 42
- 102000003702 retinoic acid receptors Human genes 0.000 claims description 40
- 108090000064 retinoic acid receptors Proteins 0.000 claims description 40
- 102000009310 vitamin D receptors Human genes 0.000 claims description 37
- 108050000156 vitamin D receptors Proteins 0.000 claims description 37
- 239000013522 chelant Substances 0.000 claims description 35
- 238000012258 culturing Methods 0.000 claims description 32
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 claims description 31
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 claims description 31
- 239000005557 antagonist Substances 0.000 claims description 30
- 102000040430 polynucleotide Human genes 0.000 claims description 27
- 108091033319 polynucleotide Proteins 0.000 claims description 27
- 239000002157 polynucleotide Substances 0.000 claims description 27
- 230000019491 signal transduction Effects 0.000 claims description 24
- 102000034527 Retinoid X Receptors Human genes 0.000 claims description 23
- 108010038912 Retinoid X Receptors Proteins 0.000 claims description 23
- 239000002207 metabolite Substances 0.000 claims description 21
- 238000002360 preparation method Methods 0.000 claims description 21
- 239000013598 vector Substances 0.000 claims description 21
- 238000002054 transplantation Methods 0.000 claims description 19
- 210000001185 bone marrow Anatomy 0.000 claims description 18
- 210000005259 peripheral blood Anatomy 0.000 claims description 18
- 239000011886 peripheral blood Substances 0.000 claims description 18
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 claims description 17
- 229930002330 retinoic acid Natural products 0.000 claims description 17
- 229960001727 tretinoin Drugs 0.000 claims description 17
- 229930003316 Vitamin D Natural products 0.000 claims description 16
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 claims description 16
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims description 16
- 229940044551 receptor antagonist Drugs 0.000 claims description 16
- 239000002464 receptor antagonist Substances 0.000 claims description 16
- 235000019166 vitamin D Nutrition 0.000 claims description 16
- 239000011710 vitamin D Substances 0.000 claims description 16
- 150000003710 vitamin D derivatives Chemical class 0.000 claims description 16
- 229940046008 vitamin d Drugs 0.000 claims description 16
- 230000001965 increasing effect Effects 0.000 claims description 15
- 230000010261 cell growth Effects 0.000 claims description 14
- 230000002452 interceptive effect Effects 0.000 claims description 14
- 108091027757 Deoxyribozyme Proteins 0.000 claims description 13
- 229940122756 Retinoic acid receptor antagonist Drugs 0.000 claims description 13
- 229910052723 transition metal Inorganic materials 0.000 claims description 13
- 150000003624 transition metals Chemical class 0.000 claims description 13
- 238000003556 assay Methods 0.000 claims description 12
- 210000004700 fetal blood Anatomy 0.000 claims description 12
- 230000000644 propagated effect Effects 0.000 claims description 12
- 229940116327 Retinoid X receptor antagonist Drugs 0.000 claims description 11
- 230000000692 anti-sense effect Effects 0.000 claims description 11
- 230000003834 intracellular effect Effects 0.000 claims description 11
- 239000013603 viral vector Substances 0.000 claims description 11
- 108090001005 Interleukin-6 Proteins 0.000 claims description 10
- 102000004889 Interleukin-6 Human genes 0.000 claims description 10
- 102000036693 Thrombopoietin Human genes 0.000 claims description 10
- 108010041111 Thrombopoietin Proteins 0.000 claims description 10
- 239000003937 drug carrier Substances 0.000 claims description 10
- 229940100601 interleukin-6 Drugs 0.000 claims description 10
- 230000035755 proliferation Effects 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 230000015556 catabolic process Effects 0.000 claims description 8
- 230000024245 cell differentiation Effects 0.000 claims description 8
- 238000006731 degradation reaction Methods 0.000 claims description 8
- 238000009169 immunotherapy Methods 0.000 claims description 8
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 7
- 150000004492 retinoid derivatives Chemical class 0.000 claims description 7
- KXDAEFPNCMNJSK-UHFFFAOYSA-N Benzamide Chemical compound NC(=O)C1=CC=CC=C1 KXDAEFPNCMNJSK-UHFFFAOYSA-N 0.000 claims description 6
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 claims description 6
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 6
- 108700014844 flt3 ligand Proteins 0.000 claims description 6
- 230000009368 gene silencing by RNA Effects 0.000 claims description 6
- 108020004999 messenger RNA Proteins 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 102000000646 Interleukin-3 Human genes 0.000 claims description 5
- 108010002386 Interleukin-3 Proteins 0.000 claims description 5
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 claims description 5
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 5
- 230000003247 decreasing effect Effects 0.000 claims description 5
- 102000003951 Erythropoietin Human genes 0.000 claims description 4
- 108090000394 Erythropoietin Proteins 0.000 claims description 4
- 102000000588 Interleukin-2 Human genes 0.000 claims description 4
- 108010002350 Interleukin-2 Proteins 0.000 claims description 4
- 108091030071 RNAI Proteins 0.000 claims description 4
- 230000003167 anti-vitamin Effects 0.000 claims description 4
- 229940105423 erythropoietin Drugs 0.000 claims description 4
- 210000003714 granulocyte Anatomy 0.000 claims description 4
- 238000012544 monitoring process Methods 0.000 claims description 4
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 claims description 4
- QDLHCMPXEPAAMD-QAIWCSMKSA-N wortmannin Chemical compound C1([C@]2(C)C3=C(C4=O)OC=C3C(=O)O[C@@H]2COC)=C4[C@@H]2CCC(=O)[C@@]2(C)C[C@H]1OC(C)=O QDLHCMPXEPAAMD-QAIWCSMKSA-N 0.000 claims description 4
- QDLHCMPXEPAAMD-UHFFFAOYSA-N wortmannin Natural products COCC1OC(=O)C2=COC(C3=O)=C2C1(C)C1=C3C2CCC(=O)C2(C)CC1OC(C)=O QDLHCMPXEPAAMD-UHFFFAOYSA-N 0.000 claims description 4
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 3
- 101150021185 FGF gene Proteins 0.000 claims description 3
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 claims description 3
- 102100021866 Hepatocyte growth factor Human genes 0.000 claims description 3
- 102000000589 Interleukin-1 Human genes 0.000 claims description 3
- 108010002352 Interleukin-1 Proteins 0.000 claims description 3
- 102000003814 Interleukin-10 Human genes 0.000 claims description 3
- 108090000174 Interleukin-10 Proteins 0.000 claims description 3
- 102000013462 Interleukin-12 Human genes 0.000 claims description 3
- 108010065805 Interleukin-12 Proteins 0.000 claims description 3
- 102000015336 Nerve Growth Factor Human genes 0.000 claims description 3
- 108010025020 Nerve Growth Factor Proteins 0.000 claims description 3
- 101710098940 Pro-epidermal growth factor Proteins 0.000 claims description 3
- 206010054094 Tumour necrosis Diseases 0.000 claims description 3
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 3
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 3
- 230000005764 inhibitory process Effects 0.000 claims description 3
- 229940076144 interleukin-10 Drugs 0.000 claims description 3
- 229940117681 interleukin-12 Drugs 0.000 claims description 3
- 229940076264 interleukin-3 Drugs 0.000 claims description 3
- 238000012423 maintenance Methods 0.000 claims description 3
- HFZXBZIYMINYKS-UHFFFAOYSA-N n-sulfanylpyridine-3-carboxamide Chemical compound SNC(=O)C1=CC=CN=C1 HFZXBZIYMINYKS-UHFFFAOYSA-N 0.000 claims description 3
- 229960003512 nicotinic acid Drugs 0.000 claims description 3
- 235000001968 nicotinic acid Nutrition 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- 230000006698 induction Effects 0.000 claims description 2
- 230000003021 clonogenic effect Effects 0.000 claims 2
- 230000002035 prolonged effect Effects 0.000 claims 2
- 102100029938 Serine/threonine-protein kinase SMG1 Human genes 0.000 claims 1
- 210000001671 embryonic stem cell Anatomy 0.000 claims 1
- 210000001161 mammalian embryo Anatomy 0.000 claims 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 111
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 111
- -1 for example Chemical compound 0.000 description 111
- 229920000768 polyamine Polymers 0.000 description 73
- 125000002947 alkylene group Chemical group 0.000 description 57
- 125000000217 alkyl group Chemical group 0.000 description 51
- 108091034117 Oligonucleotide Proteins 0.000 description 41
- 125000004432 carbon atom Chemical group C* 0.000 description 40
- 239000000047 product Substances 0.000 description 40
- 125000003118 aryl group Chemical group 0.000 description 36
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 32
- 108020004414 DNA Proteins 0.000 description 27
- 125000004122 cyclic group Chemical group 0.000 description 27
- 230000007774 longterm Effects 0.000 description 25
- 239000005711 Benzoic acid Substances 0.000 description 24
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 22
- 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 22
- 239000002253 acid Substances 0.000 description 22
- 125000003277 amino group Chemical group 0.000 description 22
- 235000010233 benzoic acid Nutrition 0.000 description 21
- 210000004369 blood Anatomy 0.000 description 21
- 239000008280 blood Substances 0.000 description 21
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 20
- 230000001105 regulatory effect Effects 0.000 description 20
- 238000000338 in vitro Methods 0.000 description 19
- 102000004169 proteins and genes Human genes 0.000 description 19
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 18
- 125000000753 cycloalkyl group Chemical group 0.000 description 18
- 239000012634 fragment Substances 0.000 description 18
- 102000005962 receptors Human genes 0.000 description 18
- 108020003175 receptors Proteins 0.000 description 18
- 125000005842 heteroatom Chemical group 0.000 description 17
- 238000001727 in vivo Methods 0.000 description 17
- 239000002585 base Substances 0.000 description 16
- 229910052799 carbon Inorganic materials 0.000 description 16
- 125000001072 heteroaryl group Chemical group 0.000 description 16
- 239000002935 phosphatidylinositol 3 kinase inhibitor Substances 0.000 description 16
- 238000001415 gene therapy Methods 0.000 description 15
- 108090000765 processed proteins & peptides Proteins 0.000 description 15
- 229910052717 sulfur Inorganic materials 0.000 description 15
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 14
- 229940116355 PI3 kinase inhibitor Drugs 0.000 description 14
- 230000001404 mediated effect Effects 0.000 description 14
- 125000001424 substituent group Chemical group 0.000 description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 12
- UORVGPXVDQYIDP-UHFFFAOYSA-N borane Chemical compound B UORVGPXVDQYIDP-UHFFFAOYSA-N 0.000 description 12
- 229910052739 hydrogen Inorganic materials 0.000 description 12
- 125000005647 linker group Chemical group 0.000 description 12
- 230000004048 modification Effects 0.000 description 12
- 238000012986 modification Methods 0.000 description 12
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 12
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 12
- 230000001413 cellular effect Effects 0.000 description 11
- 150000002430 hydrocarbons Chemical group 0.000 description 11
- 239000001257 hydrogen Substances 0.000 description 11
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 11
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 11
- 125000002813 thiocarbonyl group Chemical group *C(*)=S 0.000 description 11
- 238000001890 transfection Methods 0.000 description 11
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 10
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 10
- 241000700605 Viruses Species 0.000 description 10
- 150000001721 carbon Chemical group 0.000 description 10
- 125000004430 oxygen atom Chemical group O* 0.000 description 10
- 125000004434 sulfur atom Chemical group 0.000 description 10
- 230000003612 virological effect Effects 0.000 description 10
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 9
- 150000001408 amides Chemical class 0.000 description 9
- 150000001412 amines Chemical class 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 108091007433 antigens Proteins 0.000 description 9
- 102000036639 antigens Human genes 0.000 description 9
- 229910001431 copper ion Inorganic materials 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 230000001177 retroviral effect Effects 0.000 description 9
- 125000000475 sulfinyl group Chemical group [*:2]S([*:1])=O 0.000 description 9
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 description 9
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 8
- 108060003951 Immunoglobulin Proteins 0.000 description 8
- 125000003545 alkoxy group Chemical group 0.000 description 8
- 125000004104 aryloxy group Chemical group 0.000 description 8
- 210000000601 blood cell Anatomy 0.000 description 8
- 238000003776 cleavage reaction Methods 0.000 description 8
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 8
- 230000002950 deficient Effects 0.000 description 8
- 125000005843 halogen group Chemical group 0.000 description 8
- 102000018358 immunoglobulin Human genes 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 230000007017 scission Effects 0.000 description 8
- 125000005190 thiohydroxy group Chemical group 0.000 description 8
- 229940126634 CD38 inhibitor Drugs 0.000 description 7
- VJQALSOBHVEJQM-UHFFFAOYSA-N COCCOC1CCC(CC1)Nc1cc(=O)n(C)c2ccc(cc12)-c1cncs1 Chemical compound COCCOC1CCC(CC1)Nc1cc(=O)n(C)c2ccc(cc12)-c1cncs1 VJQALSOBHVEJQM-UHFFFAOYSA-N 0.000 description 7
- 150000004660 O-thiocarbamates Chemical class 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 239000004202 carbamide Substances 0.000 description 7
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 7
- 230000001939 inductive effect Effects 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 7
- 239000003446 ligand Substances 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 229940124530 sulfonamide Drugs 0.000 description 7
- 150000003568 thioethers Chemical class 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- 241001430294 unidentified retrovirus Species 0.000 description 7
- ZAXCZCOUDLENMH-UHFFFAOYSA-N 3,3,3-tetramine Chemical compound NCCCNCCCNCCCN ZAXCZCOUDLENMH-UHFFFAOYSA-N 0.000 description 6
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- XTEGARKTQYYJKE-UHFFFAOYSA-M Chlorate Chemical compound [O-]Cl(=O)=O XTEGARKTQYYJKE-UHFFFAOYSA-M 0.000 description 6
- RGSFGYAAUTVSQA-UHFFFAOYSA-N Cyclopentane Chemical compound C1CCCC1 RGSFGYAAUTVSQA-UHFFFAOYSA-N 0.000 description 6
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 6
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 6
- 108700019146 Transgenes Proteins 0.000 description 6
- 238000013459 approach Methods 0.000 description 6
- 125000004429 atom Chemical group 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 229910000085 borane Inorganic materials 0.000 description 6
- DKSMCEUSSQTGBK-UHFFFAOYSA-M bromite Chemical compound [O-]Br=O DKSMCEUSSQTGBK-UHFFFAOYSA-M 0.000 description 6
- 125000004093 cyano group Chemical group *C#N 0.000 description 6
- 125000001559 cyclopropyl group Chemical group [H]C1([H])C([H])([H])C1([H])* 0.000 description 6
- 150000002148 esters Chemical class 0.000 description 6
- 230000011132 hemopoiesis Effects 0.000 description 6
- DMEGYFMYUHOHGS-UHFFFAOYSA-N heptamethylene Natural products C1CCCCCC1 DMEGYFMYUHOHGS-UHFFFAOYSA-N 0.000 description 6
- JGJLWPGRMCADHB-UHFFFAOYSA-N hypobromite Chemical compound Br[O-] JGJLWPGRMCADHB-UHFFFAOYSA-N 0.000 description 6
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 230000000670 limiting effect Effects 0.000 description 6
- 239000002502 liposome Substances 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 230000037361 pathway Effects 0.000 description 6
- LLYCMZGLHLKPPU-UHFFFAOYSA-M perbromate Chemical compound [O-]Br(=O)(=O)=O LLYCMZGLHLKPPU-UHFFFAOYSA-M 0.000 description 6
- 125000005328 phosphinyl group Chemical group [PH2](=O)* 0.000 description 6
- XYFCBTPGUUZFHI-UHFFFAOYSA-O phosphonium Chemical compound [PH4+] XYFCBTPGUUZFHI-UHFFFAOYSA-O 0.000 description 6
- 125000005499 phosphonyl group Chemical group 0.000 description 6
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 6
- 230000011664 signaling Effects 0.000 description 6
- 125000004469 siloxy group Chemical group [SiH3]O* 0.000 description 6
- MDDUHVRJJAFRAU-YZNNVMRBSA-N tert-butyl-[(1r,3s,5z)-3-[tert-butyl(dimethyl)silyl]oxy-5-(2-diphenylphosphorylethylidene)-4-methylidenecyclohexyl]oxy-dimethylsilane Chemical compound C1[C@@H](O[Si](C)(C)C(C)(C)C)C[C@H](O[Si](C)(C)C(C)(C)C)C(=C)\C1=C/CP(=O)(C=1C=CC=CC=1)C1=CC=CC=C1 MDDUHVRJJAFRAU-YZNNVMRBSA-N 0.000 description 6
- 125000005309 thioalkoxy group Chemical group 0.000 description 6
- 125000005296 thioaryloxy group Chemical group 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- IMNIMPAHZVJRPE-UHFFFAOYSA-N triethylenediamine Chemical compound C1CN2CCN1CC2 IMNIMPAHZVJRPE-UHFFFAOYSA-N 0.000 description 6
- 241000701161 unidentified adenovirus Species 0.000 description 6
- 229920002307 Dextran Polymers 0.000 description 5
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 5
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 102000057297 Pepsin A Human genes 0.000 description 5
- 108090000284 Pepsin A Proteins 0.000 description 5
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Natural products P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 150000001336 alkenes Chemical class 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 230000001332 colony forming effect Effects 0.000 description 5
- 230000001186 cumulative effect Effects 0.000 description 5
- 239000012990 dithiocarbamate Substances 0.000 description 5
- 238000001476 gene delivery Methods 0.000 description 5
- 125000000623 heterocyclic group Chemical group 0.000 description 5
- 238000002513 implantation Methods 0.000 description 5
- 230000000977 initiatory effect Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 5
- 230000002441 reversible effect Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 230000003442 weekly effect Effects 0.000 description 5
- LHUPKWKWYWOMSK-UHFFFAOYSA-N 4-[2-[4-(4-ethylphenyl)-2,2-dimethylthiochromen-6-yl]ethynyl]benzoic acid Chemical compound C1=CC(CC)=CC=C1C1=CC(C)(C)SC2=CC=C(C#CC=3C=CC(=CC=3)C(O)=O)C=C12 LHUPKWKWYWOMSK-UHFFFAOYSA-N 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 4
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 4
- 108091093037 Peptide nucleic acid Proteins 0.000 description 4
- ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical compound OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 4
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 4
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 4
- 108020004459 Small interfering RNA Proteins 0.000 description 4
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 4
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical compound C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 4
- ORILYTVJVMAKLC-UHFFFAOYSA-N adamantane Chemical compound C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 description 4
- 125000003342 alkenyl group Chemical group 0.000 description 4
- 125000000304 alkynyl group Chemical group 0.000 description 4
- 125000000751 azo group Chemical group [*]N=N[*] 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 230000032823 cell division Effects 0.000 description 4
- 238000002512 chemotherapy Methods 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- MGNZXYYWBUKAII-UHFFFAOYSA-N cyclohexa-1,3-diene Chemical compound C1CC=CC=C1 MGNZXYYWBUKAII-UHFFFAOYSA-N 0.000 description 4
- LPIQUOYDBNQMRZ-UHFFFAOYSA-N cyclopentene Chemical compound C1CC=CC1 LPIQUOYDBNQMRZ-UHFFFAOYSA-N 0.000 description 4
- 229940104302 cytosine Drugs 0.000 description 4
- 230000002222 downregulating effect Effects 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 150000002466 imines Chemical class 0.000 description 4
- 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 4
- 238000002955 isolation Methods 0.000 description 4
- AWJUIBRHMBBTKR-UHFFFAOYSA-N isoquinoline Chemical compound C1=NC=CC2=CC=CC=C21 AWJUIBRHMBBTKR-UHFFFAOYSA-N 0.000 description 4
- 210000000265 leukocyte Anatomy 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229940111202 pepsin Drugs 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- SIOXPEMLGUPBBT-UHFFFAOYSA-N picolinic acid Chemical compound OC(=O)C1=CC=CC=N1 SIOXPEMLGUPBBT-UHFFFAOYSA-N 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 102000027483 retinoid hormone receptors Human genes 0.000 description 4
- 108091008679 retinoid hormone receptors Proteins 0.000 description 4
- 150000003456 sulfonamides Chemical class 0.000 description 4
- 150000007970 thio esters Chemical class 0.000 description 4
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 229940035893 uracil Drugs 0.000 description 4
- 229910052725 zinc Inorganic materials 0.000 description 4
- 239000011701 zinc Substances 0.000 description 4
- VILCJCGEZXAXTO-UHFFFAOYSA-N 2,2,2-tetramine Chemical compound NCCNCCNCCN VILCJCGEZXAXTO-UHFFFAOYSA-N 0.000 description 3
- 125000001397 3-pyrrolyl group Chemical group [H]N1C([H])=C([*])C([H])=C1[H] 0.000 description 3
- 229930024421 Adenine Natural products 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 3
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 3
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 108090000994 Catalytic RNA Proteins 0.000 description 3
- 102000053642 Catalytic RNA Human genes 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 3
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 3
- RPNUMPOLZDHAAY-UHFFFAOYSA-N Diethylenetriamine Chemical compound NCCNCCN RPNUMPOLZDHAAY-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical group NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 229910002651 NO3 Inorganic materials 0.000 description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 3
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 3
- GNVMUORYQLCPJZ-UHFFFAOYSA-M Thiocarbamate Chemical compound NC([S-])=O GNVMUORYQLCPJZ-UHFFFAOYSA-M 0.000 description 3
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 description 3
- 229960000643 adenine Drugs 0.000 description 3
- 125000004450 alkenylene group Chemical group 0.000 description 3
- 125000003282 alkyl amino group Chemical group 0.000 description 3
- 150000004789 alkyl aryl sulfoxides Chemical class 0.000 description 3
- 150000001356 alkyl thiols Chemical class 0.000 description 3
- 125000004419 alkynylene group Chemical group 0.000 description 3
- 230000000735 allogeneic effect Effects 0.000 description 3
- LHIJANUOQQMGNT-UHFFFAOYSA-N aminoethylethanolamine Chemical compound NCCNCCO LHIJANUOQQMGNT-UHFFFAOYSA-N 0.000 description 3
- 125000001769 aryl amino group Chemical group 0.000 description 3
- 125000005361 aryl sulfoxide group Chemical group 0.000 description 3
- 150000001504 aryl thiols Chemical class 0.000 description 3
- 125000000732 arylene group Chemical group 0.000 description 3
- 229940072107 ascorbate Drugs 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 3
- SXDBWCPKPHAZSM-UHFFFAOYSA-M bromate Inorganic materials [O-]Br(=O)=O SXDBWCPKPHAZSM-UHFFFAOYSA-M 0.000 description 3
- SXDBWCPKPHAZSM-UHFFFAOYSA-N bromic acid Chemical compound OBr(=O)=O SXDBWCPKPHAZSM-UHFFFAOYSA-N 0.000 description 3
- 125000001246 bromo group Chemical group Br* 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 229910001919 chlorite Inorganic materials 0.000 description 3
- 229910052619 chlorite group Inorganic materials 0.000 description 3
- QBWCMBCROVPCKQ-UHFFFAOYSA-N chlorous acid Chemical compound OCl=O QBWCMBCROVPCKQ-UHFFFAOYSA-N 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 210000004748 cultured cell Anatomy 0.000 description 3
- XLJMAIOERFSOGZ-UHFFFAOYSA-M cyanate Chemical compound [O-]C#N XLJMAIOERFSOGZ-UHFFFAOYSA-M 0.000 description 3
- 125000006310 cycloalkyl amino group Chemical group 0.000 description 3
- 210000004443 dendritic cell Anatomy 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 210000002308 embryonic cell Anatomy 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 125000005241 heteroarylamino group Chemical group 0.000 description 3
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- ICIWUVCWSCSTAQ-UHFFFAOYSA-M iodate Chemical compound [O-]I(=O)=O ICIWUVCWSCSTAQ-UHFFFAOYSA-M 0.000 description 3
- 239000012948 isocyanate Substances 0.000 description 3
- 150000002513 isocyanates Chemical class 0.000 description 3
- 150000002540 isothiocyanates Chemical class 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 125000002950 monocyclic group Chemical group 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 239000002777 nucleoside Substances 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 3
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 3
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- ACVYVLVWPXVTIT-UHFFFAOYSA-M phosphinate Chemical compound [O-][PH2]=O ACVYVLVWPXVTIT-UHFFFAOYSA-M 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 229910052697 platinum Inorganic materials 0.000 description 3
- 210000001778 pluripotent stem cell Anatomy 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000003793 prenatal diagnosis Methods 0.000 description 3
- 125000004368 propenyl group Chemical group C(=CC)* 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 108091092562 ribozyme Proteins 0.000 description 3
- 238000004904 shortening Methods 0.000 description 3
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 3
- 239000011593 sulfur Substances 0.000 description 3
- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 125000004953 trihalomethyl group Chemical group 0.000 description 3
- 239000001226 triphosphate Substances 0.000 description 3
- 235000011178 triphosphate Nutrition 0.000 description 3
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- UHGJTMGZWSGZJO-XXUKQAADSA-N (2e,4e,6e)-7-[3-tert-butyl-5-(1-phenylethenyl)phenyl]-3-methylocta-2,4,6-trienoic acid Chemical compound CC(C)(C)C1=CC(C(/C)=C/C=C/C(/C)=C/C(O)=O)=CC(C(=C)C=2C=CC=CC=2)=C1 UHGJTMGZWSGZJO-XXUKQAADSA-N 0.000 description 2
- HNODNXQAYXJFMQ-LQUSFLDPSA-N (2e,4e,6z)-3-methyl-7-(5,5,8,8-tetramethyl-3-propoxy-6,7-dihydronaphthalen-2-yl)octa-2,4,6-trienoic acid Chemical compound CC1(C)CCC(C)(C)C2=C1C=C(\C(C)=C/C=C/C(/C)=C/C(O)=O)C(OCCC)=C2 HNODNXQAYXJFMQ-LQUSFLDPSA-N 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- QBPPRVHXOZRESW-UHFFFAOYSA-N 1,4,7,10-tetraazacyclododecane Chemical compound C1CNCCNCCNCCN1 QBPPRVHXOZRESW-UHFFFAOYSA-N 0.000 description 2
- MDAXKAUIABOHTD-UHFFFAOYSA-N 1,4,8,11-tetraazacyclotetradecane Chemical compound C1CNCCNCCCNCCNC1 MDAXKAUIABOHTD-UHFFFAOYSA-N 0.000 description 2
- BGVLBVASHIQNIO-UHFFFAOYSA-N 1,4,8,11-tetrazacyclotetradecane-5,7-dione Chemical compound O=C1CC(=O)NCCNCCCNCCN1 BGVLBVASHIQNIO-UHFFFAOYSA-N 0.000 description 2
- KUFDRRWNPNXBRF-UHFFFAOYSA-N 1,4,8,12-tetrazacyclopentadecane Chemical compound C1CNCCCNCCNCCCNC1 KUFDRRWNPNXBRF-UHFFFAOYSA-N 0.000 description 2
- PWJHXHMUGFXPSN-UHFFFAOYSA-N 1,7-dioxa-4,10-diazacyclododecane Chemical compound C1COCCNCCOCCN1 PWJHXHMUGFXPSN-UHFFFAOYSA-N 0.000 description 2
- IFHMELMOPNZNGD-UHFFFAOYSA-N 1-(3-hydroxy-5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)ethanone Chemical compound CC1(C)CCC(C)(C)C2=C1C=C(O)C(C(=O)C)=C2 IFHMELMOPNZNGD-UHFFFAOYSA-N 0.000 description 2
- NLINGGCQUVARNS-UHFFFAOYSA-N 1-oxa-4,7,10-triazacyclododecane Chemical compound C1CNCCOCCNCCN1 NLINGGCQUVARNS-UHFFFAOYSA-N 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N 1H-pyrrole Natural products C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- VOZKAJLKRJDJLL-UHFFFAOYSA-N 2,4-diaminotoluene Chemical group CC1=CC=C(N)C=C1N VOZKAJLKRJDJLL-UHFFFAOYSA-N 0.000 description 2
- FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 2
- OYIFNHCXNCRBQI-UHFFFAOYSA-N 2-aminoadipic acid Chemical compound OC(=O)C(N)CCCC(O)=O OYIFNHCXNCRBQI-UHFFFAOYSA-N 0.000 description 2
- QRPOZDMXOXTFSH-UHFFFAOYSA-N 3-(4-methoxyphenyl)sulfanyl-3-methylbutanoic acid Chemical compound COC1=CC=C(SC(C)(C)CC(O)=O)C=C1 QRPOZDMXOXTFSH-UHFFFAOYSA-N 0.000 description 2
- PLRKLUBDNPSNCP-UHFFFAOYSA-N 3-(5,5,8,8-tetramethyl-3-propoxy-6,7-dihydronaphthalen-2-yl)but-2-enal Chemical compound CC1(C)CCC(C)(C)C2=C1C=C(C(C)=CC=O)C(OCCC)=C2 PLRKLUBDNPSNCP-UHFFFAOYSA-N 0.000 description 2
- QADGBOQVBUXZKO-UHFFFAOYSA-N 4-(3,5,5,8,8-pentamethyl-6,7-dihydronaphthalene-2-carbonyl)benzoic acid Chemical compound CC1=CC(C(CCC2(C)C)(C)C)=C2C=C1C(=O)C1=CC=C(C(O)=O)C=C1 QADGBOQVBUXZKO-UHFFFAOYSA-N 0.000 description 2
- SZWKGOZKRMMLAJ-UHFFFAOYSA-N 4-{[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]amino}benzoic acid Chemical compound C=1C=C2C(C)(C)CCC(C)(C)C2=CC=1C(=O)NC1=CC=C(C(O)=O)C=C1 SZWKGOZKRMMLAJ-UHFFFAOYSA-N 0.000 description 2
- SLXTWXQUEZSSTJ-UHFFFAOYSA-N 6-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl]pyridine-3-carboxylic acid Chemical compound CC1=CC(C(CCC2(C)C)(C)C)=C2C=C1C1(C=2N=CC(=CC=2)C(O)=O)CC1 SLXTWXQUEZSSTJ-UHFFFAOYSA-N 0.000 description 2
- KGBIPJPRSGFZRG-UHFFFAOYSA-N 6-[1-(3,5,5,8,8-pentamethyl-6,7-dihydronaphthalen-2-yl)ethyl]pyridine-3-carboxylic acid Chemical compound C=1C(C(CCC2(C)C)(C)C)=C2C=C(C)C=1C(C)C1=CC=C(C(O)=O)C=N1 KGBIPJPRSGFZRG-UHFFFAOYSA-N 0.000 description 2
- PEHVGBZKEYRQSX-UHFFFAOYSA-N 7-deaza-adenine Chemical compound NC1=NC=NC2=C1C=CN2 PEHVGBZKEYRQSX-UHFFFAOYSA-N 0.000 description 2
- HCGHYQLFMPXSDU-UHFFFAOYSA-N 7-methyladenine Chemical compound C1=NC(N)=C2N(C)C=NC2=N1 HCGHYQLFMPXSDU-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 239000013607 AAV vector Substances 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 241001529297 Coregonus peled Species 0.000 description 2
- PMPVIKIVABFJJI-UHFFFAOYSA-N Cyclobutane Chemical compound C1CCC1 PMPVIKIVABFJJI-UHFFFAOYSA-N 0.000 description 2
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 2
- 108010069091 Dystrophin Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108091029865 Exogenous DNA Proteins 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 101100220044 Homo sapiens CD34 gene Proteins 0.000 description 2
- 101001078143 Homo sapiens Integrin alpha-IIb Proteins 0.000 description 2
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- VSNHCAURESNICA-UHFFFAOYSA-N Hydroxyurea Chemical compound NC(=O)NO VSNHCAURESNICA-UHFFFAOYSA-N 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 102100025306 Integrin alpha-IIb Human genes 0.000 description 2
- 102100032352 Leukemia inhibitory factor Human genes 0.000 description 2
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 2
- 108090001060 Lipase Proteins 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 2
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 2
- 108091061960 Naked DNA Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 102000043276 Oncogene Human genes 0.000 description 2
- 101710160107 Outer membrane protein A Proteins 0.000 description 2
- ZCQWOFVYLHDMMC-UHFFFAOYSA-N Oxazole Chemical compound C1=COC=N1 ZCQWOFVYLHDMMC-UHFFFAOYSA-N 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- WTKZEGDFNFYCGP-UHFFFAOYSA-N Pyrazole Chemical compound C=1C=NNC=1 WTKZEGDFNFYCGP-UHFFFAOYSA-N 0.000 description 2
- 238000010240 RT-PCR analysis Methods 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 2
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 2
- 108091008605 VEGF receptors Proteins 0.000 description 2
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 210000004504 adult stem cell Anatomy 0.000 description 2
- 125000002877 alkyl aryl group Chemical group 0.000 description 2
- 125000005360 alkyl sulfoxide group Chemical group 0.000 description 2
- HSFWRNGVRCDJHI-UHFFFAOYSA-N alpha-acetylene Natural products C#C HSFWRNGVRCDJHI-UHFFFAOYSA-N 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- IMUDHTPIFIBORV-UHFFFAOYSA-N aminoethylpiperazine Chemical compound NCCN1CCNCC1 IMUDHTPIFIBORV-UHFFFAOYSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 125000002178 anthracenyl group Chemical group C1(=CC=CC2=CC3=CC=CC=C3C=C12)* 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Chemical compound BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- FAKRSMQSSFJEIM-RQJHMYQMSA-N captopril Chemical compound SC[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O FAKRSMQSSFJEIM-RQJHMYQMSA-N 0.000 description 2
- 229960000830 captopril Drugs 0.000 description 2
- 230000004700 cellular uptake Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical group 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
- 229940001468 citrate Drugs 0.000 description 2
- 229910017052 cobalt Inorganic materials 0.000 description 2
- 239000010941 cobalt Substances 0.000 description 2
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 2
- 230000002281 colonystimulating effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000001351 cycling effect Effects 0.000 description 2
- 125000002993 cycloalkylene group Chemical group 0.000 description 2
- CHVJITGCYZJHLR-UHFFFAOYSA-N cyclohepta-1,3,5-triene Chemical compound C1C=CC=CC=C1 CHVJITGCYZJHLR-UHFFFAOYSA-N 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- ZFZSCWASFPMMOX-UHFFFAOYSA-N ethyl 4-[2-(2,2-dimethyl-4-oxo-3h-thiochromen-6-yl)ethynyl]benzoate Chemical compound C1=CC(C(=O)OCC)=CC=C1C#CC1=CC=C(SC(C)(C)CC2=O)C2=C1 ZFZSCWASFPMMOX-UHFFFAOYSA-N 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 125000002534 ethynyl group Chemical group [H]C#C* 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
- 230000009395 genetic defect Effects 0.000 description 2
- 208000016361 genetic disease Diseases 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 208000024908 graft versus host disease Diseases 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 210000000777 hematopoietic system Anatomy 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 125000000814 indol-3-yl group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C([*])C2=C1[H] 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 229940043355 kinase inhibitor Drugs 0.000 description 2
- 150000002596 lactones Chemical class 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 235000019421 lipase Nutrition 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000008774 maternal effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 150000002739 metals Chemical class 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 210000003098 myoblast Anatomy 0.000 description 2
- KLRVODKPWQTPFG-UHFFFAOYSA-N n-(1-aminoethoxy)-4-(3,5,5,8,8-pentamethyl-6,7-dihydronaphthalene-2-carbonyl)benzamide Chemical compound C1=CC(C(O)=NOC(N)C)=CC=C1C(=O)C1=CC(C(CCC2(C)C)(C)C)=C2C=C1C KLRVODKPWQTPFG-UHFFFAOYSA-N 0.000 description 2
- 125000001624 naphthyl group Chemical group 0.000 description 2
- 229910052759 nickel Inorganic materials 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- IAAPVNQZSBLWKH-UHFFFAOYSA-N octatrienoic acid Natural products CC=CC=CC=CC(O)=O IAAPVNQZSBLWKH-UHFFFAOYSA-N 0.000 description 2
- 210000000287 oocyte Anatomy 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 125000002524 organometallic group Chemical group 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 229910052763 palladium Inorganic materials 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 125000004437 phosphorous atom Chemical group 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000003757 phosphotransferase inhibitor Substances 0.000 description 2
- 125000003367 polycyclic group Chemical group 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000009696 proliferative response Effects 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 150000003230 pyrimidines Chemical class 0.000 description 2
- 125000000168 pyrrolyl group Chemical group 0.000 description 2
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 229910052703 rhodium Inorganic materials 0.000 description 2
- 239000010948 rhodium Substances 0.000 description 2
- MHOVAHRLVXNVSD-UHFFFAOYSA-N rhodium atom Chemical compound [Rh] MHOVAHRLVXNVSD-UHFFFAOYSA-N 0.000 description 2
- 229910052707 ruthenium Inorganic materials 0.000 description 2
- 229960001860 salicylate Drugs 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-M salicylate Chemical compound OC1=CC=CC=C1C([O-])=O YGSDEFSMJLZEOE-UHFFFAOYSA-M 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000004055 small Interfering RNA Substances 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 238000011476 stem cell transplantation Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 229940086735 succinate Drugs 0.000 description 2
- OKQKDCXVLPGWPO-UHFFFAOYSA-N sulfanylidenephosphane Chemical compound S=P OKQKDCXVLPGWPO-UHFFFAOYSA-N 0.000 description 2
- 150000003462 sulfoxides Chemical class 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- FAGUFWYHJQFNRV-UHFFFAOYSA-N tetraethylenepentamine Chemical compound NCCNCCNCCNCCN FAGUFWYHJQFNRV-UHFFFAOYSA-N 0.000 description 2
- 150000003573 thiols Chemical class 0.000 description 2
- 229930192474 thiophene Natural products 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N tryptophan Chemical compound C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 229960005356 urokinase Drugs 0.000 description 2
- 229940124676 vascular endothelial growth factor receptor Drugs 0.000 description 2
- QDZOEBFLNHCSSF-PFFBOGFISA-N (2S)-2-[[(2R)-2-[[(2S)-1-[(2S)-6-amino-2-[[(2S)-1-[(2R)-2-amino-5-carbamimidamidopentanoyl]pyrrolidine-2-carbonyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-N-[(2R)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2S)-1-amino-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]pentanediamide Chemical compound C([C@@H](C(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(N)=O)NC(=O)[C@@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](N)CCCNC(N)=N)C1=CC=CC=C1 QDZOEBFLNHCSSF-PFFBOGFISA-N 0.000 description 1
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 description 1
- QGVQZRDQPDLHHV-DPAQBDIFSA-N (3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthrene-3-thiol Chemical compound C1C=C2C[C@@H](S)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 QGVQZRDQPDLHHV-DPAQBDIFSA-N 0.000 description 1
- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 description 1
- UTZAFOQPCXRRFF-RKBILKOESA-N (beta-D-glucosyl)-O-mycofactocinone Chemical compound CC1(C(NC(=O)C1=O)CC2=CC=C(C=C2)O[C@H]3[C@@H]([C@H]([C@@H]([C@H](O3)CO)O)O)O)C UTZAFOQPCXRRFF-RKBILKOESA-N 0.000 description 1
- FYADHXFMURLYQI-UHFFFAOYSA-N 1,2,4-triazine Chemical class C1=CN=NC=N1 FYADHXFMURLYQI-UHFFFAOYSA-N 0.000 description 1
- ITWBWJFEJCHKSN-UHFFFAOYSA-N 1,4,7-triazonane Chemical compound C1CNCCNCCN1 ITWBWJFEJCHKSN-UHFFFAOYSA-N 0.000 description 1
- UHUHBFMZVCOEOV-UHFFFAOYSA-N 1h-imidazo[4,5-c]pyridin-4-amine Chemical compound NC1=NC=CC2=C1N=CN2 UHUHBFMZVCOEOV-UHFFFAOYSA-N 0.000 description 1
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 1
- PIINGYXNCHTJTF-UHFFFAOYSA-N 2-(2-azaniumylethylamino)acetate Chemical group NCCNCC(O)=O PIINGYXNCHTJTF-UHFFFAOYSA-N 0.000 description 1
- FDZGOVDEFRJXFT-UHFFFAOYSA-N 2-(3-aminopropyl)-7h-purin-6-amine Chemical compound NCCCC1=NC(N)=C2NC=NC2=N1 FDZGOVDEFRJXFT-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- FZZMTSNZRBFGGU-UHFFFAOYSA-N 2-chloro-7-fluoroquinazolin-4-amine Chemical group FC1=CC=C2C(N)=NC(Cl)=NC2=C1 FZZMTSNZRBFGGU-UHFFFAOYSA-N 0.000 description 1
- NEAQRZUHTPSBBM-UHFFFAOYSA-N 2-hydroxy-3,3-dimethyl-7-nitro-4h-isoquinolin-1-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)N(O)C(C)(C)CC2=C1 NEAQRZUHTPSBBM-UHFFFAOYSA-N 0.000 description 1
- RTXITYKPQXMDII-UHFFFAOYSA-N 3-(5,5,8,8-tetramethyl-3-propoxy-6,7-dihydronaphthalen-2-yl)but-2-enenitrile Chemical compound CC1(C)CCC(C)(C)C2=C1C=C(C(C)=CC#N)C(OCCC)=C2 RTXITYKPQXMDII-UHFFFAOYSA-N 0.000 description 1
- IQIDRTFMVGFIEL-UHFFFAOYSA-N 4-[1-(3,5,5,8,8-pentamethyl-6,7-dihydronaphthalen-2-yl)cyclopropyl]benzoic acid Chemical compound CC1=CC(C(CCC2(C)C)(C)C)=C2C=C1C1(C=2C=CC(=CC=2)C(O)=O)CC1 IQIDRTFMVGFIEL-UHFFFAOYSA-N 0.000 description 1
- NCEQLLNVRRTCKJ-UHFFFAOYSA-N 4-[2-[5,5-dimethyl-8-(4-methylphenyl)-6h-naphthalen-2-yl]ethynyl]benzoic acid Chemical compound C1=CC(C)=CC=C1C1=CCC(C)(C)C2=CC=C(C#CC=3C=CC(=CC=3)C(O)=O)C=C12 NCEQLLNVRRTCKJ-UHFFFAOYSA-N 0.000 description 1
- OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-UHFFFAOYSA-N 0.000 description 1
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 1
- ZLAQATDNGLKIEV-UHFFFAOYSA-N 5-methyl-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CC1=CNC(=S)NC1=O ZLAQATDNGLKIEV-UHFFFAOYSA-N 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- 108091027075 5S-rRNA precursor Proteins 0.000 description 1
- KXBCLNRMQPRVTP-UHFFFAOYSA-N 6-amino-1,5-dihydroimidazo[4,5-c]pyridin-4-one Chemical compound O=C1NC(N)=CC2=C1N=CN2 KXBCLNRMQPRVTP-UHFFFAOYSA-N 0.000 description 1
- DCPSTSVLRXOYGS-UHFFFAOYSA-N 6-amino-1h-pyrimidine-2-thione Chemical compound NC1=CC=NC(S)=N1 DCPSTSVLRXOYGS-UHFFFAOYSA-N 0.000 description 1
- LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 description 1
- NALREUIWICQLPS-UHFFFAOYSA-N 7-imino-n,n-dimethylphenothiazin-3-amine;hydrochloride Chemical compound [Cl-].C1=C(N)C=C2SC3=CC(=[N+](C)C)C=CC3=NC2=C1 NALREUIWICQLPS-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical group [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- HRYKDUPGBWLLHO-UHFFFAOYSA-N 8-azaadenine Chemical compound NC1=NC=NC2=NNN=C12 HRYKDUPGBWLLHO-UHFFFAOYSA-N 0.000 description 1
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 1
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 102000055025 Adenosine deaminases Human genes 0.000 description 1
- 108090000644 Angiozyme Proteins 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 102000005427 Asialoglycoprotein Receptor Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 1
- 101100297347 Caenorhabditis elegans pgl-3 gene Proteins 0.000 description 1
- 101100258233 Caenorhabditis elegans sun-1 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 108010006303 Carboxypeptidases Proteins 0.000 description 1
- 102000005367 Carboxypeptidases Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 1
- KZBUYRJDOAKODT-UHFFFAOYSA-N Chlorine Chemical compound ClCl KZBUYRJDOAKODT-UHFFFAOYSA-N 0.000 description 1
- 101800001982 Cholecystokinin Proteins 0.000 description 1
- 102100025841 Cholecystokinin Human genes 0.000 description 1
- 239000004380 Cholic acid Substances 0.000 description 1
- 108010038061 Chymotrypsinogen Proteins 0.000 description 1
- 108010003422 Circulating Thymic Factor Proteins 0.000 description 1
- 102100022641 Coagulation factor IX Human genes 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 108010042126 Creatine kinase Proteins 0.000 description 1
- BQOHYSXSASDCEA-KEOHHSTQSA-N Cyclic ADP-Ribose Chemical compound C([C@@H]1[C@H]([C@H]([C@@H](O1)N1C=2N=CN3C(C=2N=C1)=N)O)O)OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H]3O1 BQOHYSXSASDCEA-KEOHHSTQSA-N 0.000 description 1
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 1
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 102000001039 Dystrophin Human genes 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 108010074604 Epoetin Alfa Proteins 0.000 description 1
- 206010015108 Epstein-Barr virus infection Diseases 0.000 description 1
- 108010076282 Factor IX Proteins 0.000 description 1
- 108010054218 Factor VIII Proteins 0.000 description 1
- 102000001690 Factor VIII Human genes 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- 102100028652 Gamma-enolase Human genes 0.000 description 1
- 101710191797 Gamma-enolase Proteins 0.000 description 1
- 102400000921 Gastrin Human genes 0.000 description 1
- 108010052343 Gastrins Proteins 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- 241000701109 Human adenovirus 2 Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- LCWXJXMHJVIJFK-UHFFFAOYSA-N Hydroxylysine Natural products NCC(O)CC(N)CC(O)=O LCWXJXMHJVIJFK-UHFFFAOYSA-N 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 1
- 102000012745 Immunoglobulin Subunits Human genes 0.000 description 1
- 108010079585 Immunoglobulin Subunits Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 102100020880 Kit ligand Human genes 0.000 description 1
- SNDPXSYFESPGGJ-BYPYZUCNSA-N L-2-aminopentanoic acid Chemical compound CCC[C@H](N)C(O)=O SNDPXSYFESPGGJ-BYPYZUCNSA-N 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- SNDPXSYFESPGGJ-UHFFFAOYSA-N L-norVal-OH Natural products CCCC(N)C(O)=O SNDPXSYFESPGGJ-UHFFFAOYSA-N 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
- 108010007622 LDL Lipoproteins Proteins 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
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 101150076359 Mhc gene Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101100335081 Mus musculus Flt3 gene Proteins 0.000 description 1
- 101100024583 Mus musculus Mtf1 gene Proteins 0.000 description 1
- 210000004460 N cell Anatomy 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 1
- ABDMQSFNJPYOLA-UHFFFAOYSA-N NS(=O)(=O)[N+]([O-])=O Chemical compound NS(=O)(=O)[N+]([O-])=O ABDMQSFNJPYOLA-UHFFFAOYSA-N 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108020005497 Nuclear hormone receptor Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- BZQFBWGGLXLEPQ-UHFFFAOYSA-N O-phosphoryl-L-serine Natural products OC(=O)C(N)COP(O)(O)=O BZQFBWGGLXLEPQ-UHFFFAOYSA-N 0.000 description 1
- REYJJPSVUYRZGE-UHFFFAOYSA-N Octadecylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 206010033661 Pancytopenia Diseases 0.000 description 1
- 206010034016 Paronychia Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 241001505332 Polyomavirus sp. Species 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 208000007660 Residual Neoplasm Diseases 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 229930001406 Ryanodine Natural products 0.000 description 1
- 125000005631 S-sulfonamido group Chemical group 0.000 description 1
- 238000011579 SCID mouse model Methods 0.000 description 1
- 108010086019 Secretin Proteins 0.000 description 1
- 102100037505 Secretin Human genes 0.000 description 1
- 102000005157 Somatostatin Human genes 0.000 description 1
- 108010056088 Somatostatin Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 108010039445 Stem Cell Factor Proteins 0.000 description 1
- 102400000096 Substance P Human genes 0.000 description 1
- 101800003906 Substance P Proteins 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- NAVMQTYZDKMPEU-UHFFFAOYSA-N Targretin Chemical compound CC1=CC(C(CCC2(C)C)(C)C)=C2C=C1C(=C)C1=CC=C(C(O)=O)C=C1 NAVMQTYZDKMPEU-UHFFFAOYSA-N 0.000 description 1
- 108010017842 Telomerase Proteins 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
- 102000007238 Transferrin Receptors Human genes 0.000 description 1
- 108010033576 Transferrin Receptors Proteins 0.000 description 1
- 108090001027 Troponin Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 108010027252 Trypsinogen Proteins 0.000 description 1
- 102000018690 Trypsinogen Human genes 0.000 description 1
- RLXCFCYWFYXTON-JTTSDREOSA-N [(3S,8S,9S,10R,13S,14S,17R)-3-hydroxy-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-16-yl] N-hexylcarbamate Chemical group C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC(OC(=O)NCCCCCC)[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 RLXCFCYWFYXTON-JTTSDREOSA-N 0.000 description 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 1
- USDJGQLNFPZEON-UHFFFAOYSA-N [[4,6-bis(hydroxymethylamino)-1,3,5-triazin-2-yl]amino]methanol Chemical compound OCNC1=NC(NCO)=NC(NCO)=N1 USDJGQLNFPZEON-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- XVIYCJDWYLJQBG-UHFFFAOYSA-N acetic acid;adamantane Chemical compound CC(O)=O.C1C(C2)CC3CC1CC2C3 XVIYCJDWYLJQBG-UHFFFAOYSA-N 0.000 description 1
- 208000037919 acquired disease Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 description 1
- 125000005600 alkyl phosphonate group Chemical group 0.000 description 1
- 238000011316 allogeneic transplantation Methods 0.000 description 1
- SJNALLRHIVGIBI-UHFFFAOYSA-N alpha-vinylacetonitrile Natural products C=CCC#N SJNALLRHIVGIBI-UHFFFAOYSA-N 0.000 description 1
- 125000004103 aminoalkyl group Chemical group 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 102000025171 antigen binding proteins Human genes 0.000 description 1
- 108091000831 antigen binding proteins Proteins 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 108010006523 asialoglycoprotein receptor Proteins 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000037429 base substitution Effects 0.000 description 1
- 229940054066 benzamide antipsychotics Drugs 0.000 description 1
- 150000003936 benzamides Chemical class 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000010836 blood and blood product Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 210000003995 blood forming stem cell Anatomy 0.000 description 1
- 229940125691 blood product Drugs 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](C(=O)NCC(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 description 1
- 239000013000 chemical inhibitor Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 229960003677 chloroquine Drugs 0.000 description 1
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 description 1
- 229940107137 cholecystokinin Drugs 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 1
- 235000019416 cholic acid Nutrition 0.000 description 1
- 229960002471 cholic acid Drugs 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- NKKMVIVFRUYPLQ-NSCUHMNNSA-N crotononitrile Chemical compound C\C=C\C#N NKKMVIVFRUYPLQ-NSCUHMNNSA-N 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007402 cytotoxic response Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- YSMODUONRAFBET-UHFFFAOYSA-N delta-DL-hydroxylysine Natural products NCC(O)CCC(N)C(O)=O YSMODUONRAFBET-UHFFFAOYSA-N 0.000 description 1
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 229950006137 dexfosfoserine Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-N dithiophosphoric acid Chemical class OP(O)(S)=S NAGJZTKCGNOGPW-UHFFFAOYSA-N 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000007824 enzymatic assay Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical compound NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- WZBQXHJJWMLVIS-UHFFFAOYSA-N ethyl 6-[1-(3,5,5,8,8-pentamethyl-6,7-dihydronaphthalen-2-yl)ethenyl]pyridine-3-carboxylate Chemical compound N1=CC(C(=O)OCC)=CC=C1C(=C)C1=CC(C(CCC2(C)C)(C)C)=C2C=C1C WZBQXHJJWMLVIS-UHFFFAOYSA-N 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 229960004222 factor ix Drugs 0.000 description 1
- 229960000301 factor viii Drugs 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N ferric oxide Chemical compound O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000003827 glycol group Chemical group 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 125000005549 heteroarylene group Chemical group 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- QJHBJHUKURJDLG-UHFFFAOYSA-N hydroxy-L-lysine Natural products NCCCCC(NO)C(O)=O QJHBJHUKURJDLG-UHFFFAOYSA-N 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000002743 insertional mutagenesis Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000005865 ionizing radiation Effects 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- RGXCTRIQQODGIZ-UHFFFAOYSA-O isodesmosine Chemical compound OC(=O)C(N)CCCC[N+]1=CC(CCC(N)C(O)=O)=CC(CCC(N)C(O)=O)=C1CCCC(N)C(O)=O RGXCTRIQQODGIZ-UHFFFAOYSA-O 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 150000002632 lipids Chemical group 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 230000032575 lytic viral release Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 210000003593 megakaryocyte Anatomy 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- QYXAQSHRHYYLII-UHFFFAOYSA-N methyl 6-[1-(3,5,5,8,8-pentamethyl-6,7-dihydronaphthalen-2-yl)cyclopropyl]pyridine-3-carboxylate Chemical compound N1=CC(C(=O)OC)=CC=C1C1(C=2C(=CC3=C(C(CCC3(C)C)(C)C)C=2)C)CC1 QYXAQSHRHYYLII-UHFFFAOYSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- HVHMXMSNIOHNTP-UHFFFAOYSA-N n-hydroxy-3-methylbut-2-enamide Chemical compound CC(C)=CC(=O)NO HVHMXMSNIOHNTP-UHFFFAOYSA-N 0.000 description 1
- MSAHDRAAPGQPBL-UHFFFAOYSA-N n-hydroxy-3-methylbut-2-enamide;4-(3,5,5,8,8-pentamethyl-6,7-dihydronaphthalene-2-carbonyl)benzoic acid Chemical compound CC(C)=CC(O)=NO.CC1=CC(C(CCC2(C)C)(C)C)=C2C=C1C(=O)C1=CC=C(C(O)=O)C=C1 MSAHDRAAPGQPBL-UHFFFAOYSA-N 0.000 description 1
- 210000003061 neural cell Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000003448 neutrophilic effect Effects 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 102000006255 nuclear receptors Human genes 0.000 description 1
- 108020004017 nuclear receptors Proteins 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- ONTNXMBMXUNDBF-UHFFFAOYSA-N pentatriacontane-17,18,19-triol Chemical compound CCCCCCCCCCCCCCCCC(O)C(O)C(O)CCCCCCCCCCCCCCCC ONTNXMBMXUNDBF-UHFFFAOYSA-N 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 150000008298 phosphoramidates Chemical class 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- USRGIUJOYOXOQJ-GBXIJSLDSA-N phosphothreonine Chemical compound OP(=O)(O)O[C@H](C)[C@H](N)C(O)=O USRGIUJOYOXOQJ-GBXIJSLDSA-N 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 125000004289 pyrazol-3-yl group Chemical group [H]N1N=C(*)C([H])=C1[H] 0.000 description 1
- XQWBMZWDJAZPPX-UHFFFAOYSA-N pyridine-3-carbothioamide Chemical class NC(=S)C1=CC=CN=C1 XQWBMZWDJAZPPX-UHFFFAOYSA-N 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 230000014493 regulation of gene expression Effects 0.000 description 1
- 230000002629 repopulating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- JJSYXNQGLHBRRK-SFEDZAPPSA-N ryanodine Chemical compound O([C@@H]1[C@]([C@@]2([C@]3(O)[C@]45O[C@@]2(O)C[C@]([C@]4(CC[C@H](C)[C@H]5O)O)(C)[C@@]31O)C)(O)C(C)C)C(=O)C1=CC=CN1 JJSYXNQGLHBRRK-SFEDZAPPSA-N 0.000 description 1
- 229960002101 secretin Drugs 0.000 description 1
- OWMZNFCDEHGFEP-NFBCVYDUSA-N secretin human Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)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](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)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)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(N)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)C1=CC=CC=C1 OWMZNFCDEHGFEP-NFBCVYDUSA-N 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000007781 signaling event Effects 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 1
- 229960000553 somatostatin Drugs 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- IIACRCGMVDHOTQ-UHFFFAOYSA-N sulfamic acid Chemical group NS(O)(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-N 0.000 description 1
- 125000005420 sulfonamido group Chemical group S(=O)(=O)(N*)* 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 150000003457 sulfones Chemical group 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000003319 supportive effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- ZEMGGZBWXRYJHK-UHFFFAOYSA-N thiouracil Chemical compound O=C1C=CNC(=S)N1 ZEMGGZBWXRYJHK-UHFFFAOYSA-N 0.000 description 1
- 230000002992 thymic effect Effects 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
- 210000001541 thymus gland Anatomy 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- ZMANZCXQSJIPKH-UHFFFAOYSA-O triethylammonium ion Chemical compound CC[NH+](CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-O 0.000 description 1
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 1
- 101150016042 udp gene Proteins 0.000 description 1
- 125000002948 undecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
Description
EX-VIVO EXPANSION OF HEMATOPOIETIC STEM CELL POPULATIONS IN
MONONUCLEAR CELL CULTURES i FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to methods of ex-vivo expansion (self-renewal) of hematopoietic stem cells present in the hematopoietic mononuclear cells fraction of a blood sample and to expanded (self-renewed) populations of hematopoietic stem cells obtained thereby. The present invention further relates to therapeutic applications in which these methods and/or the expanded hematopoietic stem cell populations obtained thereby are utilized.
An increasing need for ex-vivo cultures of hematopoietic stem cells has arisen, in particular for purposes such as stem cell expansion and retroviral-mediated gene transduction. Methods for generating ex-vivo cultures of stem cells, however, typically result in a rapid decline in stem cell population activity, further resulting in a markedly impaired self-renewal potential and diminished transplantability of the cultured cell populations. The need to improve such methods is widely acknowledged. Additionally, applications in gene therapy using rctroviral vectors necessitate the use of proliferating hematopoietic stem cells, yet require that these cells remain undifferentiated while in culture, in order to maintain long-term expression of the transduced gene. Thus, the ability to maintain ex-vivo cultures of hematopoictic stem cell populations with long-term, self-renewal capacity 1s of critical importance for a wide array of medical therapeutic applications.
Presently, expansion of renewable stem cells have been achieved either by growing the stem cells over a feeder layer of fibroblast cells, or by growing the cells in the presence of the early acting cytokines thrombopoietin (TPO), interleukin-6 (IL- 6), an FLT-3 ligand and stem cell factor (SCF) (Madlambayan GJ et al. (2001) J
Hematother Stem Cell Res 10: 481, Punzel M et al. (1999) Leukemia 13: 92, and
Lange W et al. (1996) Leukemia 10: 943). While expanding stem cells over a feeder ¢ layer results in vast, substantially endless cell expansion, expanding stem cells without a feeder layer, in the presence of the early acting cytokines listed above, g results in an elevated degree of differentiation (see controls described in the Examples section and Leslie NR et al. (Blood (1998) 92: 4798), Petzer AL et al. (1996) J Exp
Med Jun 183: 2551, Kawa Y et al. (2000) Pigment Cell Res 8: 73).
Hence, self-renewal (expansion) of hemopoietic stem and progenitor cells, both in vivo and in vitro, is limited by cell differentiation. Differentiation in the hematopoietic system involves, among other changes, altered expression of surface antigens (Sieff C, Bicknell D, Caine G, Robinson J, Lam G, Greaves MF (1982) ! 5 Changes in cell surface antigen expression during hematopoietic differentiation.
Blood 60:703). In normal human, most of the hematopoictic pluripotent stem cells and the lineage committed progenitor cells are CD34+. The majority of cells are
CD34+CD38+, with a minority of cells (< 10 %) being CD34+CD38-. The
CD34+CD38- phenotype appears to identify the most immature hematopoietic cells, which are capable of self-renewal and multilineage differentiation. The
CD34+CD38- cell fraction contains more long-term culture initiating cells (LTC-IC) pre-CFU and exhibits longer maintenance of their phenotype and delayed proliferative response to cytokines as compared with CD34+CD38+ cells. CD34+CD38- cells can give rise to lymphoid and myeloid cells in vitro and have an enhanced capacity to repopulate SCID mice (Bhatia M, Wang JCY, Kapp U, Bonnet D, Dick JE (1997)
Purification of primitive human hematopoietic cells capable of repopulating immune- deficient mice. Proc Natl Acad Sci USA 94:5320). Moreover, in patients who received autologous blood cell transplantation, the number of CD34+CD38- cells infused correlates positively with the speed of hematopoietic recovery. In line with these functional features, CD34+CD38- cells have been shown to have detectable levels of telomerase.
The presently published works on ex-vivo expansion of hematopoietic stem and progenitor cells involve starting inoculums of cells, which are highly enriched with progenitor cells that express CD34 or, the even earlier, AC133 antigens [Dexter, T.M., T.D. Allen, and L.G. Lajtha, Conditions controlling the proliferation of haemopoietic stem cells in vitro. J.Cell Physiol, 1977. 91(3): p. 335-44; Muench,
M.O., J.G. Schneider, and M.A. Moore, Interactions among colony-stimulating ) factors, IL-1 beta, IL-6, and kit-ligand in the regulation of primitive murine hematopoietic cells. Exp. Hematol., 1992. 20(3): p. 339-49; Verfaillie, C.M., Direct “ 30 contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long-term in vitro hematopoiesis. Blood, 1992. 79(11): p. 2821-26;
Migliaccio, G., A.R. Migliaccio, M.L. Druzin, P.J. Giardina, K.M. Zsebo, and JW,
Adamson, Long-term generation of colony-forming cells in liquid culture of CD34+ cord blood cells in the presence of recombinant human stem cell factor. Blood, 1992. 79(10): p. 2620-27; Purdy, M.H., C.J. Hogan, L. Hami, 1. McNiece, W. Franklin, R.B. - Jones, S.I. Bearman, R.J. Berenson, P.J. Cagnoni, and S. Heimfeld, Large volume ex- vivo expansion of CD34-positive hematopoietic progenitor cells for transplantation. J. * 5 Hematother., 1995. 4(6): p. 515-25; McNiece, I., R. Andrews, M. Stewart, S. Clark,
T. Boone, and P. Quesenberry, Action of interleukin-3, G-CSF, and GM-CSF on highly enriched human hematopoictic progenitor cells: synergistic interaction of GM-
CSF plus G-CSF. Blood, 1989. 74(1): p. 110-14; Colter, M., M. Jones, and S.
Heimfeld, CD34+ progenitor cell selection: clinical transplantation, tumor cell purging, gene therapy, ex-vivo expansion, and cord blood processing. J Hematother, 1996. 5(2): p. 179-84; Kohler, T., R. Plettig, W. Wetzstein, B. Schaffer, R. Ordemann,
H.O. Nagels, G. Ehninger, and M. Bornhauser, Defining optimum conditions for the ex-vivo expansion of human umbilical cord blood cells. Influences of progenitor enrichment, interference with feeder layers, early-acting cytokines and agitation of culture vessels. Stem Cells, 1999. 17(1): p. 19-24].
As it was shown that initiation of ex-vivo cultures with the entire mononuclear cells (MNC) fraction in the presence of cytokines led to expansion of CFUc during the first weeks of culturing, followed by a rapid deterioration of the cultures, 1t has been widely accepted heretofore that purification of CD34+ (or AC 133) cells is a prerequisite for achieving successful ex-vivo expansion of hematopoietic stem cells [Briddell, R., Keren, B.P., Zilm, K.L., et al. Purification of CD34+ cell is essential for optimal ex-vivo expansion of umbilical cord blood cells. J. Hematother. 6:145, 1997;
Ian K McNiece, Gregory B. Stoney, Brent P. Keren, and Robert A. Briddell CD34+ cell selection from frozen cord blood products using Isolex 300i and cliniMACS™ selection device. Journal of hematotherapy 7:457-461 (1998)] as well as long-term culture colony forming cells (LTC-CFUc).
WO 99/40783, WO 00/18885 and Peled et al, Brit. J. Haematol. 116:655 2002, - all of which are incorporated by reference as if fully set forth herein, teach the effect of free copper present in cells on the modulation of the balance between self-renewal " 30 and differentiation of hematopoietic progenitor cells. These references teach that the addition of agents that are capable of reducing the cell copper content, along with early acting cytokines, to CD34+ cell cultures results in long term CD34+ cell expansion ex-vivo in culture. According to teachings of these references, such agents preferably include transition metal chelators that are capable of binding copper, such as, for example, linear polyamines (e.g., tetracthylenepentamine, TEPA). Hence, it 1s - shown in these references that the addition of 5-10 uM TEPA to CD34+ cell cultures in the presence of early acting cytokines reduced cell copper content by 30 % (as ’ 5 measured by atomic absorption), and extended the duration of the long-term cultures in terms of long-term CFU and CD34+ cell expansion.
However, the methods disclosed in these references also involve purification of stem or progenitor cells prior to their expansion in cultures.
Thus, using present day technology, stem cells cannot be expanded unless first substantially enriched or isolated to homogeneity and therefore the presently known methods of ex-vivo expanding stem cell populations are limited by the laborious and costly process of stem cells enrichment prior to initiation of cultures.
There is thus a widely recognized need for, and it would be highly advantageous to have, methods of ex-vivo expanding hematopoictic stem cells without prior stem cells enrichment.
The present invention discloses the use of various agents in expanding hematopoietic stem cells present in the hematopoietic mononuclear cells fraction of a blood sample, without the use of a prior stem cells enrichment procedure, to expanded (self-rencwed) populations of hematopoietic stem cells obtained thereby and to their uses.
According to one aspect of the present invention there is provided a method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
In one embodiment, the method comprises providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells . and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing an - 30 expression and/or activity of CD38, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
bY
In another embodiment the method comprises providing the hematopoietic mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at . the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, thereby expanding the * 5 population of the hematopoictic stem cells while at the same time, substantially inhibiting differentiation of the stem cells ex-vivo.
In still another embodiment the method comprises providing the hematopoietic mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor, retinoid-X receptor and/or Vitamin D receptor, thereby expanding the population of the hematopoietic stem cells while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
In yet another embodiment the method comprises providing the hematopoietic mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving PI 3-kinase, thereby expanding the population of the hematopoietic stem cells while at the same time, substantially . inhibiting differentiation of the hematopoietic stem cells ex-vivo.
In still another embodiment, the method comprises providing the hematopoietic mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, with nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, thereby expanding the population of the hematopoietic stem cells while at the same time, substantially inhibiting differentiation of the hematopoictic stem cells ex-vivo.
In yet another embodiment, the method comprises providing the hematopoietic . mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, with a PI 3-kinase inhibitor, thereby expanding the population of the - 30 hematopoietic stem cells while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
In still another embodiment, the method comprises providing the hematopoietic mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, with one or more copper chelator(s) or copper chelate(s), thereby expanding the population of the hematopoietic stem cells while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo. ' 5 Further according to the present invention, there are provided ex-vivo expanded populations of hematopoietic stem cells, obtained by the methods described hereinabove.
According to another aspect of the present invention there is provided a method of hematopoietic cells transplantation or implantation.
In one embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing an expression and/or activity of
CD38, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex- vivo; and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
In another embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (¢) transplanting or implanting the hematopoietic stem cells to a recipient.
In yet another embodiment, the method comprises (a) obtaining hematopoietic . mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) ‘ 30 providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to responding to signaling pathways involving the retinoic acid receptor, the retinoid X receptor and/or the Vitamin D receptor, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) . transplanting or implanting the hematopoictic stem cells to a recipient.
In still another embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to responding to signaling pathways involving PI-3 kinase, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
In yet another embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo, and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
In still yet another embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with a Pl 3-kinase inhibitor, thereby expanding a . population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) transplanting . 30 or implanting the hematopoietic stem cells to a recipient.
In yet another embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b)
providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing the expression and/or activity of PI 3- . kinase, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoictic stem cells ex- : S vivo; and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
In still yet another embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with one or more copper chelator(s) or chelatc(s), thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo;, and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
The donor and the recipient in the methods above can be a single individual or different individuals, for example, allogeneic or xenogeneic individuals.
According to still another aspect of the present invention there are provided transplantable hematopoietic cell preparations.
In one embodiment, a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent for reducing an expression and/or activity of CD38, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
In another embodiment, a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells , propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of a 30 hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent for reducing an expression and/or activity of PI 3-kinase, while at the same time, substantially inhibiting differentiation of said hematopoictic stem cells, and a pharmaceutically acceptable carrier.
In still another embodiment, a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells } propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of : 5 hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent, the agent reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
In yet another embodiment, a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an 1S agent, the agent reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid receptor, retinoid X receptor and/or Vitamin D receptor signaling, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
In still another embodiment, a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoictic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent, the agent reducing a capacity of the hematopoietic mononuclear cells in responding to PI 3-kinase signaling, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier. - In yet another embodiment, a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells - 30 propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent selected from the group consisting of nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative and a nicotinamide or a nicotinamide analog metabolite, while at the same time, substantially inhibiting . differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier. : 5 In still another embodiment, a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of a 10 PI 3-kinase inhibitor, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
In yet another embodiment, a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to 1S expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of one : or more copper chelator(s) or copper chelate(s), while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically : acceptable carrier.
According to an additional aspect of the present invention there is provided a method of adoptive immunotherapy.
In one embodiment the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) 25s providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing an expression and/or activity of
CD38, thereby expanding a population of the hematopoietic stem cells, while at the . same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient. g 30 In another embodiment the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cel proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, thereby . expanding a population of the stem cells, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting : 5 differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient.
In still another embodiment the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor and/or the retinoid X receptor and/or the Vitamin D receptor, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient.
In yet another embodiment the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell ~~ * proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving PI 3-kinase, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient.
In still another embodiment the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells } and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell i 30 proliferation and with nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient. } In yet another embodiment the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells : 5 and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with a PI 3-kinase inhibitor, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c¢) transplanting said hematopoietic stem cells to the recipient.
In still another embodiment the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with one or more copper chelator(s) or chelate(s), thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient.
Further according to an aspect of the present invention, there is provided a method of genetically modifying stem cells with an exogene.
In one embodiment, the method comprises (a) obtaining hcmatopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing an expression and/or activity of CD38, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (¢) . genetically modifying said hematopoietic stem cells with the exogene.
In another embodiment, the method comprises (a) obtaining hematopoietic : 30 mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing an expression and/or activity of Pl 3-kinase,
thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo, ) and (c) genetically modifying said hematopoietic stem cells with the exogene.
In still another embodiment, the method comprises (a) obtaining : 5 hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
In yet another embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor and/or the retinoid X receptor and/or the Vitamin D rcceptor, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
In still another embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving PI 3-kinase, thereby expanding a population of the hematopoietic stem cells, while at the same time, . 30 substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
In yet another embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation . and with nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, thereby : 5 expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
In still another embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoictic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with a Pl 3-kinase inhibitor, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
In yet another embodiment, the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the : hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with one or more copper chelator(s) or chelate(s), thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
In a preferred embodiment, genetically modifying the cells is effected by a vector which comprises the exogene, which vector is, for example, a viral vector or a nucleic acid vector.
According to still a further aspect of the present invention there is provided a ) hematopoietic stem cells collection/culturing bag supplemented with an effective amount of a retinoic acid receptor antagonist, a retinoid X receptor antagonist and/or a : 30 Vitamin D receptor antagonist, with an effective amount of nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative and a nicotinamide or a nicotinamide analog metabolite, with an effective amount of a PI 3- kinase inhibitor, or with an effective amount of a copper chelator or chelate, each of which substantially inhibits cell differentiation of a hematopoietic stem cells fraction of hematopoietic mononuclear cells which comprise a major fraction of hematopoietic : committed cells and a minor fraction of hematopoietic stem and progenitor cells.
According to an additional aspect of the present invention, there is provided an ' 5 assay of determining whether an agent/molecule is an effective hematopoietic stem cell expansion agent. The assay comprises culturing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cclls and a minor fraction of hematopoietic stem and progenitor cells in the presence of tested agent/molecule and monitoring expansion of the hematopoietic stem cells, wherein if increased expansion and decreased differentiation of the hematopoietic stem cells occurs, as compared to non-treated hematopoietic mononuclear cells, the tested agent/molecule is an effective hematopoietic stem cell expansion agent.
The agent/molecule can be a retinoic acid receptor antagonist, a retinoid X receptor antagonist, a Vitamin D receptor antagonist, nicotinamide and an analog, a derivative and a metabolite thereof, a PI 3-kinase inhibitor, a copper chelator and a copper chelate.
According to further features in preferred embodiments of the invention described below, reducing the expression and/or activity of CD38 1s effected by an : agent that downregulates CD38 expression. :
According to still further features in the descnbed preferred embodiments the agent that downregulates CD38 expression is selected from the group consisting of a retinoic acid receptor antagonist, a retinoid X receptor antagonist and a Vitamin D receptor antagonist. Alternatively, this agent is an antagonist for reducing a capacity of the stem cells in responding to retinoic acid, retinoid and/or Vitamin D. Further alternatively, the agent that downregulates CD38 expression is a PI 3-kinase inhibitor.
According to still further features in the described preferred embodiments the agent that downregulates CD38 expression is a polynucleotide. ) According to still further fcatures in the described preferred embodiments the polynucleotide encodes an anti CD38, an anti retinoic acid receptor, an anti retinoid X receptor, an anti Vitamin D receptor or an anti PI 3-kinase antibody or intracellular antibody.
According to still further features in the described preferred embodiments the polynucleotide is a small interfering polynucleotide molecule directed to cause intracellular CD38, retinoic acid receptor, retinoid X receptor, Vitamin D receptor or
PI 3-kinase mRNA degradation. . According to still further features in the described preferred embodiments the small interfering polynucleotide molecule is selected from the group consisting of an
RNAI molecule, an anti-sense molecule, a rybozyme molecule and a DNAzyme molecule.
According to further features in preferred embodiments of the invention described below, reducing the expression and/or activity of CD38 is effected by an agent that inhibits CD38 activity. The agent can be, for example, nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite. The nicotinamide analog 1s preferably selected from the group consisting of benzamide, nicotinethioamide, nicotinic acid and a-amino-3-indolepropionic acid.
According to further features in preferred embodiments of the invention described below, reducing the expression and/or activity of CD38 is effected by an agent that inhibits PI 3-kinase activity.
According to further features in preferred embodiments of the invention described below, providing the stem cells with the conditions for ex-vivo cell proliferation comprises providing the cells with nutrients and with cytokines.
According to still further features in the described preferred embodiments the cytokines are early acting cytokines, such as, but not limited to, stem cell factor, FLT3 ligand, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-10, interleukin-12, tumor necrosis factor-a and thrombopoietin.
According to still further features in the described preferred embodiments the cytokines are late acting cytokines, such as, but not limited to, granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor, erythropoietin,
FGF, EGF, NGF, VEGF, LIF, Hepatocyte growth factor and macrophage colony . stimulating factor.
According to still further features in the described preferred embodiments the hematopoietic mononuclear cells are derived from a source selected from the group consisting of bone marrow, peripheral blood and neonatal umbilical cord blood.
According to still further features in the described preferred embodiments reducing the capacity of the hematopoietic mononuclear cells in responding to . signaling pathways is reversible, e.g., inherently reversible.
According to still further features in the described preferred embodiments ’ 5 reducing the capacity of the hematopoietic mononuclear cells in responding to the above antagonists and/or signaling pathways of the above receptors is by ex-vivo culturing the hematopoietic mononuclear cells in a presence of an effective amount of at least one retinoic acid receptor antagonist, at least one retinoid X receptor antagonist and/or at lcast one Vitamin D receptor antagonist, preferably, for a time period of 0.1-50 %, preferably, 0.1-25 %, more preferably, 0.1-15 %, of an entire ex- vivo culturing period of the hematopoietic mononuclear cells.
According to still further features in the described preferred embodiments, the retinoic acid receptor antagonist is selected from the group consisting of:
AGN 194310; AGN 193109; 3—(4-Methoxy-phenylsulfanyl)-3-methyl-butyric acid; 6-Methoxy-2,2-dimethvl-thiochroman-4-one,2,2-Dimethyl-4-oxo-thiochroman-6- yltrifluoromethane-sulfonate; Ethyl 4-((2,2 dimethyl-4-oxo-thiochroman-6- yl)ethynyl)-benzoate; Ethyl 4-((2,2-dimethy 1-4-triflouromethanensulfonyloxy -(2H)- thiochromen-6-yl)ethynyl)-benzoate(41); Thiochromen-6-yl]-ethynyl]-benzoate(yl); (p-[(E)-2-[3'4'-Dihydro-4,4'-dimethyl-7'-(heptyloxy)-2'H-1-benzothiopyran-6'yl] propenyl] benzoic acid 1'I'-dioxide; 2E,4E,6E-[7-(3,5-Di-t-butyl-4-n-butoxyphenyl)- : 3-methyl]-octa-2,4,6-trienoic acid; 2E,4E,6E-[7-(3,5-Di-t-butyl-4-n-propoxyphenyl)- 3-methyl]-octa-2,4,6-trienoic acid; 2E,4E,6E-[7-(3,5-Di-t-butyl-4-n-pentoxyphenyl)- 3-methyl]-octa-2,4,6-trienoic acid; 2E,4E,6E-[7-(3,5-Di-t-butyl-4-n-hexoxyphenyl)-3- methyl}-octa-2,4,6-trienoic acid; 2E,4E,6E-[7-(3,5-Di-t-butyl-4-n-heptoxyphenyl)-3- methyl]-octa-2,4,6-trienoic acid; 2E,4E,6E-[7-(3,5-Di-t-butyl-4-n-octoxyphenyl)-3- methyl]-octa-2,4,6-trienoic acid; (2E,4E,6E)-7-[3-t-butyl-5-(1-phenyl-vinyl)-phenyl]- 3-methyl-octa-2,4,6-trienoic acid; 2E,4E ,6E-[7-(3,5-Di-t-butyl-4-{[4,5-.sup.3
H.sub.2]-n-pentoxy } phenyl)-3-methyl}-octa-2,4,6-trienoic acid; (2E,4E)-(1RS,2RS)- 5-[2-(3,5-di-tert.butyl-2-ethoxy-phenyl)-cyclopropyl]-3-methyl-penta-2,4-dienoic acid ethyl ester; (2E,4E)-(1RS,2RS)-5-[2-(3,5-di-tert.butyl-2-ethoxy-phenyl)-cyclopropy!]- 3-methyl-penta-2,4-dienoic acid, (2E.4E)-(IRS 2RS)-5-[2-(3,5-di-tert.butyl-2- butoxy-phenyl)-cyclopropyl]-3-methyl-penta-2,4-dienoic acid; (2E,4E,6Z)-7-[3,5-di- tert.butyl-2-ethoxyphenyl]3-methyl-2,4,6-octatrienoic acid; (2E,4E,6Z)-7-[3,5-di-
tert.butyl-2-butyloxyphenyl]-3-methyl-2 4,6-octatrienoic acid; 4-(5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-2-naphthalene-carboxamido) benzoic acid; (2E,4E)-3-methyl-5- . [(1S,2S)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-cyclopropyl]- penta-2 4-dienoic acid; p-[(E)-2-[3',4'-Dihydro-4'4'-dimethyl-7-(heptyloxy)-2'H-1- : 5 benzothiopyran-6'-yl]propenyl]benzoic acid; 1',1'-dioxide, 4-(7,7,10,10-Tetramethyl- 1-pyridin-3-ylmethyl-4,5,7,8,9,10-hexahydro- | H-naphto[2,3-g]indol-3-yl)-benzoic acid; (2E4E,6Z)-7-[3,5-di-tert.butyl-2-methoxyphenyl]-3-methyl-2,4,6-octatrienoic acid; (2E,4E,6Z)-7-[3,5-di-tert.butyl-2-ethoxyphenyl]-3-methyl-2 ,4,6-octatrienoic acid; (2E,4E,62)-7-[3,5-di-tert.butyl-2-hexyloxyphenyl]-3-methyl-2,4,6-octatrienoic acid; (2E,4E,6Z)-7-[3,5-di-tert.butyl-2-octyloxyphenyl}-3-methyl-2,4,6-octatrienoic acid; and (2E,4E)-(1RS,2RS)-5-[2-(3,5-di-tert-butyl-2-butoxy-phenyl)-cyclopropyl}- 3-methyl-penta-2,4-diencic acid ~~ (2E,4E,6Z)-7-(3-n-propoxy-5,6,7,8-tetrahydro- 5,5,8,8-tetramethylnaphthalene-2-yl)-3-methylocta-2,4,6-trienoic acid, 4-(5H-2,3(2,5 dimethyl-2,5-hexano)-5-n-propyldibenzo[b,e]{1,4]diazepin-11-yl)benzoic acid, 4- (SH-2,3-(2,5-dimethyl-2,5-hexano)-5Smethyi-8-nitrodibenzo[b,e][ 1 ,4]diazepin-11- yl)benzoic acid, 4-{[4-(4-Ethylphenyl)2,2-dimethyl-(2H)-thiochromen-6- yllethynyl}benzoic acid, 4-[4-2methyl-1,2-dicarba-closo-dodecaboran-1-yl- phenylcarbamoyl]benzoic acid, 4-[4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl-1-(3- pyridylmethyl)-anthraf 1,2-b]pyrrol-3-yl]benzoic acid, (3-pyridylmethyl)-]5- thiaanthra[2,1-b]pyrrol-3-yl)benzoic acid, and (3-pyridylmethyl)-anthra[2ml- d]pyrazol-3-yljbenzoic acid.
According to still further features in the described preferred embodiments, the retinoid X receptor antagonist is selected from the group consisting of:
LGN100572, LGN100574, 1-(3-hydroxy-5,6,7,8-tetrahydro-5,5,8,8- tetramethylnaphthalene-2-yl)ethanone, 1-(3-propoxy-5,6,7,8-tetrahydro-5,5,8,8- tetramethylnaphthalene-2-yljethanone, 3-(3-propoxy-5,6,7,8-tetrahydro-5,5,8,8- tetramethylnaphthalene-2-yl)but-2-enenitrile, 3-(3-propoxy-5,6,7,8-tetrahydro- 5,5,8,8-tetramethylnaphthalene-2-yl)but-2-enal, (2E,4E,6E)-7-3[-propoxy-5,6,7,8- tetrahydro $5,5,8,8-tetramethyl-2-naphthalene-2-yl]-3-methylocta-2,4,6-trienoic acid, : 30 4-[3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl] benzoic acid, 4-[1- (3,5, S,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl] benzoic acid, 4- [1(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)cyclopropyl] benzoic acid, 4- [1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl] benzenete trazole,
2-[1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl]pyridine-S- carboxylic acid, 2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2- naphthyl)ethyl]pyridine-5-carboxylic acid, ethyl-2-[1-(3,5,5,8, 8-pentamethyl-5,6,7,8- tetrahydro-2-naphthyl)ethenyl]pyridine-5-carboxylate, ~~ 5-[1-3,5,5,8,8-pentamethyl- . 5 5,6,7,8-tetrahydro-2-naphthyl)ethenyl]pyridine-2-carboxylic acid, 2-[1-(3,5,5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) cyclopropyl]pyridine-5-carboxylic acid, methyl 2-[1-(3,5,5,8,8-pentamethyi-5,6,7,8-tetrahydro-2- naphthyl)cyclopropyl]pyridine-5-carboxylate, 4-[1-(3,5, 5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthyl)ethenyl)-N-(4-hydroxyphenyl) benzamide, 2-[1-(3,5,5,8,8-
Pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] pyridine-5-carboxylic acid, 2-[1- (3,5,5,8,8-Pentamethyl-5, 6,7,8-tetrahydro-2-naphthyl)cyclopropyl]pyridine-5- carboxylic acid, 4-[(3,5, 5,8,8-pentamethyl-5,6,7,8-tetrahydro-2- naphthyl)carbonyllbenzoic acid butyloxime, 4-[(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthyl) ~~ carbonyl]benzoic acid propyloxime, 4-[(3,5,5,8,8- pentamethyl-5,6,7,8-terrahydro-2-naphthyl)carbonyllbenzoic acid cyanoimine, 4- [(3,5,5,8,8-pentamethyi-5,6,7,8-tetrahydro-2-naphthyl)carbonyl benzoic acid allyloxime, 4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic acid 4-(3-methylbut-2-enoic acid)oxime, and 4-[(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthyl)carbonyl]benzoic acid 1-aminoethyloxime (2E,4E,6Z)-7-(3-n- propoxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalene-2-yl)-3-methylocta-2,4,6- trienoic acid, and 4-(5SH-2,3(2,5 dimethyl-2,5-hexano)-5-n- propyldibenzo[b,e][1,4]diazepin-11-yl)benzoic acid, and 4-(5H-2,3-(2,5-dimethyl- 2,5-hexano)-5Smethyl-8-nitrodibenzo[b,e]{ 1,4]diazepin-11-yl)benzoic acid.
According to still further features in the described preferred embodiments, the
Vitamin D receptor antagonist is selected from the group consisting of: 1 alpha, 25- (OH)-D3-26,23 lactone; lalpha, 25-dihydroxyvitamin D (3); the 25-carboxylic ester
ZK 159222; (23S)- 25-dehydro-1 alpha-OH-D (3); (23R)-25-dehydro-1 alpha-OH-D (3); 1 beta, 25 (OH), Dj; 1 beta, 25(OH);-3-epi-Dj; (23S) 25-dehydro-1 alpha(OH)
D3-26,23-lactone; (23R) 25-dehydro-1 alpha(OH)D3-26,23-lactone and Butyl- . 30 (5Z,7E,22E-(1S,7E,22E-(1S,3R,24R)-1,3,24-trihydroxy-26,27-cyclo-9,10- secocholesta-5,7,10(19),22-tetraene-25-carboxylate).
According to still further features in the described preferred embodiments, the
PI 3-kinase inhibitor is selected from the group consisting of wortmannin and
LY294002.
The copper chelate(s) or chelator(s) used in the various aspects of the present : 5 invention described hereinabove preferably comprise a polyamine chelator.
According to further features in preferred embodiments of the invention described below, the polyamine chelator is capable of forming an organometallic complex with a transition metal other than copper. The transition metal can be, for example, zinc, cobalt, nickel, iron, palladium, platinum, rhodium and ruthenium.
According to still further features in the described preferred embodiments the polyamine chelator is a linear polyamine.
Preferably, the linear polyamine has a general formula I:
HX-Am-(YB:)i--(YnBn)n-ZH
Formula I wherein m is an integer from 1 to 10; n is an integer from 0 to 20; X and Z are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; Y, and Yn are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; A is an alkylene chain having between | and 10 substituted and/or non-substituted carbon atoms; and B, and
Bn are each independently an alkylene chain having between 1 and 20 substituted and/or non-substituted carbon atoms, provided that at least one of the X, Z, Y, and Yn is a -NH group and/or at least one of the carbon atoms in the alkylene chains is substituted by an amine group.
According to still further features in the described preferred embodiments, A is an alkylene chain having a general formula II:
To Re —CH-CH + CgH~—
Formula I1 wherein g is an integer that equals 0 or 3-10; and each of R;, Ry and Rg 1s . independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroalicyclic, heteroaryl, halo, amino, alkylamino, arylamino, cycloalkylamino, heteroalicyclic amino, heteroarylamino, hydroxy, alkoxy, aryloxy, azo, C-amido, N-amido, ammonium, thiohydroxy, thioalkoxy, thioaryloxy, sulfonyl, sulfinyl, N-sulfonamide, S-sulfonamide, phosphonyl, phosphinyl, phosphonium, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, C-thiocarboxy, O-thiocarboxy, N- carbamate, O-carbamate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, borate, borane, boroaza, silyl, siloxy, silaza, aquo, alcohol, peroxo, amine oxide, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanate, thiocyanate, isocyanate, isothiocyanate, cyano, alkylnitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl! sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, carboxylic acid, alkyl carboxyhc acid, aryl carboxylic acid, sulfate, sulfite, bisulfite, thiosulfate, thiosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, guanidino, S-dithiocarbamate, N- dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraarylborate, tetraalkyl borate, tartarate, salicylate, succinate, citrate, ascorbate, saccharirate, amino acid, hydroxamic acid and thiotosylate. } According to still further features in the described preferred embodiments, each of B1 and Bn is independently an alkylene chain having a general formula II:
tr Jo) —Cp—C(p+1)H -CqH—
H
Formula III wherein p is an integer that equals 0 or g+1; q is an integer from g+2 to g+20; and each of Rp, Rp+1 and Rq is independently selected from the group consisting of the substituents described hereinabove with respect to R,, Rz and Rg.
According to still further features in the described preferred embodiments at least one of Cj, C; and Cg and/or at least one of Cp, Cp+1 and Cq is a chiral carbon atom.
A preferred linear polyamine according to the present invention is tetracthylenepentamine.
According to still further features in the described preferred embodiments the polyamine chelator is a cyclic polyamine, such as cyclam.
According to still further features in the described preferred embodiments the cyclic polyamine has a general formula IV: 1
X——Am—(Y,B)),---(YnBn)n—2Z
Formula IV wherein m is an integer from 1 to 10; n is an integer from 0 to 20; X and Z are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; Y, and Yn are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; A is an alkylene chain having between | and 10 substituted and/or non-substituted carbon atoms; B, and Bn are each independently an alkylene chain having between 1 and 20 substituted and/or non-substituted carbon atoms; and D is a bridging group having a general formula V:
U-W-V
Formula V whereas U and V are each independently selected from the group consisting of substituted hydrocarbon chain and non-substituted hydrocarbon chain; and W is selected from the group consisting of amide, ether, ester, disulfide, thioether, thioester, imine and alkene,
provided that at least one of the X, Z, Y, and Yn is a -NH group and/or at least one of the carbon atoms in the alkylene chains is substituted by an amine group.
According to still further features in the described preferred embodiments, A and each of Bl and Bn in Formula IV are alkylene chains having the general formulas
Il'and III, as is described hereinabove.
According to still further features in the described preferred embodiments the cyclic polyamine has a general formula selected from the group consisting of: a
X——Am— (YB), --(YnBn)n—2ZH
Formula VI rr
HX—Am—(Y;B)),---(YnBn)n—2
Formula VII
ER A is X——Am——(Y,B,)," -(YnBn)n—ZH
Formula VII1
RE RE a b)
SE SW Sl
Formula IX [DT
HX—Am——(Y,B,),"-- (YnBn)n—ZH
Formula X wherein m is an integer from 1 to 10; n is an integer from 0 to 20; X and Z are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; Y, and Yn are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; A is an alkylene chain having between 1 and 10 substituted and/or non-substituted carbon atoms; B1 and Bn are each independently an alkylene chain having between 1 and 20 substituted and/or non-substituted carbon atoms; and D is a bridging group having a general formula V, . as described hereinabove, and further wherein should the D is attached at one end to
A (Formulas VI, VII and X), the U or the V are being attached to one carbon atom in the alkylene chain and should the D is attached at one end to Bl or Bn (Formulas
VIII, IX and X), the U or the V are being attached to one carbon atom in the alkylene chain, provided that at least one of the X, Z, Y, and Yn is a -NH group and/or at least one of the carbon atoms in the alkylene chains is substituted by an amine group.
The alkylene chains A, Bl and Bn are preferably as described hereinabove.
According to still further features in the described preferred embodiments the polyamine chelator includes at least one linear polyamine and at least one cyclic polyamine.
Such a polyamine chelator preferably has a general formula XI:
LENQ-(G I {(E)i-[Q2- (G2) +++ {(Ea)-[Qu-(Gi)o 1}
Formula XI wherein n is an integer greater than 1; each of f, g, h, 1, J, k, |, 0 and t is independently an integer from O to 10; each of E,, E; and En is independently a linear polyamine as is described hereinabove; each of G;, G, and Gn is independently a cyclic polyamine as is described hereinabove; and each of Q;, Q, and Qn is independently a linker linking between two of the polyamines, provided that at least one of the Q), Q; and Qn is an amine group and/or at least one of the linear polyamine and the cyclic polyamine is having at least one free amine group.
According to still further features in the described preferred embodiments each of Qi, Q2 and Qn is independently selected from the group consisting alkylene, alkenylene, alkynylene, arylene, cycloalkylene, hetroarylene, amine, azo, amide, : 30 sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphonium, ketoester, carbonyl, thiocarbonyl, ester, ether, thioether, carbamate, thiocarbamate, urea, thiourea, borate, borane, boroaza, silyl, siloxy and silaza.
According to still further features in the described preferred embodiments the polyamine chelator is selected from the group consisting of ethylendiamine, diethylenetriamine, triethylenetetramine, triethylenediamine, tetraethylenepentamine, aminoethylethanolamine, aminoethylpiperazine, pentaethylenechexamine, captopril, penicilamine, N,N'-bis(3-aminopropyl)-1,3-propanediamine, N,N’-Bis-(2- anmimocthyl)-1,3-propanediamine, 1,7-dioxa-4,10-diazacyclododecane, 1,4.8,11- tetraaza cyclotetradecane-5,7-dione, 1,4,7-triazacyclononane, 1-oxa-4,7,10- triazacyclododecane, 1,4,8,12-tetraazacyclopentadecane, and 1,4,7,10- tetraazacyclododecane.
The present invention successfully addresses the shortcomings of the presently known configurations by providing methods of expanding hematopoietic stem cells without first enriching hematopoietic mononuclear cells for stem cells.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the : 30 description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
In the drawings:
FIGs. la-b illustrates the effect of TEPA chelator on the expansion of CD34% hematopoietic stem cells in a culture of hematopoietic mononuclear cells. Cord-blood mononuclear cells (MNCs) were seeded in culture-bags in the presence of cytokines,
S and were cither supplemented with TEPA chelator (MNC-TEPA), or not supplemented with TEPA chelator (MNC control). For comparison, purified CD34+ cells were similarly seeded in culture-bags in the presence of cytokines with no supplementation of TEPA chelator (CD34 culture). All cultures were incubated for 12 weeks and at weekly intervals, the CD34 cells were purified from cultures using : miniMacs columns and enumerated;
FIG. 2 illustrates the FACS-analysis of the density of CD34+CD38- cells in the untreated NMCs, TEPA-treated MNCs and CD34 cell cultures described above; and
FIG. 3 presents the comparative numbers of colony-forming cells (CFUs) measured from the untreated MNCs, TEPA-treated MNCs and CD34 cell cultures described above, at weekly intervals.
The present invention is of methods of ex-vivo expanding a population of hematopoietic stem cells present, as a minor fraction, in hematopoietic mononuclear cells, without first enriching the stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells. The present invention can be used to cfficiently provide ex-vivo expanded populations of hematopoietic stem cells, using hematopoietic mononuclear cells that comprise a major fraction of hematopoietic committed cells and a minor fraction of the hematopoietic stem and progenitor cells as a source of stem cells, without prior enrichment of the hematopoietic mononuclear cells for stem cells. The expanded populations of hematopoietic stem cells of the present invention can be used in, for example, hematopoietic cell transplantation, in generation of stem cells suitable for genetic manipulations for cellular gene therapy, as well as in additional application such as, but not limited to, adoptive immunotherapy, implantation of stem cells in an in vivo cis-differentiation and trans-differentiation settings, as well as, ex-vivo tissue engineering in cis-differentiation and trans-differentiation settings.
The methods of the present invention utilize various molecules (also referred to herein as agents), that interfere with CD38 expression and/or activity and/or with intracellular copper content, for inducing the ex-vivo expansion of hematopoietic stem cell populations described above, thereby providing an efficient, simplified and yet : 5 versatile technology for ex-vivo expansion of hematopoietic stem cells.
The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions and examples.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the Examples section. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
As 1s discussed hereinabove, WO 99/40783, WO 00/18885 and Peled et al,
Brit. J. Haematol. 116:655 2002 teach that cellular copper is involved in modulating the balance between self-renewal and differentiation of hematopoietic progenitor cells. According to the teachings of these references, the addition of transition metal chelators that arc capable of binding copper, such as, for example, the linear polyamine tetraethylencpentamine, to CD34+ cell cultures in the presence of early acting cytokines reduced cell copper content by 30 % and extended the duration of the long-term cultures in terms of long-term CFU and CD34+ cell expansion. These references hence teach methods of expanding stem cell populations, ex-vivo in the presence of transition metal chelators, copper chelators in particular, and further teach the use of the obtained expanded stem cell populations in various applications.
PCT/IL03/00062 discloses that copper chelates, namely, copper chelators that are complexed with a copper 10n, also promote proliferation and inhibit differentiation of stem and progenitor cells when added to the culture media of such cells.
According to the teachings of PCT/IL03/00062, these finding suggest that this effect i 30 of copper chelates on proliferation and differentiation of stem and progenitor cells is not associated solely with the content of cellular copper but rather with additional regulatory pathways.
PCT/IL03/00064 and U.S. Provisional Patent Application No. 60/452,545, which are incorporated by reference as if fully set forth herein, disclose that a series of } molecules that are capable of interfering with CD38 expression and/or activity, repress the process of differentiation of stem cells and stimulates and prolongs, for up : 5 to 16-18 weeks, the phase of active cell proliferation and expansion (renewal) ex-vivo, in a reversible manner, Hence, these references teach methods of expanding stem cell populations ex-vivo, which involve the addition of agents that either downregulate
CD38 expression or inhibit the activity of CD38 to the culture media of stem cells.
The methods disclosed in PCT/IL03/00064 and U.S. Provisional Patent Application
No. 60/452,545, therefore utilize molecules such as retinoic acid receptor antagonists of the RAR and RXR superfamilies, Vitamin D receptor antagonists, polynucleotides encoding antibodies such as anti CD38, anti retinoic acid receptor, anti retinoid X receptor, anti Vitamin D receptor, polynucleotides that are directed to cause degradation of endogenous polynucleotides encoding for these receptors, molecules that are capable of interfering with expression and/or activity of Pl 3-kinase and
CD38 inhibitors such as nicotinamide and its related compounds.
Hence, WO 99/40783, WO 00/18885, PCT/IL03/00064 and U.S. Provisional
Patent Application No. 60/452,545 all teach the use of various molecules that modulate, via diverse pathways and/or mechanisms, the balance between self-renewal and differentiation of stem cells, hematopoietic stem cells in particular, in methods for ex-vivo expanding of stem cell populations. However, unless otherwise indicated, the regulation of self-renewal and differentiation of stem cells by these molecules is obtained, according to the teachings of these references, when the cultured cells are first enriched for stem and/or progenitor cells and hence, in line with other present day technologies in this field, require preliminary stem cells enrichment.
While reducing the present invention to practice, it was surprisingly and unexpectedly found that molecules such as copper chelators, copper chelates and retinoic acid receptor (RAR) antagonists repress differentiation and stimulate and prolong proliferation of hematopoietic stem cells when the source of cells includes the - 30 entire fraction of mononuclear blood cells, namely non-enriched stem cells.
As is described in the Background section hereinabove, although being highly advantageous, presently there is no disclosed technology by which to expand non- enriched stem cells. Therefore, the technology presented and exemplified herein,
involving methods of ex-vivo expanding hematopoietic stem cell populations devoid of prior stem cells enrichment, provides for efficient, simplified and cost-effective methods of obtaining ex-vivo expanded hematopoietic stem cell populations. The expanded hematopoietic stem cell populations obtained by the technology presented . 5 herein can be used in various application, the following lists a few:
Hematopoietic cell transplantation: Transplantation of hematopoietic cells has become the treatment of choice for a variety of inherited or malignant diseases. While early transplantation procedures utilized the entire bone marrow (BM) population, recently, more defined populations, enriched for stem cells (CD34% cells) have been used (Van Epps DE, et al. Harvesting, characterization, and culture of CD34+ cells from human bone marrow, peripheral blood, and cord blood. Blood Cells 20:411, 1994). In addition to the marrow, such cells could be derived from other sources such as peripheral blood (PB) and neonatal umbilical cord blood (CB) (Emerson SG. Ex- vivo expansion of hematopoietic precursors, progenitors, and stem cells: The next generation of cellular therapeutics. Blood 87:3082, 1996). Compared to BM, transplantation with PB cells shortens the period of pancytopenia and reduces the risks of infection and bleeding (Brugger W, et al. Reconstitution of hematopoiesis after high-dose chematotherapy by autologous progenitor cells generated in-vivo. N Engl J
Med 333:283, 1995; Williams SF, et al. Selection and expansion of peripheral blood
CD34+ cells in autologous stem cell transplantation for breast cancer. Blood 87:1687, 1996; Zimmerman RM, et al. Large-scale selection of CD34+ peripheral blood progenitors and expansion of neutrophil precursors for clinical applications. J
Heamatotherapy 5:247, 1996).
An additional advantage of using PB for transplantation is its accessibility.
The limiting factor for PB transplantation is the low number of circulating pluripotent stem/progenitor cells.
To obtain enough PB-derived stem cells for transplantation, these cells are "harvested" by repeated leukophoresis following their mobilization from the marrow into the circulation by treatment with chemotherapy and cytokines (Brugger W, et al.
Reconstitution of hematopoiesis after high-dose chematotherapy by autologous progenitor cells generated in-vivo. N Engl J Med 333:283, 1995; Williams SF, et al.
Selection and expansion of peripheral blood CD34+ cells in autologous stem cell transplantation for breast cancer. Blood 87:1687, 1996). Such treatment is obviously not suitable for normal donors.
The use of ex-vivo expanded stem cells for transplantation has the following advantages (Koller MR, Emerson SG, Palsson BO. Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures. Blood 82:378, 1993; Lebkowski JS, et al. Rapid isolation and serum-free expansion of human CD34+ cells. Blood Cells 20:404, 1994):
It reduces the volume of blood required for reconstitution of an adult hematopoietic system and may obviate the need for mobilization and leukophoresis (Brugger W, etal. N Engl J Med 333:283, 1995).
It enables storage of small number of PB or CB stem cells for potential future use.
In the case of autologous transplantation of recipients with malignancies, contaminating tumor cells in autologous infusion often contribute to the recurrence of the disease (Brugger W, et al. N Engl J Med 333:283, 1995). Selecting and expanding CD34 stem cells will reduce the load of tumor cells in the final transplant.
The cultures provide a significant depletion of T lymphocytes, which may be useful in the allogeneic transplant setting for reducing graft-versus-host disease.
Clinical studies indicate that transplantation of ex-vivo expanded cells derived from a small number of PB CD34" cells can restore hematopoiesis in recipients treated with high doses of chemotherapy, although the results do not yet allow firm conclusions about long term in-vivo hematopoietic capabilities of these cultured cells (Brugger W, et al. N Engl J Med 333:283, 1995; Williams SF, et al. Blood 87:1687, 1996).
For successful transplantation, shortening of the duration of the cytopenic phase, as well as long-term engraftment, is crucial. Inclusion of intermediate and late progenitor cells in the transplant could accelerate the production of donor-derived : mature cells thereby shortening the cytopenic phase. It is important, therefore, that ex- vivo expanded cells include, in addition to stem cells, more differentiated progenitor cells in order to optimize short-term recovery and long-term restoration of hematopoiesis. Expansion of intermediate and late progenitor cells, especially those committed to the neutrophilic and megakaryocytic lineages, concomitant with expansion of stem cells, should serve this purpose (Sandstrom CE, et al. Effects of
CD34+ cell selection and perfusion on ex-vivo expansion of peripheral blood mononuclear cells. Blood 86:958, 1995).
Such cultures may be useful in restoring hematopoiesis in recipients with completely ablated bone marrow, as well as in providing a supportive measure for : 5 shortening recipient bone marrow recovery following conventional radio- or chemotherapies.
Prenatal diagnosis of genetic defects in scarce cells: Prenatal diagnosis involves the collection of embryonic cells from a pregnant woman, in utero, and analysis thereof for genetic defects. A preferred, non-invasive, means of collecting embryonic cells involves separation of embryonic nucleated red blood cell precursors that have infiltrated into peripheral maternal circulation. However, since the quantities of these cells are quite scarce, a further application of the present invention would be the expansion of such cells according to methods described herein, prior to analysis. The present invention, therefore, offers a means to expand embryonic cells for applications in prenatal diagnosis.
Gene therapy: For successful long-term gene therapy, a high frequency of genetically modified stem cells with transgenes stably integrated within their genome, is an obligatory requirement. In BM tissue, while the majority of cells are cycling progenitors and precursors, stem cells constitute only a small fraction of the cell population and most of them are in a quiescent, non-cycling state. Viral-bascd (e.g. retroviral) vectors require active cell division for integration of the transgene into the host genome. Therefore, gene transfer into fresh BM stem cells is highly inefficient.
The ability to expand a purified population of stem cells and to regulate their cell division ex-vivo would provide for an increased probability of their genetic modification (Palmiter RD. Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1. Proc Natl Acad Sci USA 91(4): 1219-1223, 1994).
Adoptive immunotherapy: Ex-vivo-expanded, defined lymphoid : 30 subpopulations have been studied and used for adoptive immunotherapy of various _ malignancies, immunodeficiencies, viral and genetic diseases (Freedman AR, et al.
Generation of T lymphocytes from bone marrow CD34+ cells in-vitro. Nature
Medicine 2: 46, 1996; Heslop HE, et al. Long term restoration of immunity against
Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nature Medicine 2: 551, 1996; Protti MP, et al. Particulate naturally processed peptides prime a cytotoxic response against human melanoma in-vitro.
Cancer Res 56: 1210, 1996).
The treatment enhances the required immune response or replaces deficient functions. This approach was pioneered clinically by Rosenberg ef al. (Rosenberg SA, et al. Prospective randomized trial of high-dose interleukin-2 alone or in conjunction with lymphokine-activated killer cells for the treatment of patients with advanced cancer. J Natl Cancer Inst 85: 622, 1993) using a large number of autologous ex-vivo expanded non-specific killer T cells, and subscquently ex-vivo expanded specific tumor infiltrating lymphocytes.
Functionally active, antigen-presenting cells could be grown from a starting population of CD34" PB cells in cytokine-supported cultures, as well. These cells can present soluble protein antigens to autologous T cells in-vitro and, thus, offer new prospects for the immunotherapy of minimal residual disease after high dose chemotherapy. Ex-vivo expansion of antigen-presenting dendritic cells has been studied as well, and is an additional promising application of the currently proposed technology (Bernhard H, et al. Generation of immunostimulatory dendritic cells from human CD34+ hematopoietic progenitor cells of the bone marrow and peripheral blood. Cancer Res 10:99, 1995; Fisch P, et al. Generation of antigen-presenting cells for soluble protein antigens ex-vivo from peripheral blood CD34+ hematopoietic progenitor cells in cancer patients. Eur J Immunol 26: 595, 1996; Siena S, et al.
Massive ex-vivo generation of functional dendritic cells from mobilized CD34+ blood progenitors for anticancer therapy. Expt Hematol 23:1463, 1996).
As 1s discussed in brief hereinabove and is further detailed in WO 99/40783,
WO 00/18885, PCT/IL03/00064 and U.S. Provisional Patent Application No. 60/452,545, copper chelators, copper chelates and retinoid receptor antagonists, each . modulate the self-renewal of stem cells via a different pathway, effecting different cellular events that lead to reduced differentiation and extended proliferation of stem cells. These molecules therefore represent a wide variety of molecules that are capable of inducing the effect of expanding a hematopoietic stem cells population that is present in a mixed hematopoietic cells population.
Hence, according to one aspect of the present invention there is provided a method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the stem cells ex-vivo. The method according to this aspect of the present invention is effected by providing : 5 hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing an expression and/or activity of CD38, thereby expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
As used herein, the phrase “hematopoictic mononuclear cells” refers to the entire repetoir of white blood cells present in a blood sample. In a healthy human being, the white blood cells comprise a mixture of hematopoietic lincages committed and differentiated cells (typically over 99 % of the mononuclear cells are lineages committed cells) including, for example: Lineage committed progenitor cells
CD34*CD33" (myeloid committed cells), CD34'CD3" (lymphoid committed cells)
CD34'CD41" (megakaryocytic committed cells) and differentiated cells - CD34
CD33" (myeloids, such as granulocytes and monocytes), CD34 CD3", CD34CD19" (T and B cells, respectively), CD34'CD41" (megakaryocytes), and hematopoietic stem and early progenitor cells such as CD34'Lineage negative (Lin’), CD34-Lineage negative CD34" CD38 (typically less than 1 %).
The phrase ‘hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells” is used herein to describe any portion of the white blood cells fraction, in which the majority of the cells are hematopoietic committed cells, while the minority of the cells are hematopoietic stem and progenitor cells, as these terms are further defined hereinunder.
Hematopoietic mononuclear cells are typically obtained from a blood sample by applying the blood sample onto a Ficoll-Hypaque layer and collecting, following : 30 density-cussion centrifugation, the interface layer present between the F icoll-Hypaque and the blood serum, which interface layer essentially entirely consists of the white blood cells present in the blood sample.
As used herein, the phrase “hematopoietic committed cells” refers to differentiated hematopoietic cells that are committed to a certain hematopoietic cell } lineage and hence can develop under physiological conditions substantially only to this specific hematopoietic lineage. : 5 As used herein, the phrase “hematopoietic stem cells” refers to pluripotent hematopoietic cells that, given the right growth conditions, may develop to any cell lineage present blood. This phrase, as used herein, refers both to the earliest renewable hematopoietic cell populations responsible for generating cell mass in the blood (e.g., CD34/ACI133*, CD34/AC1337/Lineage’, CD34"/AC133" cells) and the very early hematopoietic progenitor cells, which are somewhat more differentiated, yet are not committed and can readily revert to become a part of the earliest renewable hematopoietic cell population (e.g., CD34" cells, especially CD34"CD38 cells).
In normal human, most of the hematopoietic pluripotent stem cells and the lineage committed progenitor cells are CD34". The majority of cells are
CD34°CD38", with a minority of cells (< 10 %) being CD34°CD38".
The CD34°CD38 stem cells fraction identifies the most immature hematopoietic cells, which are capable of self-renewal and multilineage differentiation. This fraction contains more long-term culture initiating cells (LTC-
IC) pre-CFU and exhibits longer maintenance of the stem cells and delayed proliferative response to cytokines as compared with the CD34"CD38" cell fraction.
Presently, hematopoietic stem cells are obtained by further enrichment of the hematopoietic mononuclear cells obtained by differential density centrifugation as described above. This further enrichment process is typically performed by immuno- separation such as immunomagnetic-separation or FACS and results in a cell fraction that is enriched for hematopoietic stem cells.
Hence, using hematopoietic mononuclear cells as a direct source for obtaining expanded population of hematopoietic stem cells circumvents the need for stem cell enrichment prior to expansion, thereby substantially simplifying the process in terms . 30 of both efficiency and cost.
As used herein the term "inhibiting" refers to slowing, decreasing, delaying, preventing or abolishing.
As used herein the term "differentiation" refers to relatively generalized or specialized changes during development. Cell differentiation of various lineages is a well-documented process and requires no further description herein. As used herein the term differentiation is distinct from maturation which is a process, although some : 5 times associated with cell division, in which a specific cell type mature to function and then dies, e.g., via programmed cell death.
The phrase “cell expansion” is used herein to describe a process of cell proliferation substantially devoid of cell differentiation. Cells that undergo expansion hence maintain their cell renewal properties and are oftentimes referred to herein as renewable cells, e.g., renewable stem cells.
Expansion of hematopoietic stem cells using hematopoietic mononuclear cells as a source for the hematopoietic stem cells, as taught by the present invention, therefore result in converting the minor fraction (of less than 1 %) of hematopoietic stem and progenitor cells present in the mononuclear cells into at least the major, if not the sole hematopoietic cells population post expansion, whereby in the course of stem cells expansion the committed cells are either substantially diluted and/or die.
As used herein the term "ex-vivo" refers to a process in which cells are removed from a living organism and are propagated outside the organism (e.g., in a test tube). As used herein, the term "ex-vivo", however, does not refer to a process by which cells known to propagate only in-vitro, such as various cell lines (e.g., HL-60,
MEL, HeLa, etc.) are cultured. In other words, cells expanded ex-vivo according to the present invention do not transform into cell lines in that they eventually undergo differentiation.
Providing the ex-vivo grown cells with conditions for ex-vivo cell proliferation include providing the cells with nutrients and preferably with one or more cytokines, as is further detailed hereinunder.
As mentioned hereinabove, concomitant with treating the hematopoietic mononuclear cells with conditions which allow the cells to proliferate ex-vivo, the cells are short-term treated or long-term treated to reduce the expression and/or : 30 activity of CD38.
In one embodiment of the present invention, reducing the activity of CD38 is effected by providing the cells with an agent that inhibits CD38 activity (i.e., a CD38 inhibitor).
As used herein a “CD38 inhibitor” refers to an agent which is capable of downregulating or suppressing CD38 activity in stem cells.
A CD38 inhibitor according to this aspect of the present invention can be a “direct inhibitor” which inhibits CD38 intrinsic activity or an “indirect inhibitor” : 5 which inhibits the activity or expression of CD38 signaling components (e.g., the cADPR and ryanodine signaling pathways) or other signaling pathways which are effected by CD38 activity.
According to presently known embodiments of this aspect of the present invention, nicotinamide is a possible CD38 inhibitor.
Hence, in one embodiment, the method according to this aspect of the present invention is effected by providing the hematopoietic mononuclear cells either with nicotinamide itself, or with a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog mctabolite.
As used herein, the phrase “nicotinamide analog” refers to any molecule that 1s known to act similarly to nicotinamide. Representative examples of nicotinamide analogs include, without limitation, benzamide, nicotinethioamide (the thiol analog of nicotinamide), nicotinic acid and a-amino-3-indolepropionic acid.
The phrase “a nicotinamide or a nicotinamide analog derivative” refers to any structural derivative of nicotinamide itself or of an analog of nicotinamide. Examples of such derivatives include, without limitation, substituted benzamides, substituted nicotinamides and nicotinethioamides and N-substituted nicotinamides and nicotinthioamides.
The phrase “a nicotinamide or a nicotinamide analog metabolite” refers to products that are derived from nicotinamide or from analogs thereof such as, for example, NAD, NADH and NADPH.
Alternatively, a CD38 inhibitor according to this aspect of the present invention can be an activity-neutralizing antibody that binds, for example, to the
CD38 catalytic domain, thereby inhibiting CD38 catalytic activity. It will be appreciated, though, that since CD38 is an intracellular protein measures are taken to - 30 use inhibitors which may be delivered through the plasma membrane. In this respect a fragmented antibody such as a Fab fragment (described hereinunder) is preferably used.
The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab");, and Fv that are capable of } binding to macrophages. These functional antibody fragments are defined as follows:
Fab, the fragment which contains a monovalent antigen-binding fragment of : 5 an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
Fab’, the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (Fab'),, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab’) is a dimer of two Fab' fragments held together by two disulfide bonds;
Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two 1s chains; and
Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
Methods of making these fragments are known in the art. (See for example,
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
New York, 1988, incorporated herein by reference).
Antibody fragments according to the present invention can be prepared by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab'),. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulthydryl groups resulting from cleavage of - 30 disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S.
Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R,
Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies, such as } separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
Fv fragments comprise an association of Vy and V chains. This association may be noncovalent, as described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659- 62, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the
Fv fragments comprise Vy and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the Vy and Vi domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow and
Filpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426, 1988; Pack et al,,
Bio/Technology 11:1271-77, 1993; and Ladner et al., U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells.
See, for example, Larrick and Fry, Methods, 2: 106-10, 1991.
Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as
Fv, Fab, Fab', F(ab"); or other antigen-binding subsequences of antibodies) which : 30 contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins recipient antibody in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In : 5 general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the
FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). The techniques of Cole : 30 et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human can be made by introducing of human immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, . which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach 1s described, for - 5s example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al, Nature Biotechnology 14, 845-51 (1996); Neuberger,
Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
Alternatively, the method according to this aspect of the present invention can be effected by providing the ex-vivo cultured hematopoietic mononuclear cells with an agent that downregulates CD38 expression.
An agent that downregulates CD38 expression refers to any agent which affects CD38 synthesis (decelerates) or degradation (accelerates) either at the level of the mRNA or at the level of the protein. For example, a small interfering polynucleotide molecule which is designed to down regulate the expression of CD38 can be used according to this aspect of the present invention.
An example for a small interfering polynucleotide molecule which can down- regulate the expression of CD38 is a small interfering RNA or siRNA, such as, for example, the morpholino antisense oligonucleotides described by in Munshi et al. (Munshi CB, Graeff R, Lee HC, J Biol Chem 2002 Dec 20;277(51):49453-8), which includes duplex oligonucleotides which direct sequence specific degradation of mRNA through the previously described mechanism of RNA interference (RNAi) (Hutvagner and Zamore (2002) Curr. Opin. Genetics and Development 12:225-232).
As used herein, the phrase "duplex oligonucleotide” refers to an oligonucleotide structure or mimetics thereof, which is formed by either a single self- complementary nucleic acid strand or by at least two complementary nucleic acid strands. The "duplex oligonucleotide" of the present invention can be composed of . 30 double-stranded RNA (dsRNA), a DNA-RNA hybrid, single-stranded RNA (ssRNA), isolated RNA (i.e., partially purified RNA, essentially pure RNA), synthetic RNA and recombinantly produced RNA.
Preferably, the specific small interfering duplex oligonucleotide of the present invention is an oligoribonucleotide composed mainly of ribonucleic acids. ] Instructions for generation of duplex oligonucleotides capable of mediating
RNA interference are provided in www.ambion.com. h : 5 Hence, the small interfering polynucleotide molecule according to the present invention can be an RNAI molecule (RNA interference molecule).
Alternatively, a small interfering polynucleotide molecule can be an oligonucleotide such as a CD38-specific antisense molecule or a rybozyme molecule, further described hereinunder.
Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example,
Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art.
Oligonucleotides used according to this embodiment of the present invention are those having a length selected from a range of 10 to about 200 bases preferably 15-150 bases, more preferably 20-100 bases, most preferably 20-50 bases.
The oligonucleotides of the present invention may comprise heterocyclic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
Preferably used oligonucleotides are those modified in either backbone, internucleoside linkages or bases, as is broadly described hereinunder. Such modifications can oftentimes facilitate oligonucleotide uptake and resistivity to intracellular conditions.
Specific examples of preferred oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones } 30 include those that retain a phosphorus atom in the backbone, as disclosed in U.S.
Patents Nos.:. 4,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5278302; 5,286,717; 5,321,131; 5,399,676; 5,405,939,
5,453,496; 5,455,233; 5,466, 677, 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050. } Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, : 5S aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including ~~ 3-amino phosphoramidate = and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms can also be used.
Alternatively, modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH, component parts, as disclosed in U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257, 5,466,677; 5,470,967, 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.
Other oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, i.e., the backbone, of : 30 the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target. An example for such an oligonucleotide mimetic, includes peptide nucleic acid (PNA). A PNA oligonucleotide refers to an oligonucleotide where the sugar-backbone is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. United States patents that teach the preparation of
PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat.
No: 6,303,374.
Oligonucleotides of the present invention may also include base modifications or substitutions. As used herein, "unmodified" or "natural" bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), S-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, S-halouracil and cytosine, S-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, S-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly S-bromo, S-trifluoromethyl and other 5- substituted wuracils and cytosines, 7-methylguanine and 7-methyladenine, &- azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine. Further bases include those disclosed in U.S. Pat.
No: 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And
Engineering, pages 858-859, Kroschwitz, J. 1., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al.,, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993.
Such bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include S-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2- : 30 aminopropyladenine, S-propynyluracil and S-propynylcytosine. S-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C. [Sanghvi YS et al. (1993) Antisense Research and Applications, CRC Press, Boca
Raton 276-278] and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, as disclosed in U.S. Pat. No: 6,303,374.
It is not necessary for all positions in a given oligonucleotide molecule to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.
As described hereinabove, the oligonucleotides of the present invention are preferably antisense molecules, which are chimeric molecules. "Chimeric antisense molecules" are oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target polynucleotide. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. An example for such includes RNase
H, which is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter : 30 oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region.
Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
Chimeric antisense molecules of the present invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, as } described above. Representative U.S. patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797, . 5 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein fully incorporated by reference.
The oligonucleotides of the present invention can further comprise a ribozyme sequence. Ribozymes arc being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs. Several rybozyme sequences can be fused to the oligonucleotides of the present invention. These sequences include but are not limited ANGIOZYME specifically inhibiting formation of the VEGF-R (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway, and HEPTAZYME, a rybozyme designed to selectively destroy Hepatitis C
Virus (HCV) RNA, (Rybozyme Pharmaceuticals, Incorporated - WEB home page).
Further alternatively, a small interfering polynucleotide molecule, according to the present invention can be a DNAzyme.
DNAzymes are single-stranded catalytic nucleic acid molecules. A general model (the "10-23" model) for the DNAzyme has been proposed. "10-237
DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, LM Curr Opin Mol Ther 2002;4:119-21).
Examples of construction and amplification of synthetic, engineered
DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit
Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al, 20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org). In another application, DNAzymes complementary to ber-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells,
and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL. } Alternatively, as mentioned hereinabove and is further detailed in
PCT/IL03/00064 and U.S. Provisional Patent Application No. 60/452,545, retinoid : 5 receptor superfamily inhibitors (e.g., antagonists, siRNA molecules, antisense molecules, antibodies, etc.) which downregulate or suppress retinoid receptor activity and/or expression can be used to downregulate CD38 expression.
Briefly, retinoid receptors such as retinoic acid receptor (RAR), retinoid X receptor (RXR) and vitamin D receptor (VDR) have been reported to be involved in the regulation of gene expression pathways associated with cell proliferation and differentiation and in particular in the regulation of CD38 expression [Kapil M.,,
Teresa M., Taghi M., Michael A., Steven C., Maher A.. Involvement of retinoic acid receptor mediated signaling pathway in induction of CD38 cell surface antigen, Blood. 1997; 89:3607-3614; Ueno H, Kizaki M, Matsushita H, Muto A, Yamato K, Nishihara ts T, Hida T, Yoshimura H, Koeffler HP, Ikeda Y. A novel retinoic acid receptor (RAR)- selective antagonist inhibits differentiation and apoptosis of HL-60 cells: implications of RAR alpha-mediated signals in myeloid leukemic cells. Leuk Res. 1998; 22:517- 25). Hence, preferred agents that downregulate CD38 expression according to the present invention include RAR antagonists, RXR antagonists and VDR antagonists or, alternatively, antagonists for reducing the capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoid and/or Vitamin D.
As used herein the term "antagonist" refers to an agent that counteracts or abrogates the effects of an agonist or a natural ligand of a receptor. Further features relating to such antagonists are detailed hereinunder.
Further altematively, as is described in detail in U.S. Provisional Patent
Application No. 60/452,545, down regulation of CD38 expression can be obtained by downregulating the expression and/or activity of phosphatidyl inositol 3-kinase, which is also referred to herein throughout as PI 3-kinase. Briefly, it has been reported that PI 3-kinase plays a critical function in the activation of nuclear receptors . 30 such as the retinoid receptor superfamily and the vitamin D receptor, as an obligatory factor for proper receptor signaling pathways and is hence involved in cell differentiation.
Hence, agents that interfere with PI 3-kinase expression and/or activity are also preferred agents for downregulating CD38, according to the present invention. } Representative examples of agents that inhibit PI 3-kinase activity include, but are not limited to, the known PI 3-kinase inhibitors wortmannin and LY294002, and analogs, . 5 derivatives, and metabolites thereof. Additional examples of PI 3-kinase inhibitors are described in U.S. Patent No. 5,378,725, which is incorporated by reference as if fully set forth herein. Representative examples of agents that downregulate PI 3- kinase expression according to the present invention include, but are not limited to, polynucleotides, such as small interfering RNA molecules, antisense ribozymes and
DNAzymes, as well as intracellular antibodies, using the methodologies described hereinabove with respect to downregulating CD38 expression.
Each of the agents described hereinabove may reduce the expression or activity of CD38 individually. However, the present invention aims to also encompass the use of any subcombination of these agents.
It will be appreciated that protein agents (e.g., antibodies) of the present invention can be expressed from a polynucleotide encoding same and provided to ex- vivo cultured hematopoietic mononuclear cells employing an appropriate gene delivery vehicle/method and a nucleic acid construct as is further described hereinunder.
Examples of suitable constructs include, but are not limited to pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which 1s commercially available from Invitrogen Co. (www.invitrogen.com).
Examples of retroviral vector and packaging systems are those sold by Clontech, San
Diego, Calif., including Retro-X vectors pPLNCX and pLXSN, which permit cloning into multiple cloning sites and the transgene is transcribed from CMV promoter.
Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5’LTR promoter.
As the method of ex-vivo expanding a population of hematopoietic stem cells, according to this aspect of the present invention, is effected by modulating CD38 . 30 expression and/or activity, either at the protein level, using RAR, RXR or VDR antagonists, a PI-3 kinase inhibitor or a CD38 inhibitor such as nicotinamide and analogs thereof, or at the at the expression level via genetic engineering techniques, as is detailed hereinabove, there are further provided, according to the present invention,
several preferred methods of ex-vivo expanding a population of hematopoietic stem cells of hematopoietic mononuclear cells. ) In one particular, a method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting : 5 differentiation of the hematopoietic stem cells ex-vivo is effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, so as to expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
Reducing the capacity of the cells in responding to retinoic acid, retinoids and/or Vitamin D, or to retinoic acid, retinoid X and/or Vitamin D receptor signaling may be effected, for example, by the administration of chemical inhibitors, including receptor antagonists.
In another particular, the method of ex-vivo expanding a population of stem cells, while at the same time, substantially inhibiting differentiation of the stem cells ex-vivo is effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor, retinoid-X receptor and/or Vitamin D receptor, to thereby expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
Reducing the capacity of the cells to respond to retinoic acid, retinoid X and/or
Vitamin D receptor signaling events, includes treating the cells with antagonists supplied continuously or for a short-pulse period, and is effected by a diminution or . 30 abrogation of cellular signaling pathways through their respective, cognate receptors.
As is described and exemplified in PCT/IL01/00064, reducing the capacity of hematopoietic cells in responding to the disclosed signaling pathways is reversible, e.g., inherently reversible. In other words, cells expanded using the protocols of the present invention do not transform into cell lines. Hence, by exposing the cells following sufficient expansion to growth conditions by which differentiation is . induced, one would be able to direct the ex-vivo differentiation of the cells to desired direction, including ex-vivo and in vivo cis- and trans-differentiation.
As used herein “cis-differentiation” refers to differentiation of adult stem cells into a tissue from which they were derived. For example, the differentiation of
CD34+ hematopoietic cells to different committed/mature blood cells constitutes cis- differentiation.
As used herein “trans-differentiation” refers to differentiation of adult stem cells into a tissue from which they were not derived. For example, the differentiation of CD34+ hematopoietic cells to cells of different tissue origin, c¢.g., myocites constitutes trans-differentiation.
Reducing the capacity of the hematopoietic mononuclear cells in responding to the above antagonists and/or signaling pathways of the above receptors and kinase is effected by ex-vivo culturing hematopoietic mononuclear cells in a presence of an effective amount of at least one retinoic acid receptor antagonist, at least one retinoid
X receptor antagonist and/or at least one Vitamin D receptor antagonist, preferably, for a time period of 0.1-50 %, preferably, 0.1-25 %, more preferably, 0.1-15 %, of an entire ex-vivo culturing period of the hematopoietic mononuclear cells or for the entire period. In this respect it was surprisingly uncovered that an initial pulse exposure to an antagonist is sufficient to exert cell expansion long after the antagonist was removed from the culturing set up.
Final concentrations of the antagonists may be, depending on the specific application, in the micromolar or millimolar ranges. For example, within about 0.1 p
MM to about 100 mM, preferably within about 4 uM to about S50 mM, more preferably within about 5 uM to about 40 mM.
Many antagonists to RAR, RXR and VDR, which are usable in this and other : aspects and embodiments of the present invention, are presently known.
Representative examples of such retinoic acid receptor antagonist include, without limitation, AGN 194310; AGN 109; 3-(4-Methoxy-phenylsulfanyl)-3- methyl-butyric acid; 6-Methoxy-2,2-dimethvl-thiochroman-4-one,2,2-Dimethyl-4- oxo-thiochroman-6-yltrifluoromethane-sulfonate; Ethyl 4-((2,2 dimethyl-4-oxo- thiochroman-6-yl)ethynyl)-benzoate; Ethyl 4-((2,2-dimethy 1-4-
triflouromethanensulfonyloxy ~~ -(2H)- thiochromen-6-yl)ethynyl)-benzoate(41);
Thiochromen-6-yl]-ethynyl]-benzoate(yl); (p-[(E)-2-[3'4'-Dihydro-4,4'-dimethyl-7" : (heptyloxy)-2'H-1-benzothiopyran-6'yl] propenyl] benzoic acid 1'l'-dioxide; 2E 4E,6E-[7-(3,5-Di-t-butyl-4-n-butoxyphenyl)-3-methyl}-octa-2,4,6-trienoic acid; : 5 2E,4E,6E-[7-(3,5-Di-t-butyi-4-n-propoxyphenyl)-3-methyl}-octa-2,4,6-trienoic acid; 2E,AE,6E-[7-(3,5-Di-t-butyl-4-n-pentoxyphenyl)-3-methyl}-octa-2,4,6-trienoic acid; 2E 4E 6E-[7-(3,5-Di-t-butyl-4-n-hexoxyphenyl)-3-methyl}-octa-2,4,6-trienoic acid; 2E4E,6E-[7-(3,5-Di-t-butyl-4-n-heptoxyphenyl)-3-methyl]-octa-2,4,6-triencic acid; 2E4E,6E-[7-(3,5-Di-t-butyl-4-n-octoxyphenyl)-3-methyl]-octa-2,4,6-trienoic ~~ acid; (2E,4E,6E)-7-[3-t-butyl-5-(1-phenyl-vinyl)-phenyl]-3-methyl-octa-2,4,6-trienoic acid; 2E 4E ,6E-[7-(3,5-Di-t-butyl-4-{[4,5-.sup.3 H.sub.2]-n-pentoxy } phenyl)-3-methyl]- octa-2,4,6-trienoic acid; (2E,4E)-(1RS,2RS)-5-[2-(3,5-di-tert.butyl-2-ethoxy-phenyl)- cyclopropyl]-3-methyl-penta-2,4-dienoic acid ethyl ester; (2E,4E)-(1RS,2RS)-5-[2- (3,5-di-tert.butyl-2-ethoxy-phenyl)-cyclopropyl]-3-methyl-penta-2 ,4-dienoic acid, (2E,4E)-(1RS,2RS)-5-[2-(3,5-di-tert.butyl-2-butoxy-phenyl)-cyclopropyl]-3-methyl- penta-2 4-dienoic acid; (2E,4E,6Z)-7-[3,5-di-tert.butyl-2-ethoxyphenyl]3-methyl- 2,4,6-octatrienoic acid; (2E,4E,62)-7-[3,5-di-tert.butyl-2-butyloxyphenyl]-3-methyl- 2,4,6-octatrienoic acid; 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalene- carboxamido) benzoic acid; (2E,4E)-3-methyl-5-[(1S,25)-2-(5,5,8,8-tetramethyl- 5,6,7,8-tetrahydro-naphthalen-2-yl)-cyclopropyl}-penta-2,4-dienoic acid; p-[(E)-2- [3',4'-Dihydro-4',4'-dimethyl-7'-(heptyloxy)-2'H-1-benzothiopyran-6'- yl]propenyljbenzoic acid; 1',1'-dioxide, 4-(7,7,10,10-Tetramethyl-1-pyridin-3- ylmethyl-4,5,7,8,9,10-hexahydro-1H-naphto[2,3-g]indol-3-yl)-benzoic acid; (2E4E,62)-7-[3,5-di-tert.butyl-2-methoxyphenyl]-3-methyl-2.4,6-octatrienoic acid, (2E4E,6Z)-7-(3,5-di-tert.butyl-2-ethoxyphenyl]-3-methyl-2,4,6-octatrienoic acid; (2E,4E,62)-7-[3,5-di-tert.butyl-2-hexyloxyphenyl]-3-methyl-2,4,6-octatrienoic acid; (2E,4E,6Z)-7-(3,5-di-tert.butyl-2-octyloxyphenyl]-3-methyl-2,4,6-octatrienoic acid; ] and (2E,4E)-(1RS,2RS)-5-[2-(3,5-di-tert-butyl-2-butoxy-phenyl)-cyclopropyl]}-3- methyl-penta-2,4-dienoic acid (2E,4E,6Z)-7-(3-n-propoxy-5,6,7,8-tetrahydro-5,5,8,8- tetramethylnaphthalene-2-yl)-3-methylocta-2,4,6-trienoic acid, and 4-(5H-2,3(2,5 dimethyl-2,5-hexano)-5-n-propyldibenzo[b,e][ | ,4]diazepin-11-yl)benzoic acid, and 4- (5H-2,3-(2,5-dimethyl-2,5-hexano)-5methyl-8-nitrodibenzo[b,e][ 1,4]diazepin-11- yl)benzoic acid, and 4-{[4-(4-Ethylphenyl)2,2-dimethyl-(2H)-thiochromen-6-
yllethynyl}benzoic acid, and 4-[4-2methyl-1,2-dicarba-closo-dodecaboran-1-yl- phenylcarbamoyl]benzoic acid, and 4-[4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl- 1-(3-pyridylmethyl)-anthra[1,2-b]pyrrol-3-yljbenzoic acid, and (3-pyridylmethyl)-]5- thiaanthra[2,1-b]pyrrol-3-yl)benzoic acid, and (3-pyridylmethyl)-anthra[2mI- : 5 d]pyrazol-3-yl]benzoic acid.
Representative examples of such retinoid X receptor antagonist include, without limitation, LGN100572, 1-(3-hydroxy-5,6,7,8-tetrahydro-5,5,8,8- tetramethylnaphthalene-2-yl)ethanone, 1-(3-propoxy-5,6,7,8-tetrahydro-5,5,8,8- tetramethyinaphthalene-2-yl)ethanone, 3-(3-propoxy-95,6,7,8-tetrahydro-5,5,8,8- tetramethylnaphthalene-2-yl)but-2-enenitrile, 3-(3-propoxy-5,6,7,8-tetrahydro- 5,5,8,8-tetramethylnaphthalene-2-yl)but-2-enal, (2E 4E,6E)-7-3[-propoxy-5,6,7,8- tetrahydro 5,5,8,8-tetramethyl-2-naphthalene-2-yl]-3-methylocta-2,4,6-trienoic acid, 4-[3,5,5,8,8-pentamethyl-5,6,7,8-tctrahydro-2-naphthyl)carbonyl] benzoic acid, 4-[1- (3,5, 5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl] benzoic acid, 4- [1(3,5,5,8,8-pentamethyl-5,6,7 8-tetrahydro-2-naphthyl)cyclopropyl] benzoic acid, 4- [1-(3,5,5,8,8-pentamethyl-5,6,7 8-tetrahydro-2-naphthyl)ethenyl] benzenete trazole, 2-[1-(5,5,8,8-tetramethyl-5.6,7 8-tetrahydro-2-naphthyl) ethenyl]pyridine-5- carboxylic acid, 2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2- naphthyl)ethyl]pyridine-5-carboxylic acid, ethyl-2-[1-(3,5,5,8, 8-pentamethyl-5,6,7 8- tetrahydro-2-naphthyl)ethenyl]pyridine-5-carboxylate, ~~ 5-[1-3,5,5,8,8-pentamethyl- 5,6,7,8-tetrahydro-2-naphthyl)ethenyl]pyridine-2-carboxylic acid, 2-[1-(3,5,5,8,8- pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) cyclopropyl]pyridine-5-carboxylic acid, methyl 2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2- naphthyl)cyclopropyl]pyridine-5-carboxylate, 4-[1-(3,5, 5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthyl)ethenyl]-N-(4-hydroxyphenyl) benzamide, 2-{1-(3,5,5,8,8-
Pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl] pyridine-5-carboxylic acid, 2-[1- (3,5,5,8,8-Pentamethyl-5, 6,7,8-tetrahydro-2-naphthyl)cyclopropyl]pyridine-5- ] carboxylic acid, 4-[(3,5, 5,8,8-pentamethyli-5,6,7,8-tetrahydro-2- naphthyl)carbonyl]benzoic acid butyloxime, 4-[(3,5,5,8,8-pentamethyl-5,6,7,8- - 30 tetrahydro-2-naphthyl) carbonyllbenzoic acid propyloxime, 4-[(3,5,5,8,8- pentamethyl-5,6,7,8-terrahydro-2-naphthyl)carbonyl}benzoic acid cyanoimine, 4- [(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic acid allyloxime, 4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl}benzoic acid 4-(3-methylbut-2-enoic acid)oxime, and 4-[(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthyl)carbonyl]benzoic acid 1-aminoethyloxime (2E,4E,6Z)-7-(3-n- } propoxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalene-2-yl)-3-methylocta-2,4,6- trienoic acid, and 4-(5H-2,3(2,5 dimethyl-2,5-hexano)-5-n- : 5 propyldibenzo[b,e][1,4]diazepin-11-yl)benzoic acid, and 4-(5H-2,3-(2,5-dimethyl- 2,5-hexano)-Sm.
Representative examples of such Vitamin D receptor antagonist include, without limitation: 1 alpha, 25-(OH)-D3-26,23 lactone; lalpha, 25-dihydroxyvitamin
D (3); the 25-carboxylic ester ZK159222; (23S)- 25-dehydro-1 alpha-OH-D (3); (23R)-25-dehydro-1 alpha-OH-D (3); 1 beta, 25 (OH); Ds; 1 beta, 25(OH),-3-epi-Ds; (23S) 25-dchydro-1 alpha(OH) D3-26,23-lactone; (23R) 25-dehydro-1 alpha(OH)D3- 26,23-lactone and Butyl-(5Z,7E,22E-(1S,7E,22E-(1S,3R ,24R)- 1,3,24-trihydroxy- 26,27-cyclo-9,10-secocholesta-5,7,10(19),22-tetracne-25-carboxylate).
The above listed antagonists are known for their high affinity towards their 1S respective cognate receptors. However, it may be possible for these molecules to be active towards other receptors.
Hence, in another particular, the method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoictic stem cells ex-vivo 1s effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving Pl 3-kinase, to thereby expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
In another particular, the method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo is effected by providing . 30 hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and with nicotinamide, a nicotinanmmde analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, to thereby expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting } differentiation of the hematopoietic stem cells ex-vivo.
In yet another particular, the method of ex-vivo expanding a population of : 5 hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo 1s effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and with a PI 3-kinase inhibitor, to thereby expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-
Vivo.
Final concentrations of thc nicotinamide or the analogs, derivatives or metabolites thereof and of the PI 3-kinase inhibitor are preferably, depending on the specific application, in the millimolar ranges. For example, within about 0.1 mM to about 20 mM, preferably within about 1 mM to about 10 mM, more preferably within about 5 mM to about 10 mM.
As is described hereinabove and is further exemplified in the Examples section that follows, expansion of the hematopoietic stem cells population present in hematopoietic mononuclear cells can also be effected in the presence of copper chelators or chalets. As is discussed in detail in WO 00/18885 and in
PCT/IL03/00062, addition of copper chelators or copper chelates to cells culturing media affects the cellular copper concentration, which in tum, affects signaling pathways associated with cells differentiation. According to the teachings of WO 00/18885 and PCT/IL03/00062, addition of a copper chelate to the cells culturing media maintains the free copper concentration of the cells substantially unchanged during cell expansion, while addition of a copper chelator to the cells culturing media reduces the capacity of the cells in utilizing copper.
As used herein, the phrase “copper chelator” refers to a hgand that has at least : 30 two atoms capable of coordinating with copper or a copper ion, so as to form a ring.
A copper chelator is free of] i.e, not complexed with, the copper ion. Additional features relating to chelating effects are described, for example, in PCT/IL03/00062.
As used herein throughout, the phrase “copper chelate” refers to a copper chelator, as is defined hereinabove, which is complexed with a copper ion.
Hence, according to the present invention there is provided another method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, . 5 substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo. The method, according to this aspect of the present invention is effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and with one or more copper chelator(s) or copper chelate(s), to thereby expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
The copper chelate or chelators of the present invention is oftentimes capable of forming an organometallic complex with a transition metal other than copper. As 1s metals other than copper are typically present in the cells (e.g., zinc) or can be administered to cells during therapy (e.g., platinum), it was found that copper chelates or chelators that can also interact with other metals are highly effective.
Representative examples of such transition metals include, without limitation, zinc, cobalt, nickel, iron, palladium, platinum, rhodium and ruthenium.
The copper chelates of the present invention comprise a copper ion (e.g., Cu”,
Cu*?) and one or more copper chelator(s). Preferred copper chelators according to the present invention include polyamine molecules, which can form a cyclic complex with the copper ion via two or more amine groups present in the polyamine.
Hence, the copper chelate or chelator used in the context of the different aspects and embodiments of the present invention preferably includes a polyamine chelator, namely a polymeric chain that is substituted and/or interrupted with 1-10 amine moieties, preferably 2-8 amine moieties, more preferably 4-6 amine moieties and most preferably 4 amine moieties.
The phrases “amine moiety”, “amine group” and simply “amine” are used . 30 herein to describe a -NR'R’’ group or a -NR’- group, depending on its location within the molecule, where R’ and R*’ are each independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl or heterocyclic, as these terms are defined hereinbelow.
The polyamine chelator can be a linear polyamine, a cyclic polyamine or a combination thereof. : A linear polyamine, according to the present invention, can be a polyamine that has a general formula I: : 5
HX-Am-(YB);----(YnBn)n-ZH
Formula 1 wherein m is an integer from 1 to 10; n is an integer from 0 to 20; X and Z are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; Y, and Yn are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; A is an alkylene chain having between 1 and 10 substituted and/or non-substituted carbon atoms; and B, and
Bn are each independently an alkylene chain having between 1 and 20 substituted and/or non-substituted carbon atoms, provided that at least one of X, Z, Y; and Yn 1s a -NH group and/or at least one of the carbon atoms in the alkylene chains is substituted by an amine group.
Hence, the linear polyamine, according to the present invention, is preferably comprised of one or more alkylene chains (Am, B;----Bn, in Formula I), is interrupted by one or more heteroatoms such as S, O and N (Y;--Yn in Formula I), and terminates with two such heteroatoms (X and Z in Formula I).
Alkylene chain A, as is described hereinabove, includes 1-10 substituted or non-substituted carbon atoms and is connected, at least at one end thereof, to a heteroatom (e.g., X in Formula I). Whenever there are more than one alkylene chains
A (in cases where m is greater than one), only the first alkylene chain A is connected to X. However, mis preferably 1 and hence the linear polyamine depicted in Formula
I preferably includes only one alkylene chain A.
Alkylene chain B, as is described hereinabove, includes between 1 and 20 substituted or non-substituted carbon atoms. The alkylene chain B is connected at its : 30 two ends to a heteroatom (Y,----Yn and Z in Formula I).
The preferred linear polyamine delineated in Formula I comprises between 1 and 20 alkylene chains B, denoted as B, ---- Bn, where “B, --- Bn” is used herein to describe a plurality of alkylene chains B, namely, By, Bs, Bj, ----, Bn-1 and Bn, where n equals 0-20. These alkylene chains can be the same or different. Each of B, ---- Bn is connected to the respective heteroatom Y, ---- Yn, and the last alkylene chain in the structure, Bn, is also connected to the heteroatom Z.
Tt should be noted that herein throughout, whenever an integer equals 0 or ’ 5 whenever a component of a formula is followed by the digit 0, this component is absent from the structure. For example, if n in Formula I equals 0, there is no alkylene chain B and no heteroatom Y are meant to be in the structure.
Preferably, n equals 2-10, more preferably 2-8 and most preferably 3-3.
Hence, the linear polyamine depicted in Formula I preferably includes between 3 and 5 alkylene chains B, each connected to 3-5 heteroatoms Y.
The linear polyamine depicted in Formula T must include at least one amine group, as this term is defined hereinabove, preferably at least two amine groups and more preferably at least four amine groups. The amine group can be present in the © structure as the heteroatoms X, Z or Y; ---- Yn, such that at least one of X, Z and Y,
Yn is a -NH- group, or as a substituent of one or more of the substituted carbon atoms in the alkylene chains A and B; -- Bn. The presence of these amine groups is required in order to form a stable chelate with the copper ion, as is discussed hereinabove.
The alkylene chain A preferably has a general Formula II: pb —C H-C,H + CgH—
Formula II wherein g is an integer that equals 0 or 3-10.
Hence, the alkylene chain A is comprised of a plurality of carbon atoms C,,
Cy, Cy +, Cg-1 and Cg, substituted by the respective R,, Ra, R3 ---, Rg-1 and Rg . groups. Preferably, the alkylene chain A includes 2-10 carbon atoms, more preferably, 2-6 and most preferably 2-4 carbon atoms.
As is defined hereinabove, in cases where g equals 0, the component CgH(Rg) is absent from the structure and hence the alkylene chain A comprises only 2 carbon atoms.
Rj, Ry and Rg are each a substituent attached to the carbon atoms in A. Each of Ri, Ry and Rg can independently be a substituent such as, but not limited to, ) hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroalicyclic, heteroaryl, halo, amino, alkylamino, arylamino, cycloalkylamino, heteroalicyclic amino, - 5 heteroarylamino, hydroxy, alkoxy, aryloxy, azo, C-amido, N-amido, ammonium, thiohydroxy, thioalkoxy, thioaryloxy, sulfonyl, sulfinyl, N-sulfonamide, S- sulfonamide, phosphonyl, phosphinyl, phosphonium, carbonyl, thiocarbonyl, C- carboxy, O-carboxy, C-thiocarboxy, O-thiocarboxy, N-carbamate, O-carbamate, N- thiocarbamate, O-thiocarbamate, urea, thiourea, borate, borane, boroaza, silyl, siloxy, silaza, aquo, alcohol, peroxo, amine oxide, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanate, thiocyanate, isocyanate, isothiocyanate, cyano, alkylnitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, carboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, sulfate, sulfite, bisulfite, thiosulfate, thiosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, guanidino, S-dithiocarbamate, N-dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraarylborate, tetraalkyl borate, tartarate, salicylate, succinate, citrate, ascorbate, saccharirate, amino acid, hydroxamic acid and thiotosylate.
Whenever R;, R; or Rg is hydrogen, its respective carbon atom in a non- substituted carbon atom. i 30 As used herein, the term “alkyl” is a saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms. More preferably, it is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl, thiocarbonyl, O- carbamate, N-carbamate, O-thiocarbamate, N-thiocarbamate, C-amido, N-amido, C- - 5 carboxy, O-carboxy, nitro, sulfonamide, silyl, guanidine, urea or amino, as these terms are defined hereinbelow.
The term “alkenyl” describes an alkyl group which consists of at least two carbon atoms and at least one carbon-carbon double bond.
The term “alkynyl” describes an alkyl group which consists of at least two carbon atoms and at least one carbon-carbon triple bond.
The term “cycloalkyl” describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system. Examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, 1s cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. A cycloalkyl group may be substituted or unsubstituted. When substituted, the substituent group can be, for example, alkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamate, N- carbamate, C-amido, N-amido, nitro, or amino, as these terms are defined hereinabove or hereinbelow.
The term “aryl” describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or unsubstituted. When substituted, the substituent group can be, for example, halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thiocarbonyl, C-carboxy,
O-carboxy, O-carbamate, N-carbamate, O-thiocarbamate, N-thiocarbamate, C-amido,
N-amido, sulfinyl, sulfonyl or amino, as these terms are defined hereinabove or . 30 hereinbelow.
The term “heteroaryl” describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted. When substituted, the substituent group can be, for : 5 example, alkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thiocarbonyl, sulfonamide, C-carboxy, O-carboxy, sulfinyl, sulfonyl, O- carbamate, N-carbamate, O-thiocarbamate, N-thiocarbamate, C-amido, N-amido or amino, as these terms are defined hereinabove or hereinbelow.
The term “heteroalicyclic” describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heteroalicyclic may be substituted or unsubstituted. When substituted, the substituted group can be, for example, alkyl, cycloalkyl, aryl, heteroaryl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy,
O-carboxy, O-carbamate, N-carbamate, O-thiocarbamate, N-thiocarbamate, sulfinyl, sulfonyl, C-amido, N-amido or amino, as thesc terms are defined hereinabove or hereinbelow.
The term “halo” describes a fluorine, chlorine, bromine or iodine atom.
The term “amino”, as 1s defined hereinabove with respect to an “amine” or an “amino group”, is used herein to describe an -NR’R’’, wherein R’ and R’’ are each independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl or heterocyclic, as these terms are defined hereinabove.
Hence, the terms ‘“alkylamino”, ‘‘arylamino”, “cycloalkylamino”, “heteroalicyclic amino” and “heteroarylamino” describe an amino group, as defined hereinabove, wherein at least one of R’ and R’’ thereof is alkyl, aryl, cycloalkyl, heterocyclic and heteroaryl, respectively.
The term “hydroxy” describes an -OH group.
An “alkoxy” describes both an -O-alkyl and an -O-cycloalkyl group, as - 30 defined herein.
An "aryloxy" describes both an -O-aryl and an -O-heteroaryl group, as defined herein.
The term “azo” describes a -N=N group.
A “C-amido” describes a -C(=0O)-NR’R’’ group, where R’ and R’’ are as defined hereinabove. , An “N-amido” describes a R’C(=0)-NR’’- group, where R’ and R’’ are as defined hereinabove.
An “ammonium” describes an -N"HR’R’’ group, where R’ and R”’ are as defined hereinabove.
The term “thiohydroxy” describes a -SH group.
The term “thioalkoxy” describes both a -S-alkyl group and a -S-cycloalkyl group, as defined hereinabove.
The term “thioaryloxy’ describes both a -S-aryl and a -S-heteroaryl group, as defined hereinabove.
A “sulfinyl” describes a -S(=0)-R group, where R can be, without limitation, alkyl, cycloalkyl, aryl and heteroaryl as these terms are defined hereinabove.
A “sulfonyl” describes a -S(=0),-R group, where R is as defined hereinabove.
A “S-sulfonamido” is a -S(=0),-NR’R”’ group, with R’ and R’’ as defined hereinabove.
A “N-sulfonamido” is an R’(S=0),-NR’’- group, with R’ and R’’ as defined hereinabove.
A “phosphonyl” is a -O-P(=0)(OR’)-R’’ group, with R’ and R’’ as defined hereinabove.
A “phosphinyl” is a -PR’R’’ group, with R” and R’’ as defined hereinabove.
A “phosphonium” is a -P'R’R’’R’’’, where R’ and R’> are as defined hereinabove and R’"’ 1s defined as either R’ or R’".
The term “carbonyl” describes a -C(=0)-R group, where R is hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) or heteroalicyclic (bonded through a ring carbon) as defined hereinabove.
A “thiocarbonyl” describes a -C(=S)-R group, where R is as defined hereinabove with respect to the term “carbonyl”.
A “C-carboxy” describes a -C(=0)-O-R groups, where R is as defined ‘ 30 hereinabove with respect to the term “carbonyl”.
An *“O-carboxy” group refers to a RC(=0)-O- group, where R is as defined hereinabove with respect to the term “carbonyl”.
A “carboxylic acid” is a C-carboxy group in which R is hydrogen.
A “C-thiocarboxy” is a -C(=S)-O-R groups, where R is as defined hereinabove with respect to the term “carbonyl”.
An “O-thiocarboxy” group refers to an R-C(=S)-O- group, where R is as defined hereinabove with respect to the term “carbonyl”. ‘ 5 The term *“O-carbamate™ describes an -OC(=0)-NR’R’’ group, with R’ and
R’’ as defined hereinabove.
A “N-carbamate” describes a R’-O-C(=0)-NR’’- group, with R’ and R’’ as defined hereinabove.
An “O-thiocarbamate” describes an —O-C(=S)-NR’R’’ group, with R’ and R”’ as defined hereinabove.
A “N-thiocarbamate™ describes a R’OC(=S)NR’’- group, with R’ and R’’ as defined hereinabove.
The term “urea” describes a -NR’-C(=0O)-NR’R’’ group, with R’, R” and R*”’ as defined hereinabove.
The term “thiourea” describes a -NR’-C(=S)-NR’R’’ group, with R’, R”* and
R’’* as defined hereinabove.
The term “borate” describes an -O-B-(OR); group, with R as defined hereinabove.
The term “borane” describes a -B-R’R’’ group, with R’ and R”’ as defined hereinabove.
The term “boraza” describes a -B(R’)(NR’’R’’’) group, with R’, R”” and R’”’ as defined hereinabove.
The term “silyl” describes a -SiR’R”’R’”’, with R’, R’’ and R’’’ as defined herein.
The term “siloxy” 1s a -Si-(OR)3, with R as defined hereinabove.
The term “silaza” describes a -Si-(NR’R’’);, with R’ and R’’ as defined herein.
The term *“‘aquo” describes a H,O group.
The term “alcohol” describes a ROH group, with R as defined hereinabove.
The term “peroxo” describes an -OOR group, with R as defined hereinabove.
As used herein, an “amine oxide” is a -N(=0)R’R’’R’”’ group, with R’, R>’ and R’”’ as defined herein.
A “hydrazine” is a -NR’-NR’’'R’"* group, with R’, R” and R’”’ as defined herein. . Hence, “alkyl hydrazine” and “aryl hydrazine” describe a hydrazine where R’ 1s an alkyl or an aryl, respectively, and R”’ and R’"’ are as defined hereinabove. - 5 The term “nitric oxide” is a -N=0 group.
The term “cyano” is a -C=N group.
A “cyanate” is an -O-C=N group.
A “thiocyanate” is a *“-S-C=N group.
An “isocyanate” is a -N=C=0 group.
An “isothiocyanate” is a -N=C=S group.
The terms “alkyl nitrile” and “aryl nitrile” describe a -R-C=N group, where R is an alkyl or an aryl, respectively.
The terms “alkyl isonitrile” and “aryl isonitrile” describe a R-N=C- group, where R is an alkyl or aryl, respectively.
A “nitrate” or “nitro” is a -NO, group.
A “nitrite” is an -O-N=0 group.
An “azido” is a N3* group.
An “alkyl sulfonic acid” and an “aryl sulfonic acid” describe a -R-SO,-OH group, with R being an alkyl or an aryl, respectively.
An “alkyl sulfoxide”, an “aryl sulfoxide” and an *alkyl aryl sulfoxide” describe a -R’S(=O)R”’ group, where R’ and R’’ are each an alkyl, R’ and R’’ are each an aryl and where R’ is and alkyl and R’’ is an aryl, respectively.
An “alkyl sulfenic acid” and “aryl sulfenic acid” describe a -R-S-OH group, where R is an alkyl or an aryl, respectively.
An “alkyl sulfinic acid” and “aryl sulfinic acid” describe a -R-S(=0)-OH group where R is an alkyl or an aryl, respectively.
As used herein, the terms “alkyl carboxylic acid” and “aryl carboxylic acid” . describe a -R-C(=0)-OH group, where R is an alkyl or an aryl, respectively.
An “alkyl thiol carboxylic acid” and an “aryl thiol carboxylic acid” describe a ' 30 -R-C(=0)-SH group, where R is an alkyl or an aryl, respectively.
An “alkyl thiol thiocarboxylic acid” and an “aryl thiol thiocarboxylic acid” describe a -R-C(=S)-SH group, where R is an alkyl or an aryl, respectively.
A “sulfate” is a -O-SO,-OR’ group, with R’ as defined hereinabove.
A “sulfite” group is a -O-S(=0)-OR’ group, with R’ as defined hereinabove.
A “bisulfite” is a sulfite group, where R’ is hydrogen.
A “thiosulfate” is an -O-SO,-SR’ group, with R’ as defined hereinabove.
A “thiosulfite” group is an -O-S(=0)-SR’ group, with R’ as defined : 5 hereinabove.
The terms “alkyl/aryl phosphine” describe a -R-PH, group, with R being an alkyl or an aryl, respectively, as defined above.
The terms “alkyl and/or aryl phosphine oxide” describe a -R’-PR’’3(=0) group, with R’ and R”’ being an alkyl and/or an aryl, as defined hereinabove.
The terms “alkyl and/or aryl phosphine sulfide” describe a -R’-PR’’3(=S) group, with R” and R’” being an alkyl and/or an aryl, as defined hereinabove.
The terms “alkyl/aryl phosphonic acid” describe a -R’-P(=O)(OH), group, with R’ being an alkyl or an aryl as defined above.
The terms “alkyl/aryl phosphinic acid” describes a -R’-P(OH); group, with R’ being an alkyl or an aryl as defined above.
A “phosphate” is a -O-P(=0)(OR’)(OR’’) group, with R’ and R"’ as defined hereinabove.
A “hydrogen phosphate” is a phosphate group, where R’ is hydrogen.
A “dihydrogen phosphate” is a phosphate group, where R’ and R’’ are both hydrogen.
A “thiophosphate” is a -S-P(=0)(OR’); group, with R’ as defined hereinabove.
A “phosphite” is an -O-P (OR’), group, with R’ as defined hereinabove.
A “pyrophosphite” is an -O-P-(OR’)-O-P(OR"’); group, with R’ and R" as defined hereinabove.
A “triphosphate” describes an -OP(=0)(OR’)-O-P(=0)(OR’’)-O-
P(=0)(OR’’’),, with R’, R’” and R”*’ are as defined hereinabove.
As used herein, the term “guanidine” describes a -R'NC(=N)-NR’’R’’" group, with R’, R*’ and R’"’ as defined herein.
The term “S-dithiocarbamate” describes a -SC(=S)-NR’R’’ group, with R” and - 30 Ras defined hereinabove.
The term “N-dithiocarbamate™ describes an R’SC(=S)-NR’’- group, with R’ and R’’ as defined hereinabove.
A “bicarbonate” is an -O-C(=0)-O" group.
A “carbonate” is an -O-C(=0)-OH group.
A “perchlorate” 1s an -O-Cl(=0); group. . A “chlorate” is an -O-CI(=0), group.
A “chlorite” is an -O-Cl(=0) group. : 5 A “hypochlorite” is an -OCl group.
A “perbromate’ is an -O-Br(=0); group.
A “bromate” is an -O-Br(=0), group.
A “bromite” is an -O-Br(=0) group.
A “hypobromite” is an -OBr group.
A “periodate” is an -O-1(=0); group.
A “iodate” is an -O-I(=0), group.
The term “tetrahalomanganate” describes MnCls, MnBry and Mnl,.
The term “tetrafluoroborate” describes a -BF4 group.
A “tetrafluoroantimonate” is a SbF¢ group.
A “hypophosphite” is a -P(OH); group.
The term “metaborate” describes the group
R"O :
Rg o_O
B
OR" where R’, R”* and R’’’ are as defined hereinabove.
The terms “tetraalkyl/tetraaryl borate” describe a R’B” group, with R’ being an alkyl or an aryl, respectively, as defined above.
A ““tartarate” is an -OC(=0)-CH(OH)-CH(OH)-C(=0)OH group.
A “salycilate” is the group
COO0-
Oo”
A “succinate” is an -O-C(=0)-(CH;),-COOH group. . 25 A “citrate” is an -O-C(=0)-CH,-CH(OH)(COOH)-CH,-COOH group.
An “ascorbate” 1s the group . oH
Nr
HO OH .
A “saccharirate” 1s an oxidized saccharide having two carboxylic acid group.
The term “amino acid” as used herein includes natural and modified amino acids and hence includes the 21 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, : 5 phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor- leucine and omithine. Furthermore, the term “amino acid” includes both D- and L- amino acids which are linked via a peptide bond or a peptide bond analog to at least one addition amino acid as this term is defined herein.
A “hydroxamic acid” is a -C(=0)-NH-OH group.
A “thiotosylate” is the group [o}
I. 2
Similarly, each of the alkylene chains B, ---- Bn independently has a gencral formula III: fon —Cp—C(p+1)H + CqH—
H
Formula II wherein pis an integer that equals 0 or g+1 and q is an integer from g+2 to g+20.
Hence, each of the alkylene chains B) ---- Bn is comprised of a plurality of carbon atoms Cp, Cp+l, Cp+2 --, Cg-1 and Cq, substituted by the respective Rp,
Rp+l, Rp+2 -, Rg-1 and Rq groups. Preferably, each of the alkylene chains B, --- : Bn includes 2-20 carbon atoms, more preferably 2-10, and most preferably 2-6 carbon atoms.
As is defined hereinabove, in cases where p equals 0, the component -
CpH(Rp)- is absent from the structure. In cases where p equals g+1, it can be either 1 or 4-11. The integer q can be either 2 or 5-20.
Each of the substituents Rp, Rp+1 --- Rn can be any of the substituents described hereinabove with respect to Ry, R; and Rg. } Hence, a preferred linear polyamine according to the present invention includes two or more alkylene chains. The alkylene chains are interrupted ! 5 therebetween by a heteroatom and cach is connected to a heteroatom at one end thereof. Preferably, each of the alkylene chains include at least two carbon atoms, so as to enable the formation of a stable chelate between the heteroatoms and the copper ion.
The linear polyamine delineated in Formula I preferably includes at least one chiral carbon atom. Hence, at least one of C,, C; and Cg in the alkylene chain A and/or at least one of Cp, Cp+1 and Cq in the alkylene chain B is chiral.
A preferred linear polyamine according to the present invention is tetraethylenepentamine. Other representative e¢xamples of preferred linear polyamines usable in the context of the present invention include, without limitation, ethylendiamine, diethylenetriamine, triethylenctctramine, triethylenediamine, aminoethylethanolamine, pentaethylenchexamine, triethylenetetramine, N,N'-bis(3- aminopropyl)-1,3-propanediamine, and N,N’-Bis(2-animoethyl)-1,3 propanediamine.
In cases where the polyamine chelator is a cyclic polyamine, the polyamine can have a general formula IV: 7
X—Am—(Y,B)),"--(YnBn)n—2 : Formula IV wherein m is an integer from 1 to 10; n is an integer from 0 to 20; X and Z are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; Y, and Yn are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; A is an alkylene chain having between 1 and 10 substituted and/or non-substituted carbon atoms; B; and Bn : are each independently an alkylene chain having between 1 and 20 substituted and/or non-substituted carbon atoms; and D is a bridging group having a general formula V:
U-W-V
Formula V \ whereas U and V are each independently selected from the group consisting of ¢ 5 substituted hydrocarbon chain and non-substituted hydrocarbon chain; and W is selected from the group consisting of amide, ether, ester, disulfide, thioether, thioester, imine and alkene, provided that at least one of said X, Z, Y, and Yn is a -
NH group and/or at least one of said carbon atoms in said alkylene chains is substituted by an amine group.
Optionally, the cyclic polyamine has one of the general formulas VI-X: 7
X—Am—(Y,B})---(YnBn)n—2ZH
Formula VI
TT
HX—Am—(Y,B,),---(YnBn)n—2
Formula VII
PT
X——Am=—(Y,B,);--(YnBn)n—ZH
Formula VIII
ERCEERT EPR RY p)
XA (Ybor
Formula IX ) 25 : IA
HX—Am——(YB,),---(YnBn)n—ZH
Formula X wherein m, n, X, Y, Yn, Z, A, B and D are as described above and further wherein should the bridging group D is attached at one end to A (Formulas VI, VII and X), U , or V are being attached to one carbon atom in the alkylene chain and should D is attached at one end to Bl or Bn (Formulas VIII, IX and X), U or V are being attached . 5S to one carbon atom in the alkylene chain.
Hence, a preferred cyclic polyamine according to the present invention includes two or more alkylene chains, A, B, --- Bn, as is detailed hereinabove with respect to the linear polyamine. The alkylene chains can form a cyclic structure by being connected, via the bridging group D, between the ends thereof, namely between the heteroatoms X and Z (Formula 1V). Optionally, the alkylene chains can form a conformationally restricted cyclic structure by being connected, via the bridging group D, therebetween (Formula X). Further optionally, a conformationally restricted cyclic structure can be formed by connecting one alkylene chain to one terminal heteroatom (X or Z, Formulas VI-1X).
As is described hereinabove, in cases where the cyclic structure is formed by connecting one alkylene chain to one terminal heteroatom, as is depicted in Formulas
VI-IX, the bridging group D connects a terminal heteroatom, namely X or Z, and one carbon atom in the alkylene chains A and B, ---- Bn. This carbon atom can be anyone of Cy, C,, Cg, Cp, Cp+1 and Cq described hereinabove.
As is further described hereinabove, the cyclic structure is formed by the bridging group D, which connects two components in the structure. The bridging group D has a general formula U-W-V, where each of U and V is a substituted or non- substituted hydrocarbon chain.
As used herein, the phrase “hydrocarbon chain” describes a plurality of carbon atoms which are covalently attached one to another and are substituted, inter alia, by hydrogen atoms. The hydrocarbon chain can be saturated, unsaturated, branched or unbranched and can therefore include one or more alkyl, alkenyl, alkynyl, cycloalkyl » and aryl groups and combinations thereof.
The length of the hydrocarbon chains, namely the number of carbon atoms in ~ 30 the chains, is preferably determined by the structure of the cyclic polyamine, such that on one hand, the ring tension of the formed cyclic structure would be minimized and on the other hand, an efficient chelation with the copper ion would be achieved.
When the hydrocarbon chain is substituted, the substituents can be any one or combinations of the substituents described hereinabove with respect to R;, R; and Rg
R in the linear polyamine.
The two hydrocarbon chains are connected therebetween by the group W, ‘ 5 which can be amide, ether, ester, disulfide, thioether, thioester, imine and alkene.
As uscd herein, the term “ether” is an -O- group.
The term “ester” is a -C(=0)-O- group.
A “disulfide” is a -S-S- group.
A “thioether” is a -S- group.
A “thioester” is a -C(=0)-S- group.
An “imine” 1s a -C(=NH)- group.
An “alkene” is a -CH=CH- group.
The bridging group D is typically formed by connecting reactive derivatives of the hydrocarbon chains U and V, so as to produce a bond therebetween (W), via well- known techniques, as is described, for example, in U.S. Patent No. 5,811,392.
As is described above with respect to the linear polyamine, the cyclic polyamine must include at least one amine group, preferably at least two amine groups and more preferably at least four amine groups, so as to form a stable copper chelate.
A preferred cyclic polyamine according to the present invention is cyclam (1,4,8,11-tetraazacyclotetradecane).
As 1s described hereinabove, the polyamine chelator of the present invention can further include a multimeric combination of one or more linear polyamine(s) and one or more cyclic polyamine(s). Such a polyamine chelator can therefore be comprised of any combinations of the linear and cyclic polyamines described hereinabove.
Preferably, such a polyamine chelator has a general Formula XI: {EDFQi1~(G el} v= {(E2)i-[Qa(Ga)T hie { (En)1-[Qn-(Gi)o] }1 - 30 Formula XI wherein n is an integer greater than I; each of f, g, h, 1, j, k, I, 0 and t is independently an integer from 0 to 10; each of E), E> and En is independently a linear polyamine, as is described hereinabove; each of G,, G, and Gn is independently a cyclic polyamine as is described hereinabove; and each of Q,, Q, and Qn is independently a linker . linking between two of said polyamines, provided that at least one of said Q,, Qz and
Qn is an amine group and/or at least one of said linear polyamine and said cyclic ¢ 5 polyamine has at least one free amine group.
Each of E,, E> and En in Formula XI represent a linear polyamine as is described in detail hereinabove, while each of G;, G2 and Gn represents a cyclic polyamine as is described in detail hereinabove.
The polyamine described in Formula XI can include one or more linear polyamine(s), each connected to another linear polyamine or to a cyclic polyamine.
Each of the linear or cyclic polyamines in Formula XI is connected to another polyamine via one or more linker(s), represented by Q;, Q; and Qn in Formula XI.
Each of the linker(s) Q,, Q; and Qn can be, for example, alkylene, alkenylene, alkynylene, arylene, cycloalkylene, hetroarylene, amine, azo, amide, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphonium, ketoester, carbonyl, thiocarbonyl, ester, ether, thioether, carbamate, thiocarbamate, urea, thiourea, borate, borane, boroaza, silyl, siloxy and silaza.
As used herein, the term “alkenylene” describes an alkyl group which consists of at least two carbon atoms and at least one carbon-carbon double bond.
The term “alkynylene” describes an alkyl group which consists of at least two carbon atoms and at least one carbon-carbon triple bond.
The term “cycloalkylene™ describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system. Examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. . The term “arylene” describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a . 30 completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or unsubstituted.
The term “heteroarylene” describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, x such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of ‘ 5 heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted.
As used in the context of the linker of the present invention, the term “amine” describes an -NR’-, wherein R’ can be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl or heterocyclic, as these terms are defined hereinabove.
As is further used in the context of the linker of the present invention, the term “azo” describes a -N=N- group.
The term “amide” describes a -C(=0)-NR’- group, where R’ is as defined hereinabove.
The term “ammonium” describes an -N"HR’- group, where R’ is as defined hereinabove.
The term “sulfinyl” describes a -S(=0)- group.
The term “sulfonyl” describes a -S(=0),- group.
The term “sulfonamido” describes a -S(=0),-NR’- group, with R’ as defined hereinabove.
The term “phosphonyl” describes a -O-P(=0)(OR’)- group, with R’ as defined hereinabove.
The term “phosphinyl” describes a -PR’- group, with R’ as defined hereinabove.
The term “phosphonium” is a -P"R’R’’, where R’ and R”’ are as defined hereinabove.
The term “ketoester” describes a -C(=0)-C(=0)-O- group. ] The term “carbonyl” describes a -C(=0)- group.
The term “thiocarbonyl” describes a -C(=S)- group. . 30 The term “carbamate” describes an -OC(=0)-NR’- group, with R’ as defined hereinabove.
The term “thiocarbamate” describes an -OC(=S)-NR- group, with R’ as defined hereinabove.
The term “urea” describes an -NR’-C(=0)-NR’’- group, with R” and R”’ and as defined hereinabove. . The term “thiourea” describes a -NR’-C(=S)-NR’- group, with R’ and R’’ as defined hereinabove. : 5 The term “borate” describes an -O-B-(OR)- group, with R as defined hereinabove.
The term “borane” describes a -B-R-’- group, with R as defined hereinabove.
The term “‘boraza’™ describes a -B (NR’R”’)- group, with R” and R’’ as defined hereinabove.
The term “silyl” describes a -SiR’R’’-, with R’ and R”’ as defined herein.
The term *‘siloxy” is a -Si-(OR);-, with R as defined hereinabove.
The term “silaza” describes a -Si-(NR’R’’),-, with R’ and R’’ as defined herein.
It should be noted that all the terms described hereinabove in the context of the linker of the present invention arc the same as described above with respect to the substituents. However, in distinction from the substituent groups, which are connected to a component at one end thereof, the linker groups are connected to two components at two sites thereof and hence, these terms have been redefined with respect to the linker.
As has been mentioned hereinabove, according to the presently most preferred embodiment of the present invention, the polyamine chelator is tetracthylenepentamine (TEPA). However, other preferred polyamine chelators include, without limitation, ethylendiamine, diethylenetriamine, triethylenetetramine, triethylenediamine, aminoethylethanolamine, aminoethylpiperazine, pentaecthylenchexamine, tricthylenctetramine, captopril, penicilamine, N,N'-bis(3- aminopropyl)-1,3-propanediamine, N,N’-Bis(2-animoethyl)-1,3-propanediamine, 1,7- dioxa-4,10-diazacyclododecane, 1,4,8,11-tetraazacyclotetradecane-5,7-dione, 1,4,7- . triazacyclononane, 1-oxa-4,7,10-triazacyclododecane, 1,4,8,12- tetraazacyclopentadecane and 1,4,7,10-tetraazacyclododecane. . 30 The above listed preferred chelators are known in their high affinity towards copper ions. However, these chelators are further beneficially characterized by their substantial affinity also towards other transition metals, as is described by Ross and
Frant [Ross JW and Frant MS. Chelometric indicators, titration with the solid-state cupric ion selective electrode. Analytical Chemistry 41:1900, 1969], which is incorporated by reference as if fully set forth herein. . All the polyamine chelators described hereinabove can be either commercially obtained or can be synthesized using known procedures such as described, for . 5 example, in: T.W. Greene (ed.), 1999 (“Protective Groups in Organic Synthesis” 3ed
Edition, John Wiley & Sons, Inc., New York 779 pp); or in: R.C. Larock and V.C.H.
Wioley, “Comprehensive Organic Transformations — A Guide to Functional Group
Preparations”, (1999) 2™ Edition.
A preferred procedure for preparing tetraethylenepentamine-copper chelate (TEPA-Cu) is described in PCT/IL03/00062.
The copper chelate or chelator can be provided to the cell culture medium.
The final concentrations of copper chelate may be, depending on the specific application, in the micromolar or millimolar ranges, for example, within about 0.1 uM to about 100 mM, preferably within about 4 uM to about S0 mM, more preferably within about 5 uM to about 40 mM.
The methods described hereinabove for ex-vivo expanding hematopoietic stem cell populations result, inter alia, in an expanded population of hematopoietic stem cells.
Thus, further according to an aspect of the present invention there are provided ex-vivo expanded populations of hematopoietic stem cells, obtained by any of the methods described hereinabove. The expanded populations of hematopoietic stem cells according to the present invention comprise a plurality of cells characterized by 3-20 % of the cells being reselectable CD34+ cells, of which at least 40 % of cells are
CD34+yim, i.e., fall below the median intensity in a FACS analysis, wherein, in the reselectable CD34+ cells, a majority of cells which are Lin" are also CD34+;, cells.
In one embodiment, the population of hematopoietic stem cells has a single genetic background. . In another embodiment, the ex-vivo expanded population of hematopoietic stem cells comprises at least N cells derived from a single donor, wherein N equals ) 30 the average number of CD34+ cells derived from one sample of hematopoietic mononuclear cells, multiplied by 1,000.
Cell surface expression of the CD34 and/or Lin markers can be determined, for example, via FACS analysis or immunohistological staining techniques. A self renewal potential of the hematopoietic stem cells can be determined in-vitro by long term colony formation (LTC-CFUc), as is further exemplified in the Examples section . that follows.
As is discussed in detail hereinabove, ex-vivo expansion of hematopoietic stem : 5 cells can be advantageously utilized in various applications such as, for example, hematopoietic cells transplantation or implantation, adoptive immunotherapy and gene therapy. The ability to practice the ex-vivo expansion of hematopoietic stem cells with hematopoietic mononuclear cells as the cells source substantially facilitates the utilization of the methods described hereinabove in these applications.
Hence, according to another aspect of the present invention there is provided a method of hematopoietic cells transplantation or implantation. The method according to this aspect of the present invention is effected by (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor, (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing an expression and/or activity of
CD38, so as to expand a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo, and (c) transplanting or implanting the thus obtained hematopoietic stem cells to a recipient.
As is described hereinabove, various agents can be used in the context of the different aspects of the present invention for reducing an expression and/or activity of
CD38.
Thus, in a particular embodiment of this aspect of the present invention, the 2s method is effected by providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or . Vitamin D, as is described hereinabove.
In another particular embodiment, the method is effected by providing the . 30 hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor, the retinoid
X receptor and/or the Vitamin D receptor, as is described hereinabove.
In another particular embodiment of this aspect of the present invention, the method is effected by providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving } 5 PI 3-kinase, as is described hereinabove.
In still another particular embodiment of this aspect of the present invention, the method is effected by providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, as is described hereinabove.
In another particular embodiment of this aspect of the present invention, the method is effected by providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with a PI 3-kinase inhibitor, as is described hereinabove.
In another aspect of the present invention, the method of hematopoietic cells transplantation or implantation described above is effected by providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with one or more of the copper chelator(s) or chelate(s) described hereinabove.
In any of the methods of this aspect of the present invention, the donor and the recipient can be a single individual or different individuals, for example, allogeneic or xenogeneic individuals. When allogeneic transplantation is practiced, regimes for reducing implant rejection and/or graft vs. host disease, as well know in the art, should be undertaken. Such regimes are currently practiced in human therapy. Most advanced regimes are disclosed in publications by Slavin S. et al, e.g., J Clin
Immunol (2002) 22: 64, and J Hematother Stem Cell Res (2002) 11: 265), Gur H. et al. (Blood (2002) 99: 4174), and Martelli MF et al, (Semin Hematol (2002) 39: 48), which are incorporated herein by reference.
The methods described hereinabove can be utilized to produce transplantable hematopoietic cell preparations, such that according to yet another aspect of the } 30 present invention there is provided a transplantable hematopoietic cell preparation, which comprises an expanded population of hematopoietic stem cells propagated ex- vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent for reducing the expression and/or activity of CD38, while at the same time, . substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier. ‘ 5 As is described hereinabove, various agents were found to reduce the expression and/or activity of CD38, while at the same time, substantially inhibit differentiation of the hematopoietic stem cells under these conditions.
Hence, in a particular embodiment of this aspect of the present invention, the agent described above is an agent that reduces a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, while at the same time, substantially inhibits differentiation of the hematopoietic stem cells.
In another particular embodiment of this aspect of the present invention, the agent described above is an agent that reduces a capacity of the hematopoietic mononuclear cells in responding to retinoic acid receptor, retinoid X receptor and/or
Vitamin D receptor signaling, while at the same time, substantially inhibits differentiation of the stem cells.
In yet another particular embodiment of this aspect of the present invention, the agent described above is an agent that reduces a capacity of the hematopoietic mononuclear cells in responding to PI 3-kinase signaling, while at the same time, substantially inhibits differentiation of the stem cells.
In still another particular embodiment of this aspect of the present invention, the agent described above comprises an effective amount of an agent selected from the group consisting of nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative and a nicotinamide or a nicotinamide analog metabolite.
In still another particular embodiment of this aspect of the present invention, the agent described above comprises an effective amount of a PI 3-kinase inhibitor.
According to still another aspect of the present invention there is provided a transplantable hematopoietic cell preparation, which comprises an expanded } 30 population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of at least one copper chelate or chelator, as defined hereinabove, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
As is further discussed hereinabove, the ex-vivo expansion of hematopoietic stem cells of the present invention can be utilized in adoptive immunotherapy. . 5 Similarly to the hematopoietic transplantation or implementation methods of the present invention, a method of adoptive therapy according to the present invention is effected by (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, with each of the copper chelators or chelates described hereinabove and/or each of the agents for reducing the expression and/or activity of CD38 described hereinabove, so as to expand the population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells, as is detailed hereinabove; and (c) transplanting the thus obtained hematopoietic stem cells to the recipient.
As is further detailed below, stem cells in general and hematopoietic stem cells in particular may serve to exert cellular gene therapy.
Gene therapy as used herein refers to the transfer of genetic maternal (e.g.,
DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition or phenotype. The genetic material of interest encodes a product (e.g., a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value. For review see, in general, the text "Gene Therapy" (Advanced in Pharmacology 40, Academic Press, 1997).
Two basic approaches to gene therapy have evolved: (1) ex-vivo or cellular gene therapy; and (ii) in vivo gene therapy. In ex-vivo gene therapy cells are removed from a patient, and while being cultured are treated in-vitro. Generally, a functional replacement gene is introduced into the cells via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically re-implanted cells have been shown to express the transfected genetic material in situ.
Hence, further according to an aspect of the present invention, there is provided a method of genetically modifying stem cells with an exogene. The method, according to this aspect of the present invention, is effected by (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic : 5 committed cells and a minor fraction of hematopoietic stem and progenitor cells, (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, with each of the copper chelators or chelates described hereinabove and/or each of the agents for reducing the expression and/or activity of CD38 described hereinabove, so as to expand the population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells, as is detailed hereinabove, and (c) genetically modifying the hematopoietic stem cells with the exogene.
In a preferred embodiment, genetically modifying the cells is effected by a vector, which comprises the exogene or transgene, which vector is, for example, a viral vector or a nucleic acid vector. Many viral vectors suitable for use in cellular gene therapy are known, examples are provided hereinbelow. Similarly, a range of nucleic acid vectors can be used to genetically transform the expanded cells of the invention, as is further described below.
Accordingly, the expanded cells of the present invention can be modified to express a gene product. As used herein, the phrase “gene product” refers to proteins, peptides and functional RNA molecules. Generally, the gene product encoded by the nucleic acid molecule is the desired gene product to be supplied to a subject.
Examples of such gene products include proteins, peptides, glycoproteins and lipoproteins normally produced by an organ of the recipient subject. For example, gene products which may be supplied by way of gene replacement to defective organs in the pancreas include insulin, amylase, protease, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, ribonuclease, deoxyribonuclease, , triaclyglycerol lipase, phospholipase A), elastase, and amylase; gene products normally produced by the liver include blood clotting factors such as blood clotting : 30 Factor VIII and Factor IX, UDP glucuronyl transferae, ornithine transcarbanoylase, and cytochrome p450 enzymes, and adenosine deaminase, for the processing of serum adenosine or the endocytosis of low density lipoproteins; gene products produced by the thymus include serum thymic factor, thymic humoral factor, thymopoietin, and thymosin a]; gene products produced by the digestive tract cells include gastrin, secretin, cholecystokinin, somatostatin, serotinin, and substance P.
Alternatively, the encoded gene product is one, which induces the expression of the desired gene product by the cell (e.g., the introduced genetic material encodes a . 5 transcription factor, which induces the transcription of the gene product to be supplied to the subject).
In still another embodiment, the recombinant gene can provide a heterologous protein, e.g., not native to the cell in which it is expressed. For instance, various human MHC components can be provided to non-human cells to support engraftment in a human recipient. Alternatively, the transgene is one, which inhibits the expression or action of a donor MHC gene product.
A nucleic acid molecule introduced into a cell is in a form suitable for expression in the cell of the gene product encoded by the nucleic acid. Accordingly, the nucleic acid molecule includes coding and regulatory sequences required for
I5 transcription of a gene (or portion thereof) and, when the gene product is a protein or peptide, translation of the gene acid molecule include promoters, enhancers and polyadenylation signals, as well as sequences necessary for transport of an encoded protein or peptide, for example N-terminal signal sequences for transport of proteins or peptides to the surface of the cell or secretion.
Nucleotide sequences which regulate expression of a gene product (e.g, promoter and enhancer sequences) are selected based upon the type of cell in which the gene product is to be expressed and the desired level of expression of the gene product. For example, a promoter known to confer cell-type specific expression of a gene linked to the promoter can be used. A promoter specific for myoblast gene expression can be linked to a gene of interest to confer muscle-specific expression of that gene product. Muscle-specific regulatory elements, which are known in the art, include upstream regions from the dystrophin gene (Klamut et al., (1989) Mol. Cell
Biol.9: 2396), the creatine kinase gene (Buskin and Hauschka, (1989) Mol. Cell Biol. 9: 2627) and the troponin gene (Mar and Ordahl, (1988) Proc. Natl. Acad. Sci. USA. . 30 85:6404). Regulatory elements specific for other cell types are known in the art (e.g., the albumin enhancer for liver-specific expression; insulin regulatory elements for pancreatic islet cell-specific expression; various neural cell-specific regulatory elements, including neural dystrophin, neural enolase and A4 amyloid promoters).
Alternatively, a regulatory element, which can direct constitutive expression of a gene in a variety of different cell types, such as a viral regulatory element, can be used. Examples of viral promoters commonly used to drive gene expression include those derived from polyoma virus, Adenovirus 2, cytomegalovirus and Simian Virus . 5 40, and retroviral LTRs.
Alternatively, a regulatory element, which provides inducible expression of a gene linked thereto, can be used. The use of an inducible regulatory clement (e.g., an inducible promoter) allows for modulation of the production of the gene product in the cell. Examples of potentially useful inducible regulatory systems for use in eukaryotic cells include hormone-regulated elements (e.g., see Mader, S. and White,
J.H. (1993) Proc. Natl. Acad. Sci. USA 90: 5603-5607), synthetic ligand-regulated elements (see, ¢.g., Spencer, D.M. et al. 1993) Science 262: 1019-1024) and ionizing radiation-regulated elements (e.g., see Manome, Y. Et al. (1993) Biochemistry 32: 10607-10613; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1014-10153).
Additional tissue-specific or inducible regulatory systems, which may be developed, can also be used in accordance with the invention.
There are a number of techniques known in the art for introducing genetic material into a cell that can be applied to modify a cell of the invention.
In one embodiment, the nucleic acid is in the form of a naked nucleic acid molecule. In this situation, the nucleic acid molecule introduced into a cell to be modified consists only of the nucleic acid encoding the gene product and the necessary regulatory elements.
Alternatively, the nucleic acid encoding the gene product (including the necessary regulatory elements) is contained within a plasmid vector. Examples of plasmid expression vectors include CDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman, et al. (1987) EMBO J. 6: 187-195).
In another embodiment, the nucleic acid molecule to be introduced into a cell is contained within a viral vector. In this situation, the nucleic acid encoding the gene product is inserted into the viral genome (or partial viral genome). The regulatory . 30 elements directing the expression of the gene product can be included with the nucleic acid inserted into the viral genome (i.e., linked to the gene inserted into the viral genome) or can be provided by the viral genome itself.
Naked nucleic acids can be introduced into cells using calcium phosphate mediated transfection, DEAE-dextran mediated transfection, electroporation, liposome-mediated transfection, direct injection, and receptor-mediated uptake.
Naked nucleic acid, e.g., DNA, can be introduced into cells by forming a precipitate containing the nucleic acid and calcium phosphate. For example, a
HEPES-buffered saline solution can be mixed with a solution containing calcium chloride and nucleic acid to form a precipitate and the precipitate is then incubated with cells. A glycerol or dimethyl sulfoxide shock step can be added to increase the amount of nucleic acid taken up by certain cells. CaPO4-mediated transfection can be used to stably (or transiently) transfect cells and is only applicable to in vitro modification of cells. Protocols for CaPO4-mediated transfection can be found in
Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing
Associates, (1989), Section 9.1 and in Molecular Cloning: A Laboratory Manual, 2nd
Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), Sections 16.32-16.40 or other standard laboratory manuals.
Naked nucleic acid can be introduced into cells by forming a mixture of the nucleic acid and DEAE-dextran and incubating the mixture with the cells. A dimethylsulfoxide or chloroquine shock step can be added to increase the amount of nucleic acid uptake. DEAE-dextran transfection is only applicable to in vitro modification of cells and can be used to introduce DNA transiently into cells but 1s not preferred for creating stably transfected cells. Thus, this method can be used for short-term production of a gene product but is not a method of choice for long-term production of a gene product. Protocols for DEAE-dextran-mediated transfection can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.)
Greene Publishing Associates (1989), Section 9.2 and in Molecular Cloning: A
Laboratory Manual, 2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory
Press, (1989), Sections 16.41-16.46 or other standard laboratory manuals. : Naked nucleic acid can also be introduced into cells by incubating the cells and the nucleic acid together in an appropriate buffer and subjecting the cells to a : 30 high-voltage electric pulse. The efficiency with which nucleic acid is introduced into cells by electroporation is influenced by the strength of the applied field, the length of the electric pulse, the temperature, the conformation and concentration of the DNA and the ionic composition of the media. Electroporation can be used to stably (or transiently) transfect a wide variety of cell types and is only applicable to in vitro modification of cells. Protocols for electroporating cells can be found in Current
Protocols in Molecular Biology, Ausubel F.M. et al. (eds.) Greene Publishing
Associates, (1989), Section 9.3 and in Molecular Cloning: A Laboratory Manual, 2nd : 5 Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), Sections 16.54-16.55 or other standard laboratory manuals.
Another method by which naked nucleic acid can be introduced into cells includes liposome-mediated transfection (lipofection). The nucleic acid is mixed with a liposome suspension containing cationic lipids. The DNA/liposome complex is then incubated with cells. Liposome mediated transfection can be used to stably (or transiently) transfect cells in culture in vitro. Protocols can be found in Current :
Protocols in Molecular Biology, Ausubel F.M. et al. (eds.) Greene Publishing
Associates, (1989), Section 9.4 and other standard laboratory manuals. Additionally, gene delivery in vivo has been accomplished using liposomes. See for example
Nicolau et al. (1987) Meth. Enz. 149:157-176; Wang and Huang (1987) Proc. Natl.
Acad. Sci. USA 84:7851-7855; Brigham et al. (1989) Am. J Med. Sci. 298:278; and
Gould-Fogerite et al. (1989) Gene 84:429-438.
Naked nucleic acid can also be introduced into cells by directly injecting the nucleic acid into the cells. For an in vitro culture of cells, DNA can be introduced by microinjection. Since each cell is microinjected individually, this approach is very labor intensive when modifying large numbers of cells. However, a situation wherein microinjection is a method of choice is in the production of transgenic animals (discussed in greater detail below). In this situation, the DNA is stably introduced into a fertilized oocyte, which is then allowed to develop into an animal. The resultant animal contains cells carrying the DNA introduced into the oocyte. Direct injection has also been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468). A delivery apparatus (e.g., a “gene gun”) for injecting DNA into cells in vivo can be used. Such an apparatus is commercially available (e.g., from BioRad). : 30 Naked nucleic acid can be complexed to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor to be taken up by receptor-mediated endocytosis (see for example Wu, G. and Wu, C.H. (1988) J. Biol. Chem. 263: 14621;
Wilson et al. (1992) J. Biol. Chem. 267: 963-967; and U.S. Patent No. 5,166,320).
Binding of the nucleic acid-ligand complex to the receptor facilitates uptake of the
DNA by receptor-mediated endocytosis. Receptors to which a DNA-ligand complex . has targeted include the transferrin receptor and the asialoglycoprotein receptor. A
DNA-ligand complex linked to adenovirus capsids which naturally disrupt : 5S endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8850; Cristiano et al. (1993) Proc. Natl. Acad.
Sci. USA 90: 2122-2126). Receptor-mediated DNA uptake can be used to introduce
DNA into cells either in vitro or in vivo and, additionally, has the added feature that
DNA can be selectively targeted to a particular cell type by use of a ligand which binds to a receptor selectively expressed on a target cell of interest.
Generally, when naked DNA is introduced into cells in culture (e.g., by one of the transfection techniques described above) only a small fraction of cells (about 1 out of 10%) typically integrate the transfected DNA into their genomes (i.e., the DNA is maintained in the cell episomally). Thus, in order to identify cells, which have taken up exogenous DNA, it is advantageous to transfect nucleic acid encoding a selectable marker into the cell along with the nucleic acid(s) of interest. Preferred selectable markers include those, which confer resistance to drugs such as G418, hygromycin and methotrexate. Selectable markers may be introduced on the same plasmid as the gene(s) of interest or may be introduced on a separate plasmid.
A preferred approach for introducing nucleic acid encoding a gene product into a cell 1s by use of a viral vector containing nucleic acid, e.g., a cDNA, encoding the gene product. Infection of cells with a viral vector has the advantage that a large proportion of cells receive the nucleic acid which can obviate the need for selection of cells which have received the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid and viral vector systems can be : used either in vitro or in vivo.
Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for review see Miller, A.D. (1990) Blood 76: 271). A recombinant retrovirus can be constructed having a nucleic acid encoding a gene product of interest inserted into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions, which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) . 5 Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM, which are well known to those skilled in the art. Examples of suitable packaging virus lines include yCrip, yCrip, y2 and yAm. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230: 1395-1398;
Danosand Mulligan (1988) Proc. Natl. Acad. Sci. USA 85: 6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci USA 85:3014-3018; Armentano et al., (1990) Proc. Natl.
Acad. Sci. USA 87: 6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8039-8043; Feri et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad.
Sci USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; US Patent No. 4,868,116; US Patent No. 4,980,286; PCT Application
WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and
PCT Application WO 92/07573). Retroviral vectors require target cell division in order for the retroviral genome (and foreign nucleic acid inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell.
The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988)
BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dI324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl.
Acad. Sci. USA 89: 6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl.
Acad. Sci. USA 90: 2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl.
Acad. Sci. USA 89: 2581-2584). Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra, Haj-Ahmand and Graham (1986) J. Virol 57: 267). Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral E1 and E3 genes but retain as much as 80% of the adenoviral genetic material.
Adeno-associated virus (AAV) is a naturally occurring defective virus that
I5 requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al.
Curr. Topics In Micro. And Immunol. (1992) 158: 97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flottc et al. (1992) Am. J. Respir.
Cell. Mol. Biol. 7: 349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and
McLaughlin et al. (1989) J. Virol. 62: 1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6466- 6470; Tratschin et al. (1985) Mol. Cell Biol. 4: 2072-2081; Wondisford ct al. (1988)
Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51: 61 1-619; and Flotte et al. (1993) J. Biol. Chem. 268: 3781-3790).
The efficacy of a particular expression vector system and method of introducing nucleic acid into a cell can be assessed by standard approaches routinely used in the art. For example, DNA introduced into a cell can be detected by a filter hybridization technique (e.g., Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR). The gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional : 5 assay to detect a functional activity of the gene product, such as an enzymatic assay.
If the gene product of interest to be interest to be expressed by a cell is not readily assayable, an expression system can first be optimized using a reporter gene linked to the regulatory elements and vector to be used. The reporter gene encodes a gene product, which is easily detectable and, thus, can be used to evaluate efficacy of the system. Standard reporter genes used in the art include genes encoding [- galactosidase, chloramphenicol acetyl transferase, luciferase and human growth hormone.
When the method used to introduce nucleic acid into a population of cells results in modification of a large proportion of the cells and efficient expression of the gene product by the cells (e.g., as is often the case when using a viral expression vector), the modified population of cells may be used without further isolation or subcloning of individual cells within the population. That is, there may be sufficient production of the gene product by the population of cells such that no further cell isolation is needed. Alternatively, it may be desirable to grow a homogenous population of identically modified cells from a single modified cell to isolate cells, which efficiently express the gene product. Such a population of uniform cells can be prepared by isolating a single modified cell by limiting dilution cloning followed by expanding the single cell in culture into a clonal population of cells by standard techniques.
According to a preferred embodiment of the present invention, in each of the methods described hereinabove, providing the hematopoietic mononuclear cells with conditions for ex-vivo cell proliferation is effected by providing the cells with nutrients and with cytokines. Preferably, the cytokines are early acting cytokines, such as, but not limited to, stem cell factor, FLT3 ligand, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-10, interleukin-12, tumor necrosis factor-a and thrombopoietin. It will be appreciated in this respect that novel cytokines are continuously discovered, some of which may find uses in the methods of cell expansion of the present invention.
Late acting cytokines can also be used. These include, for example, granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating } factor, erythropoietin, FGF, EGF, NGF, VEGF, LIF, Hepatocyte growth factor and macrophage colony stimulating factor. - 5 The ability of the agents of the present invention to inhibit differentiation of hematopoietic stem cells present in hematopoietic mononuclear cells can be further used in technical applications such as cells collection and cells culturing.
According to a further aspect of the present invention there is provided a hematopoietic stem cells collection/culturing bag. The cells collection/culturing bag of the present invention is supplemented with an effective amount of a retinoic acid receptor antagonist, a retinoid X receptor antagonist and/or a Vitamin D receptor antagonist, which substantially inhibits cell differentiation of a hematopoietic stem cells fraction of hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells. Altematively, the hematopoietic stem cells collection/culturing bag of the present invention is supplemented with an effective amount of nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite. Still alternatively, the hematopoietic stem cells collection/culturing bag of the present invention is supplemented with an effective amount of a PI 3-kinase inhibitor. Further altematively, the hematopoietic stem cells collection/culturing bag of the present invention is supplemented with an effective amount of one or more copper chelator(s) or chelate(s).
According to an additional aspect of the present invention, there is provided an assay of determining whether a specific molecule/agent, e.g., a retinoic acid receptor antagonist, a retinoid X receptor antagonist, a Vitamin D receptor antagonist, a CD38 inhibitor, a PI 3-kinase inhibitor, a copper chelator or a copper chelate, is an effective agent for expanding a population of hematopoietic stem cells that are present in a hematopoietic mononuclear cells fraction. . 30 The assay, according to this aspect of the present invention, is performed by culturing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells in the presence of the tested agent/molecule and monitoring expansion of the hematopoietic stem cells over time, e.g., a few weeks to a few months. If increased expansion and decreased differentiation occurs, as compared to non-treated cells, the tested agent/molecule is an effective hematopoietic stem cell expansion agent. : 5 Preferably, culturing the hematopoietic mononuclear cells is performed in a presence of an effective amount of a cytokine, preferably, an early acting cytokine or a combination of such cytokines, e.g., thrombopoietin (TPO), interleukin-6 (IL-6), an
FLT-3 ligand and stem cell factor (SCF). This assay can be used, by one ordinarily skilled in the art, to determine, for example, which of the antagonists, inhibitors or copper chelators and chelates listed above is most efficient for the purpose of implementing the various methods and preparations of the present invention described hereinabove. The assay can be further used to determine most effective concentrations and exposure time for achieving optimal results with hematopoietic mononuclear cells of different origins.
In each of the aspects of the present invention described hereinabove, the hematopoietic mononuclear cells can be obtained from any multicellular organism including both animals and plants. Preferably, the hematopoietic mononuclear cells are obtained from the bone marrow (Rowley SD et al. (1998) Bone Marrow
Transplant 21: 1253), the peripheral blood (Koizumi K, (2000) Bone Marrow
Transplant 26: 787, the liver (Petersen BE et al. (1998) Hepatology 27: 433) and neonatal umbilical cord blood.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples. : 30 EXAMPLES
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.
EXAMPLE 1
THE EFFECT OF A COPPER CHELATOR ON THE EX-VIVO EXPANSION
OF HEMATOPOIETIC STEM CELLS OF A MONONUCLEAR CELLS
CULTURE
. 5
Experimental Procedures
Sample collection and processing: Samples were obtained from umbilical cord blood after a normal full-term delivery and were frozen within 24 hours pospartum. The blood cells were thawed in Dextran buffer and incubated for 15 hours in MEM (Biological Industries, lsrael) supplemented with 10 % fetal calf serum (FCS; Biological Industries). The cells were then layered on Ficoll-Hypaque (density 1.077 gram/ml; Sigma) and centrifuged at 400 g for 30 minutes at room temperature.
The mononuclear cells in the interface layer were then collected, washed three times in phosphate-buffered saline (PBS; Biological Industries), and re-suspended in PBS {5 containing 0.5 % human serum albumin (HSA). The cells were then split into two fractions, the first being the mononuclear cells (MNC) fraction and the second fraction was used for purifying CD34 cells by immunomagnetic separation using the “MiniMACS CD34% progenitor cell isolation kit” (Miltenyi Biotec, Aubum, CA) according to the manufacturer’s recommendations. The purity of the CD34" cells obtained ranged between 95 % and 98 %, based on Flow Cytometry evaluation.
Ex-vivo expansion of hematopoietic stem cells: The Mononuclear cells (MNC), obtained as described hereinabove, were plated in 24-well Costar Cell
Clusters (Coming Inc., Corning, NY) or seeded in Culture Bags (American Fluoroseal
Corp.), with alpha minimal essential medium (a-MEM) supplemented with 10 % fetal 2s bovine serum (FBS, Biological Industries), at a concentration of about 10° cells/ml.
The purified CD34 cells were similarly plated or seeded in the Culture Bags, at a concentration of about 10° cells/ml. The media were supplemented with tetracthylpantamine (TEPA) chelator (obtained from Sigma) and/or with the following human recombinant cytokines (all obtained from Perpo Tech, Inc., Rocky
Hill, NJ): Thrombopoietin (TPO), 50 ng/ml; interleukin 6 (IL-6), 50 ng/ml; FLT-3 ligand, 50 ng/ml and a stem cell factor (SCF), 50 ng/ml; occasionally SCF was replaced by IL-3, 20 ng/ml. All cultures were incubated at 37 °C in an atmosphere of
% CO2 in air with extra humidity. At weekly intervals, the cell cultures were semi- depopulated and supplemented with fresh medium containing cytokines. Following : different incubation periods, cells were harvested, stained with trypan blue and enumerated. } 5 Morphological assessment: Morphological characterization of the resulting culture populations was accomplished on aliquots of cells deposited on glass slides via cytospin (Cytocentrifuge, Shandon, Runcorn, UK). Cells were fixed, stained with
May-Grunwald/Giemsa stain and examined microscopically.
Surface antigen analysis: Cells were harvested, washed with a PBS solution containing 1 % bovine sera albumin (BSA) and 0.1 % sodium azide (Sigma), and stained at 4 °C for 60 minutes with fluorescein isothiocyanate or phycoerythrin- conjugated antibodies (all from Immunoquality Products, the Netherlands). The cells were then washed with the same buffer and analyzed by FACS caliber or Facstarplus flow cytometers. Cells were passed at a rate of 1000 cells/second, using saline as the sheath fluid. A 488 nm argon laser beam served as the light source for excitation.
Emission of ten thousand cells was measured using logarithmic amplification, and analyzed using CellQuest software.
Determination of CD34+ cells and subsets: CD34+ surface expression on short and long-term cultures initiated either with purified CD34+ cells or the entire MNC fraction was determined as follows: CD34+ cells were positively reselected (Miltenyi kit) and counted. Purity was confirmed by subsequent FACS and cell morphology analysis, as is described hereinabove.
Reselected CD34+ cell subsets were stained for the following combination of antigens: CD34PE/CD38FITC and CD34PE/38-, 33-, 14-, 15-, 3, 4, 61, 19 (Lin)
FITC.
Cell population calculations:
FACS analysis results are given as percentage values of cells. Absolute . numbers of subsets are calculated from the absolute number of CD34+ cells.
Determination of baseline levels of CD34"/CD38- and CD34"/Lin™ cells was : 30 conducted as follows: CD34+ cells were purified from 3 thawed cord blood units and stained for the above markers. The mean of these experiments was considered as the baseline value.
Total cell counts, numbers of CD34+ cells and subsets, and CFU numbers are presented as cumulative numbers, with the assumption that the cultures had not been passaged; i.e., the number of cells per ml were multiplied by the number of passages performed. : 5 Assaying Colony Forming Unit (CFU) ability: Cells were cloned in semi- solid, methylcellulose-containing medium supplemented with 2 IU/ml erythropoietin (Eprex, Cilag AG Int., Switzerland), stem cell factor and IL-3, both at 20 ng/ml, and
G-CSF and GM-CSF, both at 10 ng/ml (all from Perpo Tech). Cultures were incubated for 14 days at 37 °C, 5 % CO, in a humidified atmosphere.
Determination of LTC-CFUc values: The ability of the cultures to maintain self-renewal was measured by determination of the content of colony forming unit cells in the long and extended long-term cultures (LTC-CFUc), as described in the references hereinabove.
Experimental Results
Mononuclear cells (MNC) were seeded in culture bags and were provided with nutrients and cytokines (50 ng/ml FLt3, IL-6, TPO and SCF) as described above.
The MNC cultures were either treated or untreated (untreated controls) with various concentrations {5-10 uM) of TEPA chelator. The treated MNC cultures were supplemented with TEPA for only the first three weeks and from week three onward were topped with chelator-free media. The pre-purified CD34™ cultures were not supplemented with TEPA and served as positive controls. The cultures were analyzed weekly during a l2-week period for the number of cells, CFUc, CD34+ and
CD34+CD?38- cells. In order to precisely determine the CD34+ cell content, CD34+ cells were weekly reselected and enumerated from each of the experimental groups (treated and untreated MNC cultures) and the positive control (CD34+ cultures).
The results, illustrated in Figures la-b, 2 and 3, show that addition of TEPA : chelator to non-purified MNC cultures, substantially and progressively increased the number of CD34™ cells, CD34" colony-forming cells and CD34TCD38- cells, over a 12-week period. Thus, in MNC cultures treated with TEPA, the cumulative number of CD34 cells increased from a non-detectable level to over 8 x 107 cells/ml, after 2 and 12 weeks, respectively (Figures la-b); the cumulative number of CD341CDsg"
cells increased from a non-detectable level to 2.5 x 10’ cells/ml, after 2 and 12 weeks, respectively (Figure 2); and the number of CD34% CFUs increased from a non- . detectable level to 3.2 x 107 cells/ml after 2 and 10 weeks, respectively (Figure 3).
On the other hand, when TEPA was not added to MNC cultures (untreated controls), ’ 5 no significant expansion of stem or progenitor cells was measured throughout the 12- week period. Furthermore, the stem and progenitor cells densities in the TEPA- treated MNC cultures, either equalized or surpassed the densities of stem and progenitor cells in pre-purified CD34 cell cultures (not treated with TEPA, positive controls). Morphological analysis of cells derived from long-term and TEPA-treated MNC cultures, revealed a high proportion of non-differentiated cells, while most of the cells derived from long-term and MNC cultures not treated with TEPA, where fully differentiated.
The results described in this Example clearly show that stem and progenitor hematopoietic cells may be substantially expanded ex-vivo, continuously over at least 12 weeks period, in a culture of mixed (mononuclear fraction) blood cells, with no prior purification of CD34" cells. The data also show that this effect resulted from supplementing the cells culture medium with TEPA chelator, only during the first three weeks of culturing.
These results indicate that short-term MNC cell cultures supplemented with
TEPA in addition to cytokines, enabled tremendous expansion of CD34+ cells and stem/early progenitor cells (CD34+38-) as compared with minimal expansion of these cells obtained in MNC cultures treated only with cytokines. Comparison experiments demonstrated that expansion of CD34+ cells and its rare CD34+CD38- cell subset continue to occur in the extended long-term cultures and is much higher as compared with that obtained from cultures initiated with highly purified CD34+ cells.
Therefore, the results may suggest that short-term treatment of MNC with TEPA potentiate the MNC cultures in a way that enables higher expansion of cells with extended self-renewal potential.
The results may also suggest that in addition to the regulatory effect on
CD34+ cells and its early subsets, the chelator may also enable ex-vivo expansion of a small subset of cells that are not co-purified with the CD34+ cell fraction. This subset of cells, which is probably in nature CD34-, may support superior expansion of
CD34+ cells and its subsets during the extended long-term cultures.
Hence, this Example illustrates a substantial ex-vivo expansion of stem and progenitor cells in a mixed mononuclear cells culture. This novel procedure . 5 circumvents the need of the laborious and costly enrichment of stem cells prior to initiation of cultures, which is currently used in the art. Hence, the use of a copper chelator, such as TEPA, can substantially simplify, reduce cost and improve efficiency of procedures for an ex-vivo expansion of stem and/or progenitor cells.
EXAMPLE 2
THE EFFECT OF A COPPER CHELATE ON THE EX-VIVO EXPANSION OF
HEMATOPOIETIC STEM CELLS OF A MONONUCLEAR CELLS CULTURE
Copper-TEPA chelate was prepared as described, for example, in
PCT/IL03/00062.
Mononuclear cells (MNC) were seeded in culture bags and were provided with nutrients and cytokines as described in Example 1 above. The mononuclear cell cultures were either untreated (control) or treated with Cu-TEPA chelate. The treated
MNC cultures were supplemented with Copper-TEPA chelate for the first three weeks and from week threc onward were topped with chelator-free media. All cultures were analyzed eight weeks after an 8-week period.
The results, presented in Table 1 below, show that addition of Copper-TEPA chelate to MNC cultures markedly increased the number of CD34% cells, the proportion of CD34% cells, and the number of CD34TCD38- cells, after an eight weeks incubation period. Thus, the cumulative number of CD347 cells per culture bag after incubation was 2.56 x 10, 12.37 x 10% or 32.85 x 10°, in the untreated cultures (cytokines only), 50 uM Copper-TEPA-treated and 100 uM Copper-TEPA- treated cultures, respectively. The cumulative number of CD34+tCD3g~ cells increased from 2.1 x 10° in the untreated control culture (cytokines only) to 6.1 x 10° in the Copper-TEPA (100 uM) treated culture.
Table 1
S of | “Portion of |B umber of i CD34+ cells | CD34+ cells | CD34/38- cells
I
Er I 50uM rs a ! 100pM | Nn
The results described in this Example demonstrate that hematopoietic stem cells may be substantially expanded ex-vivo, over at least § weeks period, in a culture of mononuclear blood cells, with no prior purification of CD34 cells, in the presence of a copper chelate such as Copper-TEPA.
EXAMPLE 3
THE EFFECT OF A RAR ANTAGONIST ON THE EX-VIVO EXPANSION OF
HEMATOPOIETIC STEM CELLS OF A MONONUCLEAR CELLS CULTURE
Materials and Experimental Methods
The high-Affinity retinoic acid receptor (RAR) antagonist 4-[[4-(4- ethylphenyl)-2,2-dimethyl- (2H)-thiochomen-6-yl)]-benzoic acid, (AGN 194310) was synthesized according to the procedure described in PCT/IL03/00064.
Mononuclear cells fraction was collected and purified as described above in
Example 1. MNC cultures were prepared and maintained as described above. AGN 194310 RAR antagonist was added to the tested cultures at concentrations ranging from 1 x 10° - 1x 107" M [or 410 pg/l to 4.1 x 10° ng/1]. The antagonist was added for a predetermined, limited period, for up to three weeks or continuously during the entire culture period. : The results, presented in Table 2, show that mononuclear cell fractions cultured in the presence of RAR antagonists and cytokines revealed a significant increase in the number of CD34+Lin- cells (78 %, 24 %) as quantitated by FACS analysis from a reselected, highly purified CD34+ cell fraction, as compared with the control untreated MNC fractions, 2 and 5 weeks (respectively), after initial seeding.
The MNC cells responded to the RAR antagonists and expanded an undifferentiated population, without prior purification of the CD34+ population. RAR antagonist treatment was sufficient to stimulate specific expansion of the stem/progenitor cell compartment, at 5 weeks post seeding. While control untreated MNCs had no detectable CD34+ population, RAR antagonist treated cultures revealed significant numbers of CD34+ cells, and those that were lineage marker deficient. Thus, any factors elaborated by the MNC culture cells that suppress CD34+ cell survival in control samples are insufficient to override the signal provided by the RAR antagonist to elaborate this compartment.
Table 2 weeks
Cytokines Cytokines +RAR
Neof CD34'/Lin X10" | xX 10% [176 | - 16 | ms | 132.5 To BN %CD34Lin | CD34/Lin’ 0 EE 184 TT l Cytokines Cytokines +RAR [a
L. = — IS BE ; — ee
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art.
Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.
In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.
The scope of the present invention and of the appended claims is not to be regarded as restricted or limited by or to any explicit or specific theory presented herein.
Claims (43)
- ® WO 2004/016731 PCT/IL2003/000618 WHAT IS CLAIMED IS:I. A method of expanding an ex-vivo population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo, the method comprising providing hematopoietic mononuclear cells: culturing said mononuclear cells ex-vivo under conditions allowing for cell proliferation and, at the same time, culturing said cells under conditions selected from the group consisting of: conditions reducing expression and/or activity of CD38 in said mononuclear cells; conditions reducing capacity of said hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D in said mononuclear cells; conditions reducing capacity of said hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor, the retinoid X receptor and/or the Vitamin D receptor in said mononuclear cells; culturing said mononuclear cells in the presence of nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite in said mononuclear cells: conditions reducing an expression and/or activity of PI 3-kinase in said mononuclear cells; and culturing said mononuclear cells in the presence of at least one copper chelator or chelate; thereby expanding a population of said hematopoietic stem cells while at the same time substantially inhibiting differentiation of said hematopoietic stem cells ex- Vivo.
- 2. Use of hematopoietic mononuclear cells in the manufacture of 4 medicament for transplanting or implanting hematopoietic mononuclear cells, wherein the hematopoictic mononuclear cells are subjected to the method of claim |. AMENDED SHEET
- 3. The use of claim 2, wherein the hematopoietic mononuclear cells are obtained from a donor who is intended to be the recipient thereof.
- 4. A method of genetically modifying hematopoietic stem cells with an exogene comprising: (a) obtaining hematopoietic mononuclear cells; (b) culturing said mononuclear cells ex vivo for cell proliferation, wherein said culturing is performed in a condition selected from the group consisting of: conditions reducing expression and/or activity of CD38 in said mononuclear cells, conditions reducing capacity of said hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D in said mononuclear cells, conditions reducing capacity of said hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor, the retinoid X receptor and/or the Vitamin D receptor in said mononuclear cells; culturing said mononuclear cells in the presence of nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite in said mononuclear cells; conditions reducing an expression and/or activity of PI 3-kinase in said mononuclear cells; and culturing said mononuclear cells in the presence of at least one copper chelator or chelate, thereby expanding a population of said hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells ex- vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
- 5. The method of claim 4, wherein genetically modifying is effected by a vector which comprises the exogene. AMENDED SHEET
- 6. The method of claim 3, wherein the vector is a viral vector or a nucleic acid vector.
- 7. Use of hematopoietic mononuclear cells in the manufacture of a medicament for adoptive immunotherapy wherein the hematopoietic mononuclear cells are subjected to the method of claim 4.
- 8. A transplantable hematopoietic cell preparation comprising an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells in the presence of an effective amount of an agent, wherein said agent has an activity selected from the group consisting of: reducing expression and/or activity of CD38 in said mononuclear cells, reducing capacity of said hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D in said mononuclear cells, reducing capacity of said hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor, the retinoid X receptor and/or the Vitamin D receptor in said mononuclear cells; and reducing an expression and/or activity of PI 3-kinase in said mononuclear cells; or wherein said agent is a copper chelator or chelate, or nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite in said mononuclear cells; while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
- 9. The method of claim 1, wherein said hematopoietic mononuclear cells are derived from a source selected from the group consisting of bone marrow, peripheral blood and neonatal umbilical cord blood. AMENDED SHEET® WO 2004/016731 PCT/IL2003/000618
- 10. The method of claim |, wherein providing said hematopoietic mononuclear cells with said conditions for ex-vivo cell proliferation comprises providing said hematopoietic mononuclear cells with nutrients and with cytokines.
- 1. The method of claim 10, wherein said cytokines are early acting cytokines.
- 12. The method of claim 11, wherein said early acting cytokines are selected from the group consisting of stem cell factor, FLT3 ligand, interleukin- 1, interleukin- 2, interleukin-3, interleukin-6, interleukin-10, interleukin-12, tumor necrosis factor-a and thrombopoietin.
- 13. The method of claim 10, wherein said cytokines are late acting cytokines.
- 14. The method of claim 13, wherein said late acting cytokines are selected from the group consisting of granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor, erythropoietin, FGF, EGF, NGF, VEGF, LIP, Hepatocyte growth factor and macrophage colony stimulating factor.
- 15. The method of claim 1, wherein providing said hematopoietic mononuclear cells with ex-vivo culture conditions for reducing said expression and/or said activity of CD38 is by providing said hematopoietic mononuclear cells with an agent that downregulates CD38 expression.
- 16. The transplantable hematopoietic cell preparation of claim 8, wherein said agent is an agent that downregulates CD38 expression.
- 17. The method of claim 15, wherein the agent that downregulates CD38 expression is selected from the group consisting of a retinoic acid receptor antagonist, a retinoid X receptor antagonist and a Vitamin D receptor antagonist. AMENDED SHEET® WO 2004016731 PCT/IL2003/000618
- 18. The method of claim 15, wherein the agent that downregulates CD38 expression is an antagonist for reducing a capacity of said hematopoietic mononuclear cells in responding to retinoic acid, retinoid and/or Vitamin D.
- 19. The method of claim 15, wherein said agent that downregulates CD38 expression is a polynucleotide.
- 20. The method of claim 19, wherein the polynucleotide encodes an anti CD38, an anti retinoic acid receptor, an anti retinoid X receptor or an anti Vitamin D receptor intracellular antibody.
- 21. The method of claim 19, wherein the polynucleotide encodes an anti CD38, an anti retinoic acid receptor, an anti retinoid X receptor or an anti Vitamin D receptor antibody.
- 22. The method of claim 19, wherein said polynucleotide is a small interfering polynucleotide molecule directed to cause intracellular CD38, retinoic acid receptor, retinoid X receptor or Vitamin D receptor mRNA degradation.
- 23. The method of claim 22, wherein said small interfering polynucleotide molecule is selected from the group consisting of an RNAi molecule, an anti-sense molecule, a rybozyme molecule and a DNAzyme molecule.
- 24. The method of claim 15, wherein said agent that downregulates CD38 expression is an agent that downregulates PI 3-kinase expression.
- 25. The method of claim 24, wherein said agent that downregulates PI 3- kinase expression is a polynucleotide. AMENDED SHEET® WO 2004/016731 PCT/IL2003/000618
- 26. The method of claim 24, wherein agent that downregulates PI 3-Kinase expression is an intracellular antibody.
- 27. The method of claim 25, wherein said polynucleotide is a small interfering polynucleotide molecule directed to cause intracellular PI 3-kinase mRNA or gene degradation.
- 28. The method of claim 27, wherein said small interfering polynucleotide molecule is selected from the group consisting of an RNAi molecule, an anti-sense molecule, a rybozyme molecule and a DNAzyme molecule.
- 29. The method of claim 15, wherein said agent that downregulates CD38 expression is an agent that inhibits PI 3-kinase activity.
- 30. The method of claim 29, wherein said agent that inhibits PI 3-kinase activity is selected from the group consisting of wortmannin and LY294002
- 31. The method of claim 1, wherein providing said hematopoietic mononuclear cells with ex-vivo culture conditions for reducing said expression and/or said activity of CD38 is by providing said hematopoietic mononuclear cells with an agent that inhibits CD38 activity.
- 32. The transplantable hematopoietic cell preparation of claim 8, wherein said agent is an agent that inhibits CD38 activity.
- 33. The method of claim 22, wherein said agent that inhibits CD38 activity is nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite. AMENDED SHEET® WO 2004/016731 PCT/IL2003/000618
- 34. The method of claim 33, wherein said nicotinamide analog is selected from the group consisting of benzamide, nicotinethioamide, nicotinic acid and o- amino-3-indolepropionic acid.
- 35. The method of claim 1, wherein providing said hematopoietic mononuclear cells with ex-vivo culture conditions for reducing said expression and/or said activity of CD38 is by providing said hematopoietic mononuclear cells with an agent that inhibits PI 3-kinase activity.
- 36. The transplantable hematopoietic cell preparation of claim 8, wherein said agent is an agent that inhibits PI 3-kinase activity.
- 37. The method of claim 36, wherein said agent that inhibits PI 3-kinase activity is selected from the group consisting of wortmannin and LY 294002.
- 38. The method of claim !, wherein said hematopoietic mononuclear cells are not enriched prior to culturing ex-vivo under conditions allowing for cell proliferation.
- 39. The method of claim I, wherein said hematopoietic cells comprise a : major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells.
- 40. An assay for determining whether a transition metal chelate or chelator causes substantial inhibition or induction of differentiation of hematopoietic stem cells, the assay comprising: culturing hematopoietic mononuclear cells in the presence of the transition metal chelate or chelator and monitoring differentiation of said hematopoietic stem cells, wherein if ditferentiation is increased as is compared to non-treated AMENDED SHEET hematopoietic mononuclear cells, said transition metal chelate induces differentiation, whereas if differentiation is decreased as is compared to non-treated hematopoietic mononuclear cells, or if differentiation is absent altogether, said transition metal chelate inhibits differentiation.
- 41. An assay for identifying an effective hematopoietic stem cell expansion agent, the assay comprising culturing hematopoietic mononuclear cells in the presence of a compound selected from the group consisting of: a retinoic acid receptor antagonist; retinoid X receptor antagonist, vitamin D receptor antagonist; agent that inhibits PI 3-kinase activity; and a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, and monitoring expansion of said hematopoietic stem cells, wherein if increased expansion and decreased differentiation of said hematopoietic stem cells occurs, as compared to non-treated hematopoietic mononuclear cells, the compound is an effective hematopoietic stem cell expansion agent.
- 42. A hematopoietic stem cells collection/culturing bag supplemented with an effective amount of a compound selected from the group consisting of: a retinoic acid receptor antagonist, a retinoid X receptor antagonist and/or a Vitamin D receptor antagonist, nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite; or an agent that inhibits PI 3-kinase activity, which substantially inhibits cell differentiation of a hematopoietic stem cells fraction of hematopoietic mononuclear cells.
- 43. An ex-vivo expanded population of hematopoietic stem cells, obtained by the method of claim 1. AMENDED SHEET® WO 2004/016731 PCT/IL2003/000618+4. A method of preparing expanded, undifferentiated hematopoietic cells for transplantation into a recipient, the method comprising: (a) expanding and inhibiting differentiation of said hematopoietic cells by: (1) providing said hematopoietic cells with conditions for cell proliferation; (ii) contacting said hematopoietic cells with at least one transition metal chelator, said chelator having an affinity for copper and being capable of prolonging expansion of clonogenic cells in the presence of early acting cytokines, wherein said chelator and said proliferation conditions result in (i) prolonged active proliferation; (i) prolonged expansion of clonogenic cells (CFUc): and (iii) maintenance of undifferentiated cells in their undifferentiated state: thereby inhibiting differentiation and expanding said hematopoietic cells ex-vivo, and (b) isolating said expanded, undifferentiated hematopoietic cells, with the proviso that said hematopoietic cells are not human embryonic stem cells obtained from a human embryo. AMENDED SHEET
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US40413702P | 2002-08-19 | 2002-08-19 |
Publications (1)
Publication Number | Publication Date |
---|---|
ZA200502111B true ZA200502111B (en) | 2005-09-14 |
Family
ID=36675119
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
ZA200502111A ZA200502111B (en) | 2002-08-19 | 2005-03-14 | Ex-vivo expansion of hematopoietic stem cell popu lations in mononuclear cell cultures. |
Country Status (1)
Country | Link |
---|---|
ZA (1) | ZA200502111B (en) |
-
2005
- 2005-03-14 ZA ZA200502111A patent/ZA200502111B/en unknown
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8080417B2 (en) | Methods of ex vivo hematopoietic stem cell expansion by co-culture with mesenchymal cells | |
JP4738738B2 (en) | Expansion of renewable stem cell population | |
US7655225B2 (en) | Methods of expanding stem and progenitor cells and expanded cell populations obtained thereby | |
AU2004217699B2 (en) | Expansion of renewable stem cell populations using modulators of PI 3-kinase | |
US20060205071A1 (en) | Methods for ex-vivo expanding stem/progenitor cells | |
US8986992B2 (en) | Expansion of stem/progenitor cells by inhibition of enzymatic reactions catalyzed by the Sir2 family of enzymes | |
US20050054097A1 (en) | EX-VIVO expansion of hematopoietic system cell populations in mononuclear cell cultures | |
EP1648397A2 (en) | Ex vivo progenitor and stem cell expansion for use in the treatment of disease of endodermally-derived organs | |
WO2004016731A2 (en) | Ex-vivo expansion of hematopoietic stem cell populations in mononuclear cell cultures | |
AU2005200679B2 (en) | Ex-Vivo Expansion of Hematopoietic Stem Cell Populations in Mononuclear Cell Cultures | |
ZA200502111B (en) | Ex-vivo expansion of hematopoietic stem cell popu lations in mononuclear cell cultures. | |
US20180320139A1 (en) | Expansion of renewable stem cell populations | |
ZA200405901B (en) | Expansion of renewable stem cell populations | |
ZA200407797B (en) | Methods of inducing differentiation in ex vivo expanded stem cells. |