WO2014066505A1 - Double knockout (gt/cmah-ko) pigs, organs and tissues - Google Patents
Double knockout (gt/cmah-ko) pigs, organs and tissues Download PDFInfo
- Publication number
- WO2014066505A1 WO2014066505A1 PCT/US2013/066387 US2013066387W WO2014066505A1 WO 2014066505 A1 WO2014066505 A1 WO 2014066505A1 US 2013066387 W US2013066387 W US 2013066387W WO 2014066505 A1 WO2014066505 A1 WO 2014066505A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- base pair
- cmah
- cells
- pig
- human
- Prior art date
Links
- 210000000056 organ Anatomy 0.000 title claims abstract description 63
- 241000282887 Suidae Species 0.000 title abstract description 50
- 238000000034 method Methods 0.000 claims abstract description 58
- 230000014509 gene expression Effects 0.000 claims abstract description 48
- 101000902205 Homo sapiens Inactive cytidine monophosphate-N-acetylneuraminic acid hydroxylase Proteins 0.000 claims abstract description 18
- 230000009261 transgenic effect Effects 0.000 claims abstract description 17
- 102100022247 Inactive cytidine monophosphate-N-acetylneuraminic acid hydroxylase Human genes 0.000 claims abstract description 11
- 241000282898 Sus scrofa Species 0.000 claims description 116
- 241000282414 Homo sapiens Species 0.000 claims description 110
- 238000012217 deletion Methods 0.000 claims description 63
- 230000037430 deletion Effects 0.000 claims description 63
- 210000004185 liver Anatomy 0.000 claims description 54
- 210000002966 serum Anatomy 0.000 claims description 41
- FDJKUWYYUZCUJX-UHFFFAOYSA-N N-glycolyl-beta-neuraminic acid Natural products OCC(O)C(O)C1OC(O)(C(O)=O)CC(O)C1NC(=O)CO FDJKUWYYUZCUJX-UHFFFAOYSA-N 0.000 claims description 38
- 230000003247 decreasing effect Effects 0.000 claims description 26
- 238000003780 insertion Methods 0.000 claims description 26
- 230000037431 insertion Effects 0.000 claims description 26
- 108090000623 proteins and genes Proteins 0.000 claims description 24
- 102000003886 Glycoproteins Human genes 0.000 claims description 19
- 108090000288 Glycoproteins Proteins 0.000 claims description 19
- 238000002689 xenotransplantation Methods 0.000 claims description 19
- 239000012578 cell culture reagent Substances 0.000 claims description 16
- 210000003491 skin Anatomy 0.000 claims description 16
- 238000006467 substitution reaction Methods 0.000 claims description 16
- 208000024891 symptom Diseases 0.000 claims description 15
- 238000002054 transplantation Methods 0.000 claims description 15
- 239000000463 material Substances 0.000 claims description 12
- 238000004113 cell culture Methods 0.000 claims description 11
- 101150113197 CMAH gene Proteins 0.000 claims description 10
- 102000030902 Galactosyltransferase Human genes 0.000 claims description 10
- 108060003306 Galactosyltransferase Proteins 0.000 claims description 10
- 210000003734 kidney Anatomy 0.000 claims description 10
- 230000002829 reductive effect Effects 0.000 claims description 10
- 210000002216 heart Anatomy 0.000 claims description 8
- 210000004072 lung Anatomy 0.000 claims description 6
- 206010043554 thrombocytopenia Diseases 0.000 claims description 6
- 239000000427 antigen Substances 0.000 claims description 5
- 108091007433 antigens Proteins 0.000 claims description 5
- 102000036639 antigens Human genes 0.000 claims description 5
- 210000000496 pancreas Anatomy 0.000 claims description 5
- 239000006143 cell culture medium Substances 0.000 claims description 4
- 230000001965 increasing effect Effects 0.000 claims description 4
- 230000035755 proliferation Effects 0.000 claims description 4
- 210000001685 thyroid gland Anatomy 0.000 claims description 4
- 102000015081 Blood Coagulation Factors Human genes 0.000 claims description 3
- 108010039209 Blood Coagulation Factors Proteins 0.000 claims description 3
- 102000004127 Cytokines Human genes 0.000 claims description 3
- 108090000695 Cytokines Proteins 0.000 claims description 3
- 239000000654 additive Substances 0.000 claims description 3
- 230000000996 additive effect Effects 0.000 claims description 3
- 239000003114 blood coagulation factor Substances 0.000 claims description 3
- 239000003102 growth factor Substances 0.000 claims description 3
- 239000005556 hormone Substances 0.000 claims description 3
- 229940088597 hormone Drugs 0.000 claims description 3
- FDJKUWYYUZCUJX-AJKRCSPLSA-N N-glycoloyl-beta-neuraminic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@@H]1O[C@](O)(C(O)=O)C[C@H](O)[C@H]1NC(=O)CO FDJKUWYYUZCUJX-AJKRCSPLSA-N 0.000 claims 5
- 101000718529 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) Alpha-galactosidase Proteins 0.000 claims 2
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims 2
- 210000004027 cell Anatomy 0.000 description 191
- 101710168055 Cytidine monophosphate-N-acetylneuraminic acid hydroxylase Proteins 0.000 description 144
- 210000001519 tissue Anatomy 0.000 description 82
- 210000003754 fetus Anatomy 0.000 description 55
- 210000001772 blood platelet Anatomy 0.000 description 42
- 241001465754 Metazoa Species 0.000 description 32
- 210000002950 fibroblast Anatomy 0.000 description 30
- 101000856513 Homo sapiens Inactive N-acetyllactosaminide alpha-1,3-galactosyltransferase Proteins 0.000 description 28
- 230000035772 mutation Effects 0.000 description 28
- 102100025509 Inactive N-acetyllactosaminide alpha-1,3-galactosyltransferase Human genes 0.000 description 27
- 238000010374 somatic cell nuclear transfer Methods 0.000 description 23
- 239000000047 product Substances 0.000 description 22
- 238000000684 flow cytometry Methods 0.000 description 21
- 230000001605 fetal effect Effects 0.000 description 20
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 20
- 108090001090 Lectins Proteins 0.000 description 19
- 102000004856 Lectins Human genes 0.000 description 19
- 108700028369 Alleles Proteins 0.000 description 17
- 238000002474 experimental method Methods 0.000 description 17
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 16
- 238000004458 analytical method Methods 0.000 description 16
- 239000002523 lectin Substances 0.000 description 16
- 210000003743 erythrocyte Anatomy 0.000 description 15
- 108091033409 CRISPR Proteins 0.000 description 13
- 241000124008 Mammalia Species 0.000 description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 12
- 238000003556 assay Methods 0.000 description 12
- 230000010412 perfusion Effects 0.000 description 12
- 239000012981 Hank's balanced salt solution Substances 0.000 description 11
- 230000027455 binding Effects 0.000 description 11
- 230000035935 pregnancy Effects 0.000 description 11
- 241001504519 Papio ursinus Species 0.000 description 10
- 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 10
- 210000000287 oocyte Anatomy 0.000 description 10
- 239000002953 phosphate buffered saline Substances 0.000 description 10
- 238000010186 staining Methods 0.000 description 10
- 210000004369 blood Anatomy 0.000 description 9
- 239000008280 blood Substances 0.000 description 9
- 231100000135 cytotoxicity Toxicity 0.000 description 9
- 230000003013 cytotoxicity Effects 0.000 description 9
- 239000012091 fetal bovine serum Substances 0.000 description 9
- 239000002773 nucleotide Substances 0.000 description 9
- 125000003729 nucleotide group Chemical group 0.000 description 9
- CHADEQDQBURGHL-UHFFFAOYSA-N (6'-acetyloxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl) acetate Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(OC(C)=O)C=C1OC1=CC(OC(=O)C)=CC=C21 CHADEQDQBURGHL-UHFFFAOYSA-N 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 8
- 210000002919 epithelial cell Anatomy 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 239000013642 negative control Substances 0.000 description 8
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 241000283074 Equus asinus Species 0.000 description 7
- 108091027544 Subgenomic mRNA Proteins 0.000 description 7
- 210000000988 bone and bone Anatomy 0.000 description 7
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 7
- 230000002950 deficient Effects 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 239000002981 blocking agent Substances 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 210000005003 heart tissue Anatomy 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 210000005228 liver tissue Anatomy 0.000 description 6
- 210000005084 renal tissue Anatomy 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 238000001712 DNA sequencing Methods 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 206010052779 Transplant rejections Diseases 0.000 description 5
- 230000004075 alteration Effects 0.000 description 5
- 238000004624 confocal microscopy Methods 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 210000001616 monocyte Anatomy 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 4
- 208000032843 Hemorrhage Diseases 0.000 description 4
- 241000282577 Pan troglodytes Species 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- BABWHSBPEIVBBZ-UHFFFAOYSA-N diazete Chemical compound C1=CN=N1 BABWHSBPEIVBBZ-UHFFFAOYSA-N 0.000 description 4
- 210000002257 embryonic structure Anatomy 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 238000002513 implantation Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- IJRKANNOPXMZSG-SSPAHAAFSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC(=O)CC(O)(C(O)=O)CC(O)=O IJRKANNOPXMZSG-SSPAHAAFSA-N 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- 101150042360 GGTA1 gene Proteins 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 241000282520 Papio Species 0.000 description 3
- 238000012300 Sequence Analysis Methods 0.000 description 3
- 238000000692 Student's t-test Methods 0.000 description 3
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 210000003850 cellular structure Anatomy 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 238000002784 cytotoxicity assay Methods 0.000 description 3
- 231100000263 cytotoxicity test Toxicity 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 210000004207 dermis Anatomy 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 210000002510 keratinocyte Anatomy 0.000 description 3
- 210000003292 kidney cell Anatomy 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 239000012114 Alexa Fluor 647 Substances 0.000 description 2
- 206010053567 Coagulopathies Diseases 0.000 description 2
- 102000012422 Collagen Type I Human genes 0.000 description 2
- 108010022452 Collagen Type I Proteins 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 230000004568 DNA-binding Effects 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 206010037549 Purpura Diseases 0.000 description 2
- 238000010459 TALEN Methods 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 208000034158 bleeding Diseases 0.000 description 2
- 230000000740 bleeding effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000004413 cardiac myocyte Anatomy 0.000 description 2
- 210000000845 cartilage Anatomy 0.000 description 2
- 210000001612 chondrocyte Anatomy 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 210000001771 cumulus cell Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000002500 effect on skin Effects 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 239000012997 ficoll-paque Substances 0.000 description 2
- 238000012239 gene modification Methods 0.000 description 2
- 230000005017 genetic modification Effects 0.000 description 2
- 235000013617 genetically modified food Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 210000004153 islets of langerhan Anatomy 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 210000001865 kupffer cell Anatomy 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 210000002751 lymph Anatomy 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 210000000663 muscle cell Anatomy 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 229910052754 neon Inorganic materials 0.000 description 2
- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 230000001817 pituitary effect Effects 0.000 description 2
- 210000004508 polar body Anatomy 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 210000002027 skeletal muscle Anatomy 0.000 description 2
- 210000002460 smooth muscle Anatomy 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 238000012353 t test Methods 0.000 description 2
- 210000002435 tendon Anatomy 0.000 description 2
- 210000003932 urinary bladder Anatomy 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-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
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 206010000060 Abdominal distension Diseases 0.000 description 1
- 208000010444 Acidosis Diseases 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 241000321096 Adenoides Species 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 208000002109 Argyria Diseases 0.000 description 1
- 102100026292 Asialoglycoprotein receptor 1 Human genes 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 101100124609 Caenorhabditis elegans zyg-12 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 229920002567 Chondroitin Polymers 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 206010011703 Cyanosis Diseases 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010014080 Ecchymosis Diseases 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 206010048748 Graft loss Diseases 0.000 description 1
- 241000219726 Griffonia simplicifolia Species 0.000 description 1
- 108020005004 Guide RNA Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 208000034507 Haematemesis Diseases 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 101000785944 Homo sapiens Asialoglycoprotein receptor 1 Proteins 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 235000019687 Lamb Nutrition 0.000 description 1
- 206010024264 Lethargy Diseases 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- SUHQNCLNRUAGOO-UHFFFAOYSA-N N-glycoloyl-neuraminic acid Natural products OCC(O)C(O)C(O)C(NC(=O)CO)C(O)CC(=O)C(O)=O SUHQNCLNRUAGOO-UHFFFAOYSA-N 0.000 description 1
- FDJKUWYYUZCUJX-KVNVFURPSA-N N-glycolylneuraminic acid Chemical compound OC[C@H](O)[C@H](O)[C@@H]1O[C@](O)(C(O)=O)C[C@H](O)[C@H]1NC(=O)CO FDJKUWYYUZCUJX-KVNVFURPSA-N 0.000 description 1
- 208000036830 Normal foetus Diseases 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 206010030302 Oliguria Diseases 0.000 description 1
- 206010053159 Organ failure Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 101100248830 Pelargonium hortorum rpl22-B gene Proteins 0.000 description 1
- KMNTUASVUKNVJS-UHFFFAOYSA-N Ponceau S (acid form) Chemical compound OC1=C(S(O)(=O)=O)C=C2C=C(S(O)(=O)=O)C=CC2=C1N=NC(C(=C1)S(O)(=O)=O)=CC=C1N=NC1=CC=C(S(O)(=O)=O)C=C1 KMNTUASVUKNVJS-UHFFFAOYSA-N 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 241001672981 Purpura Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000282894 Sus scrofa domesticus Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- GBOGMAARMMDZGR-UHFFFAOYSA-N UNPD149280 Natural products N1C(=O)C23OC(=O)C=CC(O)CCCC(C)CC=CC3C(O)C(=C)C(C)C2C1CC1=CC=CC=C1 GBOGMAARMMDZGR-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000007950 acidosis Effects 0.000 description 1
- 208000026545 acidosis disease Diseases 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 210000002534 adenoid Anatomy 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 210000002403 aortic endothelial cell Anatomy 0.000 description 1
- 210000003433 aortic smooth muscle cell Anatomy 0.000 description 1
- 238000002617 apheresis Methods 0.000 description 1
- 206010003549 asthenia Diseases 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000003443 bladder cell Anatomy 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 208000015294 blood coagulation disease Diseases 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 210000000254 ciliated cell Anatomy 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000000942 confocal micrograph Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- GBOGMAARMMDZGR-JREHFAHYSA-N cytochalasin B Natural products C[C@H]1CCC[C@@H](O)C=CC(=O)O[C@@]23[C@H](C=CC1)[C@H](O)C(=C)[C@@H](C)[C@@H]2[C@H](Cc4ccccc4)NC3=O GBOGMAARMMDZGR-JREHFAHYSA-N 0.000 description 1
- GBOGMAARMMDZGR-TYHYBEHESA-N cytochalasin B Chemical compound C([C@H]1[C@@H]2[C@@H](C([C@@H](O)[C@@H]3/C=C/C[C@H](C)CCC[C@@H](O)/C=C/C(=O)O[C@@]23C(=O)N1)=C)C)C1=CC=CC=C1 GBOGMAARMMDZGR-TYHYBEHESA-N 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007402 cytotoxic response Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000004002 dopaminergic cell Anatomy 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000005069 ears Anatomy 0.000 description 1
- 229960001484 edetic acid Drugs 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000005168 endometrial cell Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000007824 enzymatic assay Methods 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 208000001780 epistaxis Diseases 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 230000012173 estrus Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 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 1
- 238000007421 fluorometric assay Methods 0.000 description 1
- 210000004186 follicle cell Anatomy 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000003633 gene expression assay Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 210000005086 glomerual capillary Anatomy 0.000 description 1
- 210000002175 goblet cell Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 210000002503 granulosa cell Anatomy 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 210000002768 hair cell Anatomy 0.000 description 1
- 210000002064 heart cell Anatomy 0.000 description 1
- 210000003709 heart valve Anatomy 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 208000006750 hematuria Diseases 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 230000002607 hemopoietic effect Effects 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000009851 immunogenic response Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 210000002660 insulin-secreting cell Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000002332 leydig cell Anatomy 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000000088 lip Anatomy 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 210000004216 mammary stem cell Anatomy 0.000 description 1
- 210000003593 megakaryocyte Anatomy 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 210000001809 melena Anatomy 0.000 description 1
- 230000005906 menstruation Effects 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 210000004925 microvascular endothelial cell Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 244000309715 mini pig Species 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 210000003550 mucous cell Anatomy 0.000 description 1
- 210000004457 myocytus nodalis Anatomy 0.000 description 1
- 210000000282 nail Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000002988 nephrogenic effect Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000003061 neural cell Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 230000000474 nursing effect Effects 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 210000004409 osteocyte Anatomy 0.000 description 1
- 230000002188 osteogenic effect Effects 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 210000002741 palatine tonsil Anatomy 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000000849 parathyroid Effects 0.000 description 1
- 210000003899 penis Anatomy 0.000 description 1
- 206010034754 petechiae Diseases 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 210000000680 phagosome Anatomy 0.000 description 1
- 210000003800 pharynx Anatomy 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000002600 positron emission tomography Methods 0.000 description 1
- 238000010149 post-hoc-test Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 210000005267 prostate cell Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 210000001187 pylorus Anatomy 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 235000002020 sage Nutrition 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 210000004116 schwann cell Anatomy 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 210000001625 seminal vesicle Anatomy 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000000717 sertoli cell Anatomy 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 210000004085 squamous epithelial cell Anatomy 0.000 description 1
- 238000000551 statistical hypothesis test Methods 0.000 description 1
- 238000012066 statistical methodology Methods 0.000 description 1
- 210000004500 stellate cell Anatomy 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 230000001732 thrombotic effect Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000002105 tongue Anatomy 0.000 description 1
- 210000000515 tooth Anatomy 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 210000000626 ureter Anatomy 0.000 description 1
- 210000003708 urethra Anatomy 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 201000010653 vesiculitis Diseases 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/37—Digestive system
- A61K35/407—Liver; Hepatocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
- C12N15/877—Techniques for producing new mammalian cloned embryos
- C12N15/8778—Swine embryos
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/108—Swine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
- A01K2267/025—Animal producing cells or organs for transplantation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
Definitions
- the present invention is generally in the field of xenotransplantation and genetic modification to produce transgenic animals, orga ns tissue, or cells suita ble for transplantation into a hu man.
- transplants from one animal into another animal of the same species are a routine treatment option for many serious conditions, including kidney, heart, lung, liver and other organ disease.
- transplants from one animal into another animal of the same species are a routine treatment option for many serious conditions, including kidney, heart, lung, liver and other organ disease.
- there are not enough suita ble organs availa ble for transplant to meet current or expected clinical demands for organ transplants.
- Xenotransplantation the transplant of organs, tissue or cells from one animal into another animal of a different species, such as the transplantation of a pig organ into a human recipient, has the potential to eliminate the shortage of organs ava ila ble for transplant, potentially helping hundreds of thousands of people worldwide. For instance, suita ble organs for transplant from non-human donors, such as from a pig, could help keep seriously ill patients alive, either permanently or temporarily, until a suita ble human organ is availa ble for transplant. [0007] While many mammalian animals may be suitable candidates for xenotransplantation, much of the current focus is on the pig.
- pig organs tissue or cells for xenotransplantaion offers many advantages over other non-human mammalian donors. For instance, pigs are easily obtainable, they are inexpensive to breed and maintain, and, most importantly, many pig organs are similar to humans in size, shape and function.
- xenotransplantation using standard, unmodified pig tissue into a human (or other primate) is accompanied by severe rejection of the transplanted tissue.
- the rejection may be a hyperacute rejection, an acute rejection or a chronic rejection.
- the hyperacute response to the pig antibodies present on the transplanted tissue is so strong that the transplant is typically damaged by the human immune system within minutes or hours of transplant into the human recipient.
- Pig cells express al,3 galactosyltransferase (aGal) and cytidine monophosphate-N- acetylneuraminic acid hydroxylase (CMAH), which are not found in human cells.
- the aGal enzyme produces the aGal epitope.
- CMAH converts the sialic acid N-acetylneuraminic acid (Neu5Ac) to N- glycolylneuraminic acid (Neu5Gc). Accordingly, when pig tissue is transplanted into a human, these epitopes elicit an antibody-mediated rejection from the human patient immediately following implantation.
- the antibodies are present in the patient's blood prior to implantation of the tissue, resulting in the intense and immediate rejection of the implanted tissue.
- GTKO pig may have eliminated anti-aGal antibodies as a barrier to xenotransplantation
- studies using GTKO cardiac and renal xenografts in baboons show that the GTKO organs still trigger an immunogenic response, resulting in rejection or damage to the transplanted organ.
- Baboons transplanted with GTKO kidneys and treated with two different immunosuppressive regimens died within 16 days of surgery. Chen et al. concluded "genetic depletion of Gal antigens does not provide a major benefit in xenograft survival" (Chen et al., 2005, Nature Med. 11(12):1295-1298.
- Basnet et al examined the cytotoxic response of human serum to CMAH-/- mouse cells. Basnet et al. concluded "the anti-NeuGc Ab-mediated immune response may be significantly involved in graft loss in xenogeneic cell transplantation, but not in organ transplantation" (Basnet et al., 2010, Xenotransplantation, 17(6):440-448).
- This disclosure relates generally to methods of making porcine organs, tissues or cells for transplantation into a human that do not express aGal and CMAH.
- the present disclosure provides, in one embodiment, a knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)- galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and when tissue from said pig is transplanted into a human, hyperacute rejection is decreased as compared to when tissue from a wild-type pig is transplanted into a human.
- the specification provides porcine organs, tissue or cells for transplantation into a human having reduced expression of aGal and CMAH on the porcine organs, tissue or cells.
- the specification provides a method for modifying a porcine organs, tissue or cells for transplantation into a human, the method comprising removing or reducing expression of aGal and CMAH on the porcine organs, tissue or cells.
- the porcine organs, tissue, or cells may be selected from the group consisting of red blood cells, skin, heart, livers, kidneys, lung, pancreas, thyroid, small bowel, and components thereof.
- the specification provides a method for making porcine organs, tissue or cells for transplantation into a human, the method comprising reducing expression of aGal and CMAH on the porcine organs, tissue or cells.
- the porcine organs, tissue, or cells may be selected from the group consisting of red blood cells, skin, heart, liver, kidneys, lung, pancreas, small bowel, and components thereof.
- the specification provides a knockout pig comprising disrupted a(l,3)- galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and wherein when tissue from a knockout pig is transplanted into a human, thrombocytopenia is decreased as compared to when tissue from a wild-type pig is transplanted into a human.
- the specification provides a knockout pig comprising disrupted a(l,3)- galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and wherein a liver from said pig exhibits reduced uptake of human platelets when said liver is exposed to said human platelets.
- the specification provides a method of increasing the duration of the period between when a human subject is identified as a subject in need of human liver transplant and when said human liver transplant occurs, said method comprising providing a liver from a knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type swine and surgically attaching said liver from said knockout pig to said human su bject in a therapeutically effective manner.
- the liver from the knockout pig may be internal or external to the human subject, and may be directly or indirectly attached to the human subject.
- the specification provides a method of preparing organs, tissues, or cells for xenotransplantation into human patients with reduced rejection, the method comprising providing a transgenic pig as a source of transplant material wherein the transplant material is selected from the group consisting of organs, tissues, or cells, and wherein the pig masks or reduces the expression of at least two xenoreactive antigens on the transplant material.
- At least two xenoreactive antigens may be aGal and Neu5Gc.
- the specification provides a knockout pig comprising disrupted a(l,3)- galactosyltransferase and CMAH genes, wherein the disruption of said a(l,3)-galactosyltransferase gene is selected from the group of disruptions comprising a 3 base pair deletion adjacent to a G to A substitution, a single base pair deletion, a single base pair insertion, a six base pair deletion, a two base pair insertion, a ten base pair deletion, a seven base pair deletion, and an eight base pair substitution for a five base pair sequence; wherein the disruption of said CMAH gene is selected from the group of disruptions comprising a four base pair insertion, a two base pair deletion, a single base pair insertion, an eight base pair deletion, a five base pair deletion, a three base pair deletion, a two base pair substitution for a single base pair, and a twenty base pair deletion; and wherein expression of functional a(l,3)-galacto
- the specification provides a method of improving symptoms of hyperacute rejection in a patient comprising transplanting porcine organs, tissue or cells having reduced expression of aGal and CMAH on the porcine organs, tissue or cells into a human, wherein the symptoms of hyperacute rejection are improved as compared to tissue from a wild-type swine when transplanted into a human.
- the disclosure provides a cell culture reagent derived from a knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein the disruption of said a(l,3)-galactosyltransferase gene is selected from the group of disruptions comprising a 3 base pair deletion adjacent to a G to A substitution, a single base pair deletion, a single base pair insertion, a six base pair deletion, a two base pair insertion, a ten base pair deletion, a seven base pair deletion, and an eight base pair substitution for a five base pair sequence; wherein the disruption of said CMAH gene is selected from the group of disruptions comprising a four base pair insertion, a two base pair deletion, a single base pair insertion, an eight base pair deletion, a five base pair deletion, a three base pair deletion, a two base pair substitution for a single base pair, and a twenty base pair deletion; and wherein expression of functional
- a cell culture reagent may be selected from the group of cell culture reagents comprising cell culture media, cell culture serum, a cell culture additive and an isolated cell capable of proliferation.
- the invention provides a method of producing a glycoprotein of interest comprising the step of incubating an isolated cell capable of expressing the glycoprotein of interest with a cell culture reagent derived from a knockout pig comprising disrupted a(l,3)- galactosyltransferase and CMAH genes, wherein the amount of Neu5Gc or aGal epitopes on the glycoprotein of interest is lower than the amount of Neu5Gc or aGal epitopes on the glycoprotein of interest when an isolated cell capable of expressing said glycoprotein of interest is incubated with a cell culture reagent derived from a wild-type pig.
- the glycoprotein of interest may be selected from the group comprising an antibody, growth factor, cytokine, hormone and clotting factor.
- the disruption of the a(l,3)-galactosyltransferase gene is selected from the group of disruptions comprising a 3 base pair deletion adjacent to a G to A substitution, a single base pair deletion, a single base pair insertion, a six base pair deletion, a two base pair insertion, a ten base pair deletion, a seven base pair deletion, and an eight base pair substitution for a five base pair sequence;
- the disruption of said CMAH gene is selected from the group of disruptions comprising a four base pair insertion, a two base pair deletion, a single base pair insertion, an eight base pair deletion, a five base pair deletion, a three base pair deletion, a two base pair substitution for a single base pair, and a twenty base pair deletion; and expression of the functional a(l,3) galactosyltransferase and CMAH in the
- Figure 1 depicts a schematic of a protocol used to develop the double knock-out CMAH/GGTA1- pigs.
- Step a) shows the delivery of CMAH ZFNs to liver derived cells.
- Step b) illustrates identified CMAH mutant cells by screening individual clones.
- Step c) illustrates the delivery of GGTA1 ZFNs to CMAH KO cells.
- Step d) illustrates the counterselection of Gal negative cells.
- Step e) illustrates CMAH and GGTA1 double KO cells.
- Step f) illustrates somatic cell nuclear transfer (SCNT) resulting in CMAH and GGTAl double KO cells.
- SCNT somatic cell nuclear transfer
- Figure 2 provides genotype and phenotype analysis of double-KO fetuses.
- Panel A presents a photographic image of harvested double-KO fetuses.
- Panel B provides representative electropherograms of the mutations found in the CMAH gene in the double knockout fetuses. Mutations are underlined while the DNA binding sites of the ZFN are italicized.
- Panel C provides representative electropherograms of the mutations found in the GGTAl gene in the double knockout fetuses. Mutations are underlined while the DNA binding sites of the ZFN are italicized.
- Panel D presents data obtained from flow cytometric analysis of red blood cells (RBC) obtained from six month old wild-type piglets (WT), six-month-old GGTA1-KO (GGTA1-KO) piglets, double-KO fetuses and adult humans showing cells stained with an antibody recognizing Neu5GC.
- Panel E presents data obtained from flow cytometric analysis of red blood cells (RBC) obtained from six month old wild-type piglets (WT), six-month-old GGTA1-KO (GGTA1-KO) piglets, double-KO fetuses and adult humans showing cells stained with fluorescently labeled IB4 lectin to measure the level of the Gal epitopes. Unstained RBC were used as negative controls for IB4 lectin staining and an isotype matched control was used for Neu5Gc staining. Some negative control histograms are difficult to see because of significant overlap with the experimental group.
- Figure 3A presents a photograph of viable double-KO piglets.
- Panel B provides sequence information regarding the wild-type (WT) sequences for the CMAH and GGTAl target regions. The alterations that occur in the double-KO piglets in either the CMAH or GGTAl target region are underlined, while the binding sites are italicized.
- WT wild-type
- Figure 4 presents results from a series of experiments analyzing carbohydrate expression in genetically modified pigs.
- Panels A and B provide confocal micrographs of tissues from wild type (WT), single (GGTA1-KO) and CMAH-/-/GGTA1-/- (dou ble-KO) pigs. DAPI staining of nuclei may be visible. Heart, kidney and liver tissues were stained with anti-Neu5Gc antibody in the micrographs of panel A. Limited staining of Neu5Gc occurs in tissues from double-KO pigs. Heart, kidney and liver tissues were stained with IB4 lectin in the micrographs of panel B. Limited IB4 binding occurs in tissues from GGTA1- KO and double-KO pigs.
- Panel C presents results obtained from flow cytometry analysis of peripheral blood mononuclear cells (PBMC). Traces were obtained from cells labeled with anti-Neu5Gc antibody (left column) and IB4 lectin (right column). Unstained PBMC were the negative controls for IB4 lectin; an isotype negative control was used in the anti-Neu5Gc staining. Negative controls are shown (*) but are difficult to see in some panels because of significant overlap with the experimental group.
- PBMC peripheral blood mononuclear cells
- Figure 5 presents results from a series of experiments examining human antibody recognition of PBMC from GGTA1-KO and double-KO pigs.
- Panels A and B show IgG (A) and IgM (B) histograms of a representative sample (Subject 5) of randomly chosen normal human serum against GGTA1-KO or double-KO cells.
- Bar graphs C and D show the mean fluorescent intensities (MFI) of IgM) or IgG binding to peripheral blood monocytes (PBMCs). The results of testing ten unique human subjects are shown.
- Panels E and F illustrate example curves (Subject 3) for antibody-mediated complement-dependent cytotoxicity of normal human serum against GGTA1-KO and double KOPBMCs. Percent cytotoxicity for each serum tested (2.0% final concentration) is shown.
- Figure 6 shows flow cytometry used to analyze human antibody binding to GT-KO and GT/CMAH-KO porcine fetal fibroblasts in a dose dependent manner.
- GT/CMAH-KO cells bind less human IgM as compared to GT-KO porcine fetal fibroblasts.
- Figure 7 provides panels pertaining to the generation of GGTA1/CMAH double knockout porcine LDCs through a C ISP /Cas9 technique. LDCs that survived IB4 lectin counter-selection were expanded and stained with IB4-FITC.
- Figure 7A provides results of flow cytometry analysis of the a-Gal epitope on LDCs that survived IB4 lectin counter-selection. Also shown are unlabeled LDC and labeled, non- transfected LDC. In the particular example shown, 91.7% of the selected cells were free of the a-Gal epitope.
- Figure 7B provides an electropherogram of mutations found in Crispr-targeted GGTA1 and CMAH regions. The wild-type sequence of a portion of each targeted region is shown above the sequencing results. The top electropherogram is a portion of the GGTA1 gene; the bottom electropherogram is a portion of the CMAH gene.
- Figure 8 depicts flow cytometry results of labeled liver derived cells (LDCs) simultaneously transfected with CrispR CMAH and a-Gal targeting plasmids (CRISPR LDC); labeled, non-transfected LDC; and unlabeled LDC.
- CMAH expression in the cells was evaluated by assessing the anti-Neu5Gc antibody binding to Neu5Gc epitopes on the cell surface.
- CMAH produces Neu5Gc.
- Panel B Unselected
- a small percentage of the bulk transfected cell population shows a CMAH deficiency.
- FIG. 9 provides panels pertaining to generation of CMAH/aGal DKO fetuses from CMAH- CrispR and aGal-CrispR transfected LDC.
- Figure 9A provides a photograph of 10 fetuses harvested at day 32 of gestation after SCNT of Crispr-transfected LDC that survived IB4 counterselection and implantation of resulting embryos in recipient sows.
- DNA sequencing analysis of the GGTA1 and CMAH target regions was performed on each fetus.
- GGTA1 seven fetuses contained biallelic mutations, one fetus contained a monoallelic mutation, and two fetuses were wild-type.
- CMAH five fetuses contained biallelic mutations, one fetus contained a monoallelic mutation, and four fetuses were wild-type.
- Figure 9B provides the results of DNA sequencing analysis of the targeted GGTA1 and CMAH regions of five DKO fetuses with mutations in both alleles of both CMAH and GGTA1. If the two alleles have different mutations in a particular fetus, the sequence of both alleles is shown. If the two alleles of a fetus have the same mutation, the sequence is only shown once. The net change in nucleotide number is indicated to the right of the sequences. The wild-type sequence of the relevant portions of the GGTA1 and CMAH genes is shown above the fetal sequences. Fetal fibroblasts were derived from fetus 7.
- Panel C provides data from flow cytometry analysis of aGal and Neu5Gc on double knockout (DKO) and wild-type (WT) fetal fibroblasts. Unlabeled DKO and WT cells were also analyzed. The upper trace shows aGal results; the lower trace shows Neu5Gc results.
- DKO double knockout
- WT wild-type
- Figure 10 provides panels of flow cytometry analysis of Neu5Gc on CMAH/GAL double knockout fibroblast cells grown in media supplemented with either fetal bovine serum (FBS) or CMAH/aGAL double knockout derived serum (CMAH/Gal KO).
- Figure 10A illustrates data from an isotype control.
- Figure 10B provides Neu5Gc data. Cells grown in the CMAH deficient serum show lower anti-Neu5Gc antibody binding than cells grown in FBS.
- Figure 11 provides panels of flow cytometry analysis of Neu5Gc on double knockout (CMAH/Gal KO) fibroblasts and single aGal knockout (GGTA KO) renal cells grown with a variety of cell culture reagents.
- the upper panel provides flow cytometry results obtained from isotype control analysis of a CMAH/aGal DKO cell line (CMAH/Gal KO Fibroblast cell line) and aGall single knockout kidney cells (GGTA KO renal cells).
- the lower panel provides flow cytometry results of Neu5Gc on a CMAH/aGal DKO cell line (CMAH/Gal KO Fibroblast cell line) grown in culture media supplemented with CMAH/aGal derived serum and a GGTA-1 kidney cell line grown in culture media supplemented with FBS.
- CMAH/aGal DKO cell line CMAH/Gal KO Fibroblast cell line
- Figure 12 provides a graph of data obtained from perfusion experiments involving porcine livers perfused with human platelets.
- the post-perfusion initiation time in minutes is indicated on the x-axis (Time); the percent of human platelets remaining in the perfusion solution is indicated on the y-axis (Percent Platelets Remaining in Perfusion).
- Figure 13 provides individual curves for antibody-mediated, complement-dependent cytotoxicity of 10 randomly selected baboon serum samples against single (GGTA1-KO) and GGTA1/CMAH double (double-KO) peripheral blood monocytes (PBMCs). Results from a human serum sample against single (GGTA1-KO) and double-KO PBMC are also shown on each graph. Unexpectedly cytotoxicity of the baboon serum to the CMAH/aGAL double knockout PBMC differed widely from cytotoxicity of human serum to CMAH/aGal PBMCs.
- Cytotoxicity of baboon serum with CMAH/aGal double knockout PBMCs consistently increased as compared to cytotoxicity of baboon serum with aGal single knockout PBMCs.
- the dashed horizontal line indicates 50% killing.
- the %CTX for each sample was plotted against the log of each serum dilution and the sigmoid curve was analyzed by non-linear regression.
- Figure 14 provides histograms showing results of flow cytometry analysis of IgM ( Figure 14) and IgG ( Figure 14B) antibody recognition. Histograms show the results of 5 randomly selected human sera (1, 2, 3, 4 and 5) incu bated with GGTA1-KO pig, double-KO pig, baboon, chimpanzee and human PBMCs. Secondary only control antibody staining is shown in the bottom graph in each column. In four of the five samples shown, IgG antibody recognition for CMAH/GAL DKO PBMC and human PBMC yield almost overlaying traces.
- the IgM traces for the CMAH/GAL DKO PBMC are shifted closer to the traces for the human PBMC than the baboon, chimpanzee and single knockout (GGTA1-KO) traces.
- the present invention provides pigs and porcine organs, tissues or cells for transplantation into a human that do not express aGal and CMAH and methods of making the same.
- the invention provides a knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and when tissue from said pig is transplanted into a human, hyperacute rejection is decreased as compared to when tissue from a wild-type pig is transplanted into a human.
- the present invention provides transgenic animals suitable for use in xenotransplantation and methods of producing mammals suitable for use in xenotransplantation.
- the present invention describes the production of homozygous double knockout pigs lacking any functional expression of alpha 1,3 galactosyltransferase (aGAL) and CMAH.
- aGAL alpha 1,3 galactosyltransferase
- CMAH CMAH
- SCNT somatic cell nuclear transfer
- knockout mammal refers to a transgenic mammal wherein a given gene has been altered, removed or disrupted.
- double knockout we mean a transgenic mammal wherein two genes have been altered, removed or disrupted.
- the term is intended to include all progeny generations.
- the founder animal and all Fl, F2, F3, and so on, progeny thereof are included.
- knockout animals may have one or both copies of the gene sequence of interest disrupted. In the latter case, in which a homozygous disruption is present, the mutation is termed a "null" mutation. In the case where only one copy of the nucleic acid sequence of interest is disrupted, the knockout animal is termed a "heterozygous knockout animal".
- the knockout animals of the invention are typically homozygous for disruptions of both genes being targeted.
- chimera refers to a transgenic mammal with a knockout in some of its genome-containing cells.
- heterozygote or “heterozygotic mammal” refers to a transgenic mammal with a disruption on one of a chromosome pair in all of its genome containing cells.
- homozygote or “homozygotic mammal” refers to a transgenic mammal with a disruption on both members of a chromosome pair in all of its genome-containing cells.
- a "non-human mammal" of the invention includes mammals such as rodents, sheep, dogs, ovine such as lamb, bovine such as beef cattle and milk cows, and swine such as pigs and hogs.
- mammals such as rodents, sheep, dogs, ovine such as lamb, bovine such as beef cattle and milk cows, and swine such as pigs and hogs.
- porcine a typical non-human animal
- other mammals can similarly be genetically modified using the methods and compositions of the invention.
- a “mutation” is a detecta ble change in the genetic material in the animal, which is transmitted to the animal's progeny.
- a mutation is usually a change in one or more deoxyribonucleotides, such as, for example, adding, deleting, inverting, or substituting nucleotides.
- pig we mean any pig known to the art, including a wild pig, a domestic pig, mini pigs, a Sus scrofa pig, a Sus scrofa domesticus pig, as well as inbred pigs.
- the pig can be selected from the group consisting of, for example, Landrace, Hampshire, Duroc, Chinese Meishan, Chester White, Berkshire Goettingen, Landrace/York/Chester White, Yucatan, Bama Xiang Zhu, Wuzhishan, Xi Shuang Banna, and Pietrain pigs.
- Porcine organs, tissue or cells are organs, tissue or cells from a pig.
- the present invention provides a transgenic animal lacking any expression of functional aGal and CMAH genes.
- the animal can be any mammal suitable for xenotransplantation.
- the animal is a pig.
- CMAH/aGAL double knockouts "CMAH/aGAL DKO”, “CMAH/aGal”, “CMAH/aGal DKO”, "CMAH 7 ⁇ /GAL.
- the invention provides organs, tissue and/or cells from animals lacking any expression of functional aGal and CMAH for use as xenografts.
- the tissues from animals lacking any functional expression of the aGal and CMAH gene can be obtained from a prenatal, neonatal, immature, or fully mature animal, such as a porcine, bovine or ovine.
- the organ may be used as a temporary or permanent organ replacement for a patient in need of an organ transplant.
- Any porcine organ can be used, including but not limited to the brain, heart, lungs, eye, stomach, pancreas, kidneys, liver, intestines, uterus, bladder, skin, hair, nails, ears, glands, nose, mouth, lips, spleen, gums, teeth, tongue, salivary glands, tonsils, pharynx, esophagus, large intestine, small intestine, rectum, anus, thyroid gland, thymus gland, bones, cartilage, tendons, ligaments, suprarenal capsule, skeletal muscles, smooth muscles, blood vessels, blood, spinal cord, trachea, ureters, urethra, hypothalamus, pituitary, pylorus, adrenal glands, ovaries, oviducts, vagina, mammary glands, testes, seminal vesicles, penis, lymph, lymph nodes and lymph vessels.
- the invention provides non-human tissues that are useful for xenotransplantation.
- the non-human tissue is porcine tissue. Any porcine tissue can be used, including but not limited to epithelium, connective tissue, blood, bone, cartilage, muscle, nerve, adenoid, adipose, areolar, bone, brown adipose, cancellous, muscle, cartilaginous, cavernous, chondroid, chromaffin, dartoic, elastic, epithelial, fatty, fibrohyaline, fibrous, Gamgee, gelatinous, granulation, gut-associated lymphoid, skeletal muscle, Haller's vascular, indifferent, interstitial, investing, islet, lymphatic, lymphoid, mesenchymal, mesonephric, multilocular adipose, mucous connective, myeloid, nasion soft, nephrogenic, nodal, osteoid, osse
- the invention also provides cells and cell lines from porcine animals that lack expression of functional aGal and CMAH.
- these cells or cell lines can be used for xenotransplantation.
- Cells from any porcine tissue or organ can be used, including, but not limited to: epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, Islets of Langerhans cells, pancreatic insulin secreting cells, pancreatic alpha-2 cells, pancreatic beta cells, pancreatic alpha-1 cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endotheli
- Nonviable derivatives include tissues stripped of viable cells by enzymatic or chemical treatment these tissue derivatives can be further processed through crosslinking or other chemical treatments prior to use in transplantation.
- the derivatives include extracellular matrix derived from a variety of tissues, including skin, bone, urinary, bladder or organ submucosal tissues.
- tendons, joints, and bones stripped of viable tissue to including but not limited to heart valves and other nonviable tissues as medical devices are provided.
- serum or medium suitable for cell culture and isolated from a knockout pig of the invention are provided.
- Components of porcine knockout organs, tissues or cells are also provided.
- Components may also be modified through any means known in the art including but not limited to crosslinking and aldehyde crosslinking. Components may vary depending on the larger organ or tissue from which the component is obtained. Skin components may include but are not limited to stripped skin, collagen, epithelial cells, fibroblasts and dermis. Bone components may include but are not limited to collagen and extracellular matrix. Heart components may include but are not limited to valves and valve tissue.
- Expression of a gene product is decreased when total expression of the gene product is decreased, a gene product of an altered size is produced or when the gene product exhibits an altered functionality. Thus if a gene expresses a wild-type amount of product but the product has an altered enzymatic activity, altered size, altered cellular localization pattern, altered receptor-ligand binding or other altered activity, expression of that gene product is considered decreased.
- Expression may be analyzed by any means known in the art including, but not limited to, T-PC , Western blots, Northern blots, microarray analysis, immunoprecipitation, radiological assays, polypeptide purification, spectrophotometric analysis, Coomassie staining of acrylamide gels, ELISAs, 2-D gel electrophoresis, in situ hybridization, chemiluminescence, silver staining, enzymatic assays, ponceau S staining, multiplex T-PC , immunohistochemical assays, radioimmunoassay, colorimetric assays, immunoradiometric assays, positron emission tomography, fluorometric assays, fluorescence activated cell sorter staining of permeablized cells, radioimunnosorbent assays, real-time PCR, hybridization assays, sandwich immunoassays, flow cytometry, SAGE, differential amplification or electronic analysis.
- T-PC Western
- Expression may be analyzed directly or indirectly.
- Indirect expression analysis may include but is not limited to, analyzing levels of a product catalyzed by an enzyme to evaluate expression of the enzyme. See for example, Ausubel et al, eds (2013) Current Protocols in Molecular Biology, Wiley-lnterscience, New York, N.Y. and Coligan et al (2013) Current Protocols in Protein Science, Wiley-lnterscience New York, NY. Gene expression assays for porcine ASGR1 are commercially available (Applied BiosystemsTM, Carlsbad CA).
- As compared to is intended encompass comparing something to a similar but different thing, such as comparing a data point obtained from an experiment with a knockout pig to a data point obtained from a similar experiment with a wildtype pig.
- the word "comparing” is intended to encompass examining character, qualities, values, quantities, or ratios in order to discover resemblances or differences between that which is being compared. Comparing may reveal a significant difference in that which is being compared.
- significant difference is intended a statistically significant difference in results obtained for multiple groups such as the results for material from a knockout pig and material from a wild-type pig.
- Statistical significance is assessed by a statistical significance test such as but not limited to the student's t-test, Chi-square, one-tailed t-test, two-tailed t-test, ANOVA, Dunett's post hoc test, Fisher's test and z-test.
- a significant difference between two results may be results with a p ⁇ 0.1, p ⁇ 0.05, p ⁇ 0.04, p ⁇ 0.03, p ⁇ 0.02, p ⁇ 0.01 or greater.
- isolated is intended to encompass an entity that is physically separated from another entity or group.
- An isolated cell is physically separated from another group of cells. Examples of a group of cells include, but are not limited to, a developing cell mass, a cell culture, a cell line, a tissue, and an animal.
- isolated is intended to encompass physically separating an entity from another entity or group. Examples include physically separating a cell from other cells, physically separating a cell component from the remainder of the cell and physically separating tissue or organ from an animal.
- An isolated cell or cell component is separated by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, up to 100% of other naturally occurring cells or cell components.
- Methods for isolating one or more cells from another group of cells are known in the art. See for example Freshney (ED) Culture of Animal Cells: a manual of basic techniques (3 Ed.) 1994, Wiley-Liss; Spector et al (Eds)(1998) Cells: a Laboratory Manual (vol.1) Cold Spring Harbor Laboratory Press and Darling et al (1994) Animal Cells: culture and media John Wiley & Sons.
- Methods of isolating a tissue or an organ from an animal are known in the art and vary depending on the tissue or organ to be isolated and the desired method of transplanting the tissue or organ.
- a "skin related product” encompasses products isolated from skin and products intended for use with skin. Skin related products isolated from skin or other tissues may be modified before use with skin. Skin related products include but are not limited to replacement dressings, burn coverings, dermal products, replacement dermis, dermal fibroblasts,collagen, chondroitin, connective tissue, keratinocytes, cell-free xenodermis,cell-free pig dermis,composite skin su bstitutes and epidermis and temporary wound coverings. See for example Matou-Kovd et al (1994) Ann Med Burn Club 7:143, herein incorporated by reference in its entirety.
- Xenotransplantation encompasses any procedure that involves the transplantation, implantation or infusion of cells, tissues or organs into a recipient subject from a different species. Xenotransplantation in which the recipient is a human is particularly envisioned. Thus xenotransplantation includes but is not limited to vascularized xenotransplant, partially vascularized xenotransplant, unvascularized xenotransplant, xenodressings, xenobandages, and xenostructures.
- the invention provides a method of improving a hyperacute rejection related symptom in a patient comprising transplanting porcine organs, tissue or cells having reduced expression of aGal and Neu5Gc on the porcine organs, tissue or cells into a human, wherein the hyperacute rejection related symptoms are improved as compared to when tissue from a wild-type swine is transplanted into a human.
- a hyperacute rejection related symptom may encompass a decrease, lessening, or diminishing of an undesirable symptom.
- hyperacute rejection we mean rejection of the transplanted material or tissue occurring or beginning within the first 24 hours post-transplant involving one or more mechanisms of rejection. Hyperacute rejection encompasses but is not limited to “acute humoral rejection” and “antibody mediated rejection”.
- Hyperacute rejection related symptom is intended to encompass any symptom known to the field as related to or caused by hyperacute rejection. It is recognized that hyperacute rejection related symptoms may vary depending upon the type of organ, tissue or cell that was transplanted.
- Hyperacute rejection related symptoms may include, but are not limited to, thrombotic occlusion, hemorrhage of the graft vasculature, neutrophil influx, ischemia, mottling, cyanosis, edema, organ failure, reduced organ function, necrosis, glomerular capillary thrombosis, lack of function, hemolysis, fever, clotting, decreased bile production, asthenia, hypotension, oliguria, coagulopathy, elevated serum aminotransferase levels, elevated alkaline phosphatase levels, jaundice, lethargy, acidosis and hyperbilirubenemia and thrombocytopenia.
- Thrombocytopenia is a quantity of platelets below the normal range of 140,000 to 440,000/ ⁇ .
- Thrombocytopenia related symptoms include, but are not limited to, internal hemorrhage, intracranial bleeding, hematuria, hematemesis, bleeding gums, abdominal distension, melena, prolonged menstruation, epistaxis, ecchymosis, petechiae or purpura. Uptake of human platelets by pig livers contributes to the development of thrombocytopenia in xenograft recipients.
- Platelets also known as thrombocytes, are enucleate fragments of megakaryocytes involved in blood coagulation, hemostasis and blood thrombus formation. Human platelets are routinely isolated through a variety of methods including, but not limited to, platelet apheresis, plateletpheresis and ultracentrifugation.
- platelet uptake is intended to encompass the incorporation of a platelet into a liver or liver cell. While not being limited by mechanism, such uptake may occur through a phagocytic process. Platelet uptake may be monitored by any platelet uptake monitoring assay known in the art. Platelet uptake monitoring assays include, but are not limited to immunological methods, western blots, immunoblotting, microscopy, confocal microscopy, transmission electron microscopy and phagosome isolation. It is recognized that the appropriate platelet uptake monitoring assay may depend upon the type of label used.
- Platelet uptake may be measured as a percentage of total platelets absorbed, percentage of total platelets not absorbed, a ratio of absorbed to unabsorbed platelets, percentage of cells absorbing at least one platelet, percentage of cells not absorbing a platelet, or number of platelets absorbed per cell. It is recognized that platelet uptake by more than one cell type may contribute to the total platelet uptake of the liver.
- Total platelet uptake by an animal liver may include platelet uptake by liver sinusoidal endothelial cells, platelet uptake by Kuppffer cells, platelet uptake by LSECs and Kupffer cells and platelet uptake by additional cell types. It is recognized that platelet uptake by different cell types may contribute similar or disparate fractions of the total platelet uptake by a liver.
- an alteration, inhibition, reduction, decrease, or lowering of platelet uptake by a liver comprises an alteration, inhibition, reduction, decrease, or lowering of platelet uptake by one or more liver cell types.
- the word "providing” is intended to encompass preparing, procuring, getting ready, making ready, supplying or furnishing. It is recognized that methods of providing a cell may differ from methods of providing a subject and that methods of providing a liver may differ from methods of providing a pig.
- a cell culture reagent derived from a transgenic pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes is provided.
- Cell culture reagents are reagents utilized for tissue culture, in vitro tissue culture, microfluidic tissue culture, cell culture or other means of growing isolated cells or cell lines.
- Cell culture reagents may include but are not limited to cell culture media, cell culture serum, a cell culture additive, a feeder cell, and an isolated cell capable of proliferation.
- an isolated cell capable of proliferation is intended a cell isolated or partially isolated from other cell types or other cells wherein the cell is capable of proliferating, dividing, or multiplying into at least one additional clonal cell.
- Cells grown in culture may synthesize or meta bolically incorporate antigenic epitopes into glycoproteins secreted by the cultured cell.
- the antigenic epitopes may result in increased binding by human antibodies and decreased efficacy of the glycoprotein. See Ghaderi et al, 2010 Nature Biotechnology 28(8):863-867, herein incorporated by reference in its entirety.
- Growing the producing cell in a cell culture reagent with less aGal or Neu5Gc may reduce the amount of aGal, Neu5Gc, or both aGal and Neu5Gc epitopes on the glycoprotein of interest.
- Glycoproteins of interest may include any glycoprotein, particularly glycoproteins intended for use in human su bjects such as but not limited to, an antibody, growth factor, cytokine, hormone, or clotting factor.
- xenoantigens aGal and Neu5Gc were eliminated in transgenic pigs by genetic modification.
- Double knockout pigs (GT/CMAH-KO) were produced within 5-10 months or less.
- tissues are provided in which both the aGal and CMAH genes are rendered inactive, such that the resultant aGal and CMAH products can no longer generate alpha 1,3-galactosyl epitopes or Neu5Gc on the cell surface.
- the aGal and CMAH genes can be inactivated in such a way that no transcription of the gene occurs.
- the present invention provides a method for producing viable pigs lacking any functional expression of aGal and CMAH.
- the pigs are produced as described below. Methods of making transgenic pigs, and the challenges thereto, are discussed in Galli et al. 2010 Xenotransplantation, 17(6) p. 397-410, incorporated by reference herein for all purposes. The methods and cell cultures of the invention are further detailed below. EXAMPLES
- a pair of ZFN were designed to bind and cleave the sequence of porcine CMAH exon 9 (SEQ ID NO: 1-AAACTCCTGAACTACAAGGCTCGGCTGGTGAAGGA) beginning at position 1,341 of Ensemble transcript ENSSSCT00000001195.
- Another pair of ZFN were designed to bind and cleave the region of GGTA1 exon 8 (SEQ ID NO: 2-GTCATCTTTTACATCATGGTGGATGATATCTCCAGGATGCC) beginning at position 1165 of Ensemble transcript ENSSSCT00000006069.
- the ZFN activities were validated in yeast (Sigma-Aldrich, St. Louis, MO). Additional detail regarding the ZFN of the present invention can be found in Li et al., Journal of Surgical Research, (2012)E1-E7 Epub 2012 July 3, which is incorporated by reference herein for all purposes.
- LDC Porcine adult liver derived cells
- LDC were harvested by trypsin digestion, washed with calcium and magnesium free Dulbecco's phosphate buffered saline (DPBS) (Invitrogen) and centrifuged. 10 s cells were suspended in 120 ⁇ of resuspension buffer (Invitrogen) containing 2 ⁇ gs of each CMAH ZFN plasmid. pEGFP-N 1 (Clonetech, Mountain View, CA) was used as a control to measure transfection efficiency as well as ZFN activity. Cells were electroporated at 1300 V, 30 msec for 1 pulse.
- DPBS calcium and magnesium free Dulbecco's phosphate buffered saline
- ZFN-induced mutation was detected using the Surveyor Mutation Detection kit (Transgenomic, Omaha, NE). At day 5 post-transfection, genomic DNA from ZFN-treated and control plasmid pEGFP-Nl treated cells was extracted and PCR was performed using primers ZFN-CMAH-F (SEQ ID NO: 3-5' GGACCTGCTTTATCTTGCTCGC 3'), ZFN-CMAH-R (SEQ ID NO: 4-5' CC ATACTTGTCTG CTGGGTGGG 3').
- PCR product was denatured and annealed using the following program on a MyCycler (Bio-Rad): 95°C, 10 minutes; 95°C to 85°C, -2°C /second; 85°C to 25°C, -0.1°C /second. 200-400 ng of PCR product was digested with 1 ⁇ of Nuclease S and 1 ⁇ of enhancer (Transgenomic, Omaha, NE) at 42°C for 40 minutes. The product was separated on a 10% polyacrylamide gel and stained with SYBR Safe to assess ZFN-induced mutations.
- CMAH-S1 SEQ ID NO: 5-5' CCAAACCCTGTCATTCCAG 3' was used to sequence the ZFN targeted CMAH region. A clone with an identical mutation in both CMAH gene copies was identified.
- CMAH deficient LDC were treated with zinc finger plasmids targeting porcine 1,3 aGal.
- LDC were transfected with Crispr CMAH sgRNA expression plasmids and GGTAl sgRNA expression plasmids and grown for 48 hours as described elsewhere herein. The cells were harvested, washed and counted.
- the IB4 lectin is specific for the Gal epitope; cells that did not bind the Dynabeads remained in the supernatant.
- CMAH cells that did not bind IB4 and the Dynabeads were identified as Gal-/- cells.
- Crispr CMAH and GGTAl treated cells that did not bind IB4 and the Dynabeads were utilized in SCNT as described below herein.
- TALEN constructs are designed to bind and cleave the sequence of porcine CMAH at a suitable site.
- Additional Talen constructs are designed to bind and cleave GGTAl at a suitable site.
- Somatic Cell Nuclear Transfer was performed using in vitro matured oocytes (DeSoto Biosciences Inc., St. Seymour TN and Minitube of America, Mount Horeb, Wl). Cumulus cells were removed from the oocytes by pipetting in 0.1% hyaluronidase. Oocytes with normal morphology and a visible polar body were selected and incubated in manipulation media (calcium-free NCSU-23 with 5% fetal bovine serum (FBS) containing 5 ⁇ g/ml bisbenzimide and 7.5 ⁇ g/mL cytochalasin B for 15 minutes.
- manipulation media calcium-free NCSU-23 with 5% fetal bovine serum (FBS) containing 5 ⁇ g/ml bisbenzimide and 7.5 ⁇ g/mL cytochalasin B for 15 minutes.
- oocytes were enucleated by removing the first polar body and metaphase II plate.
- Single cells of CMAH deficient LDC that survived IB4 counterselection were injected into each enucleated oocyte.
- Crispr transfected cells that survived IB4 counterselection were injected into each enucleated oocyte.
- Electrical fusion was induced with a BTX cell electroporator (Harvard Apparatus, Holliston, MA).
- Enucleated oocytes injected with a cell (couples) were exposed to two DC pulses of 140 V for 50 ⁇ in 280 mM mannitol, 0.001 mM CaCI 2 and 0.05 mM MgCI 2 .
- reconstructed oocytes were activated by two DC pulses of 120 V for 60 ⁇ in 280 mM mannitol, 0. 1 mM CaCI 2 and 0.05 mM MgCI 2 .
- oocytes were placed in NCSU-23 medium with 0.4% bovine serum albumin (BSA) and incubated at 38.5°C, 5% C0 2 in a humidified atmosphere for less than one hour.
- BSA bovine serum albumin
- oocytes were transferred into a recipient pig.
- Recipient pigs were synchronized occidental pigs on their first day of estrus. Pregnancies were verified by ultrasound at day 25 or day 26 after embryo transfer.
- Results of SCNT using double knockout cells are summarized in Table 1.
- Results of SCNT using Crispr-transfected IB4 counterselected cells are summarized in Table 2.
- Table 1 Results of somatic cell nuclear transfer using double-KO cells.
- Dou ble-KO cells (cl22) were used as donor cells in SCNT and transferred into 2 recipients, 01 and 02. Fetal fibroblasts from fetus num ber 1 of recipient 02 were re-cloned and transferred to animals 03, 04, 05, and 06. 477 embryos were delivered to four gilts, resulting in one pregnancy and the birth of 5 piglets. The four via ble piglets appeared healthy as they were nursing since birth and needed no feeding support.
- SCNT of the selected cells deficient for CMAH and GGTAl resulted in a pregnancy that was terminated at day 30 of gestation.
- Five normal fetuses and one resorbed fetus were harvested, photogra phed and shown in Figure 1A.
- Red blood cells from the dou ble knockout fetuses were analyzed by flow cytometry as described below herein.
- Fibroblasts were grown from fetus number one. The fibroblasts were utilized in SCNT to generate 477 em bryos which were transferred into four recipient gilts. The SCNT resulted in one pregnancy and the birth of four live piglets and one still born piglet. The four via ble piglets needed no feeding support and appeared healthy. Three of the aGal/CMAH dou ble knockout pigs are shown in Figure 3A. The above described disruptions of the CMAH and GGTAl genes were validated in the four via ble pigs from the second pregnancy as described below herein. Results of somatic cell nuclear transfer using dou ble-KO cells are su mmarized in Ta ble 1 a bove.
- CMAH complementary metal-oxide-semiconductor
- five fetuses were biallelic mutations, one fetus was a monoallelic mutation and four fetuses were wild-type.
- two homozygous mutations were found in fetus 7 at the GGTA1 locus and in fetus 2 at the CMAH locus.
- the remaining mutations were heterozygous biallelic mutations. Results are summarized in Table 2 below and sequence data are presented in Figure 9B.
- Fetal fibroblasts were harvested from one dou ble knockout fetus. The fetal fibroblasts were utilized in SCNT to generate embryos which were transferred into recipient sows. SCNT of the double knockout fetal fibroblasts resulted in at least one pregnancy.
- Genomic DNA from double knockout Zn finger cloned fetuses and the four viable piglets was extracted using DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA). PCR amplification of the CMAH region was performed as described above herein. [0095] Primer CMAH-S1 (SEQ ID NO: 5-5' CCAAACCCTGTCATTCCAG 3') and GGTA1-F primer (SEQ ID NO: 6-5' CTAGAAATCCCAGAGGTTAC 3') were used to sequence the targeted CMAH region and GGTAl region, respectively.
- the GGTAl region was amplified by PCR using the GGTA1-F primer (SEQ ID NO: 6 (5' CTAGAAATCCCAGAGGTTAC 3')) and GGTA1-R primer (SEQ ID NO:7 (5'TCCTTGTCCTGGAGGATTCC3')).
- Pwo Master Roche, Indianapolis IN
- PCR conditions were as follows: 94°C, 2 min; 94°C, 15 s, 57°C, 30 s, and 72°C, 30s for 40 cycles; and a final extension step of 72°C for 5 min.
- a total of 200-400 ng of PCR product was denatured and annealed using the following program on a Mycycler (Bio-Rad): 95°C, 10 min; 95° to 85°C, -2°C/s; 85°C to 25°C, -0.1°C/s.
- One microliter of enhancer and 1 ⁇ of Nuclease S was added to each reaction and incubated at 42°C for 40 minutes. The product was separated on a 10% polyacrylamide gel and stained with SYBR Safe (Invitrogen USA Eugene OR).
- Results from an exemplary DNA sequence analysis are shown in Figure 2B.
- DNA sequence analysis confirmed homozygous alterations of the GGTAl and CMAH genes in at least one fetus.
- the CMAH gene sequence is altered by a four base pair insertion.
- the inserted sequence is "GGAA”.
- the GGTAl gene sequence is altered by a 3 base pair deletion adjacent to a G to A substitution.
- the sequences of the targeted CMAH and GGTAl regions of the genomic DNA of four viable piglets are shown in Figure 3B.
- Genomic DNA from IB4 counter-selected cells and Crispr cloned fetuses was extracted using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma Aldrich, St. Louis MO).
- the GGTAl region was amplified by PCR using the GGTA1-F seq primer (SEQ ID NO: 8 (5' CCTTAGTATCCTTCCC AACCC AG AC-3' ) ) and GGTA1-R seq primer (SEQ ID NO: 9 (5' GCTTTCTTTACGGTGTCAGTGAATCC-3')).
- the CMAH region was amplified by PCR using the CMAH-F seq primer (SEQ ID NO: 10 (5'- CTTGGAGGTGATTTGAGTTGGG-3')) and the CMAH-R seq primer (SEQ ID NO: 11 (5'- CATTTTCTTCGGAGTTGAGGGC-3')).
- GGTAl Pwo Superyield DNA polymerase (Roche, Indianapolis IN) was used and PCR conditions for GGTAl were as follows: 94°C, 2 min; 94°C, 15 s, 54°C, 30 s, and 72°C, 45s for 15 cycles; 94°C, 15 s, 54°C, 30 s, and 72°C, 45s + 5 seconds/cycle for 25 cycles; and a final extension step of 72°C for 5 min.
- CMAH CMAH-derived DNA sequencer
- GenElute Gel Extraction kit Sigma-Aldrich, St. Louis MO
- Sanger method DNA Sequencing Core Facility, Indiana University School of Medicine
- CMAH-F seq (SEQ ID NO: 10) and GGTA1-F seq primer (SEQ ID NO: 8) were used to sequence the targeted CMAH region and GGTAl region, respectively.
- both GGTAl alleles have an eight nucleotide insertion that substitute for a five nucleotide sequence.
- one CMAH allele has a double base pair deletion (deletes TG), the other allele has a single nucleotide insertion (inserts T).
- both CMAH alleles have an 8 base pair deletion.
- one CMAH allele has a 5 nucleotide deletion, the other has a 3 nucleotide deletion.
- one CMAH allele has a single base pair deletion (T), the other has single base pair deletion and a colocalized two base pair insertion.
- CMAH allele has a 5 base pair deletion, the other has a 20 base pair deletion.
- Sequences of the targeted CMAH and GGTAl regions of the genomic DNA of five fetuses are shown in Figure 9B.
- Fetal livers were removed from double knockout fetuses and incubated in RPM I1640 for
- RBCs were collected from cells released into the media after incubation of the fetal livers. RBCs were also obtained from adult human donors, six month old wild-type pigs and GGTA- knockout pigs (GGTA-KO pigs (fetal or 6 month old)). Porcine and human peripheral blood monocytes (PBMCs) were prepared using Ficoll-Paque Plus from whole blood collected in anticoagulant citrate dextrose (ACD).
- ACD anticoagulant citrate dextrose
- Neu5Gc antibody (Sialix, Vista CA). A negative control antibody for comparison with anti-Neu5Gc antibody was also used (Sialix, Vista CA). Cells were incubated with IB4 lectin for 20 minutes at 4°C. The cells were washed with blocking agent (Sialix, Vista, Ca) diluted in HBSS. Gal epitopes bind IB4 lectin. Cells were then stained with anti-Neu5Gc antibody for one hour at 4°C followed by Donkey anti-chicken DyLight 649 (Jackson ImmunoResearch Laboratories Inc, West Grove PA) for 40-60 minutes at 4°C. Cells stained with anti-Neu5Gc antibody were washed before and after secondary antibody with Sialix blocking agent diluted in PBS. In various experiments unstained BC or PBMC were used as negative controls for IB4 lectin staining. An Accuri C6 flow cytometer and CFlow Software (Accuri, Ann Arbor, Ml) were used for analysis.
- One of the four double knockout (Double-KO, DKO, CMAH-/aGAL-) piglets with mutations generated by Zn-finger targeting was euthanized.
- Liver, heart and kidney tissues were obtained from the double-ko pig.
- Liver, heart and kidney tissues were also obtained from wild-type (WT) and GGTA1 knockout (GGTA-ko) pigs. Frozen sections of each tissue were prepared. Mounted tissues were blocked in Odyssey blocking buffer (Ll-Cor Biosciences, Lincoln NE) in HBSS for one hour. The slides were then fixed in 4% paraformaldehyde for 10 minutes.
- Tissues were stained with IB4 lectin Alexa Fluor 647 (Invitrogen, Grand Island NY) to visualize the presence of the Gal epitope.
- IB4 lectin Alexa Fluor 647 Invitrogen, Grand Island NY
- tissues were stained with a chicken anti-Neu5Gc antibody or with a control antibody (Sialix, Vista, CA) for one hour.
- Tissues were washed three times with HBSS.
- Donkey anti- chicken DyLight 649 Jackson ImmunoResearch Laboratories Inc., West Grove PA
- secondary antibody was incubated with the tissue for approximately one hour.
- Tissues were washed three times with 0.1% HBSS Tween.
- DAPI stain Invitrogen, Grand Island NY was added to all the slides for 1 minute followed by two 0.1% HBSS Tween washes. Tissues were mounted in ProLong Gold (Invitrogen, Grand Island NY). Confocal microscopy was performed using an Olympus FV1000.
- Confocal microscopy indicated the presence of a-Gal and Neu5Gc in liver, heart and kidney tissues obtained from a wild-type pig.
- GGTA1-KO pigs displayed only Neu5Gc in liver, heart and kidney tissues.
- Confocal microscopy indicated the absence of a-Gal and Neu5Gc in liver, heart and kidney in tissues obtained from a double knockout piglet (exemplary fields are shown in Figure 4A and 4B).
- Example 11 Crossmatch of Human Sera with GGTA1-KO and double-KO PBMCs.
- Porcine whole blood from GGTA1-KO and dou ble-KO pigs was collected in ACD. Porcine
- PBMCs were prepared from the whole blood using Ficoll-Paque Plus. Cell viability was assessed microscopically with Trypan Blue. Sera were obtained from ten healthy human volunteers. Twenty-five percent heat-inactivated serum was prepared. Approximately 2 X 10 6 /ml GGTA-KO and double-KO PBMCs were incubated with each human serum sample for 2 hours at 4°C. After incubation of the serum and PBMCs, the PBMCs were washed three times in 0.5% PBS Sialix Blocking agent.
- PBMCs were stained with DyLight 649-conjugated Donkey anti-human IgM or DyLight 488 Donkey anti-human IgG (Jackson Immunoresearch Laboratories Inc, West Grove PA) for 1 hour at 4°C.
- PBMCs were washed three times using 0.5% PBS Sialix blocking agent.
- Analyses were performed using an Accuri C6 flow cytometer and BD CFlow Plus Software (Accuri, Ann Arbor Ml). Overlays were produced using Kaluza version 1.2 software from Beckman Coulter (Brea, CA).
- a representative histogram is shown in Figure 5A. MFI's obtained from flow cytometry crossmatch analysis of 10 unique human subjects are also shown in Figure 5A.
- MFI Mean fluorescent Intensities
- Antibody-mediated complement dependent cytotoxicity assays are known in the art.
- a modified method of Diaz et al (Diaz et al, 2004, Transplant Immunology 13(4):313-317) was performed.
- Human serum was obtained from ten healthy volunteers ( Figures 5 and 6). Twenty-five percent heat- inactivated human serum was prepared. The heat-inactivated human sera were serially diluted and 100 ⁇ of each concentration were placed in a 96 well v-bottom assay plate. Sera were mixed with a 100 ⁇ aliquot of PBMC obtained from either GGTA1-KO or double-KO pigs.
- the final concentration of PBMC in each well was 5 X 10 6 /ml; in some experiments 1 x 10 6 /ml PBMC were used.
- the serum concentrations varied from 50%, 17%, 6%, 2%, 0.6%, 0.2% and 0.07%.
- the mixtures were incubated for 30 minutes at 4°C. After 30 minutes, the plates were centrifuged for 4 minutes at 400 x g. The plates were decanted and washed with HBSS. Rabbit complement (150 ⁇ of a 1:15 dilution) was added to each well and incubated for 30 minutes at 37°C.
- PBMC peripheral blood mononuclear cells
- %CTX (%CTX eX p-%CTX S pont)/(100-%CTX S p O nt) x 100, where %CTX exp is the percentage of dead cells under the experimental condition.
- Antibody-mediated, complement dependent cytotoxicity data was also analyzed as above.
- the log of each serum dilution was plotted against the %CTX for each sample and the sigmoid curve was analyzed by non-linear regression to estimate the serum LD50.
- the %CTX of GGTAl-KO and double-KO PBMCs at each serum dilution for each sample was analyzed using the Wilcoxon matched pairs signed rank test.
- Example 14 Ex vivo Perfusion of Human Platelet through a Double KO Liver.
- a double knockout CMAH/aGal pig is anesthetized and intubated. A midline abdominal incision is made. The liver is removed and placed in a perfusion device under normothermic conditions. Humidity, temperature and air flow are maintained in the perfusion device. Human platelets obtained from healthy volunteer subjects are circulated through the double knockout liver. Platelet levels in the pre-perfusion and post-perfusion samples are evaluated. Pre and post-perfusion evaluation of the pig liver is performed.
- Example 15 Evaluation of Response to a Double Knockout (ctGal and CMAH) Xenograft.
- a porcine liver obtained from double knockout (aGal-/CMAH-) pigs is surgically transplanted into a recently deceased human cadaver using the piggyback method. After the surgery, biological samples are obtained from the human cadaver. Clinical indicia of graft rejection are monitored.
- Example 16 Evaluation of a Response to a Double Knockout (ctGal and CMAH) Xenograft.
- Porcine kidneys are obtained from double knockout (GGTA1/CMAH double-KO) pigs. A highly sensitized human subject is administered compounds to manage preexisting and de novo donor- specific antibodies. Porcine double-KO kidneys are surgically transplanted into the subject. Clinical indicia of graft rejection are monitored.
- Targeting sites for the CMAH and GGTA1 genes were identified. Forward and reverse oligonucleotides for each targeting site were obtained.
- the GGTA1 forward sgRNA sequence was CACCGAGAAAATAATGAATGTCAA (SEQ ID NO: 12); the GGTA1 reverse sgRNA sequence was AAACTTGACATTCATTATTTTCTC (SEQ ID NO: 13).
- the CMAH forward sgRNA sequence was CACCGAGTAAGGTACGTGATCTGT (SEQ ID NO: 14); the CMAH reverse sgRNA sequence was AAACACAGATCACGTACCTTACT (SEQ ID NO: 15).
- the oligonucleotide pairs were annealed together to generate short double strand DNA fragments with Bbsl compatible overhangs.
- the pX330 bicistronic expression vector expressing Cas9 and sgRNA of interest was linearized with Bbsl.
- pX330 expresses a mammalian codon optimized Cas9.
- the linearized pX330 expression vector and the double strand DNA fragments for each sg NA (GGTA1 and CMAH) were incubated with ligase.
- LDCs Liver derived cells
- LDC were cotransfected with pX330- CMAHsgRNA and pX300-GGTAlsgRNA expression plasmids using the Neon transfection system (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were harvested and used for isolectin B4 counter-selection.
- IB4 Isolectin B4 counter-selected LDC, cloned fetal fibroblasts from DKO fetuses, wild- type LDC and wild-type fetal fibroblasts were stained with IB4 conjugated with FITC (Enzo Life Science, Farmingdale NY) to assess the aGal epitope level.
- IB4 counter-selected LDC cloned fetal fibroblasts from DKO fetuses, wild-type LDC and wild-type fetal fibroblasts were stained with antiNeu5Gc antibody (Sialix, Vista CA) followed by donkey anti-chicken DyLight 649 (Jackson ImmunoResearch Laboratories Inc., West Grove PA).
- a negative control antibody for comparison with anti-Neu5Gc antibody was also used (Sialix, Vista CA).
- An Accuri C6 flow cytometer (Accuri, Ann Arbor Ml) and FlowJo software (Tree Star, Inc. Ashland OR) were used for analysis.
- Representative flow cytometry results of aGal from IB4 counter selected cells are shown in Figure 7A.
- Representative flow cytometry results of Neu5Gc on IB4 counter selected cells are shown in Figure 8A.
- Representative flow cytometry results of the aGal and Neu5Gc on fetal fibroblasts derived from fetus 7 are shown in Figure 9C.
- Example 20 Neu5Gc Levels in Cells Cultured with a DKO (aGal/CMAH) Piq Cell Culture Reagent.
- Fibroblasts were cultured from a CMAH/aGal Double knockout pig. Because the DKO fibroblasts lack CMAH, they do not produce Neu5Gc. DKO serum was isolated from the blood of CMAH/aGAL pigs. The DKO fibroblasts were cultured in cell culture media supplemented with either DKO serum or bovine serum (FBS) for six weeks. After six weeks, cells were evaluated by flow cytometry with anti-Neu5Gc antibodies. Results from one such experiment are presented in Figure 10.
- Example 21 Concordant Analysis of DKO Pig, Baboon and Chimpanzee Material.
- Antibody-mediated complement dependent cytotoxicity assays are known in the art.
- a modified method of Diaz et al (Diaz et al, 2004, Transplant Immunology 13(4):313-317) was performed. Serum samples were obtained from 10 randomly selected baboons. Twenty-five percent heat- inactivated baboon serum was prepared. The heat-inactivated baboon sera were serially diluted and 100 ⁇ of each concentration were placed in a 96 well v-bottom assay plate. Sera were mixed with a 100 ⁇ aliquot of PBMC obtained from either GGTA1-KO or double-KO (GGTA1/CMAH DKO) pigs. The final concentration of PBMC in each well was 5 X 10 6 /ml; in some experiments 1 x 1010 6 /ml PBMC were used.
- Serum was obtained from five randomly selected human donors. Twenty-five percent heat-inactivated serum was prepared. Human, baboon and chimpanzee PBMCs were obtained. PBMCs were obtained from single aGal knockout pigs (GGTA1-KO) and double CMAH/aGal (double-KO) pigs. Approximately 2 X 10 6 /ml PBMCs were incubated with each human serum sample for 2 hours at 4°C. After incu bation of the serum and PBMCs, the PBMCs were washed three times in 0.5% PBS Sialix Blocking agent.
- PBMCs were stained with DyLight 649-conjugated Donkey anti-human IgM or DyLight 488 Donkey anti-human IgG (Jackson Immunoresearch Laboratories Inc, West Grove PA) for 1 hour at 4°C.
- PBMCs were washed three times using 0.5% PBS Sialix blocking agent.
- Analyses were performed using an Accuri C6 flow cytometer and BD CFlow Plus Software (Accuri, Ann Arbor Ml). Overlays were produced using Kaluza version 1.2 software from Beckman Coulter (Brea, CA). Secondary only control antibody staining was also evaluated. Histograms from one such experiment are presented in Figure 14.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Cell Biology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Husbandry (AREA)
- Physiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Virology (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides double knockout transgenic pigs (GT/CMAH-KO pigs) lacking expression of any functional aGAL and CMAH. Double knockout GT/CMAH-KO transgenic organs, tissues and cells are also provided. Methods of making and using the GT/CMAH-KO pigs and tissue are also provided.
Description
DOUBLE KNOCKOUT (GT/CMAH-KO) PIGS, ORGANS AND TISSUES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No:61/717,845, filed October 24, 2012, and U.S. Patent Application 13/804,365, filed March 14, 2013, both of which are incorporated herein by reference in their entirety for all purposes.
INCORPORATION OF SEQUENCE LISTING
[0002] The sequence listing in text format su bmitted herewith is herein incorporated by reference in its entirety for all purposes.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0003] Not applica ble.
FIELD OF THE INVENTION
[0004] The present invention is generally in the field of xenotransplantation and genetic modification to produce transgenic animals, orga ns tissue, or cells suita ble for transplantation into a hu man.
BACKGROUND
[0005] It is well known that transplants from one animal into another animal of the same species, such as human to human, are a routine treatment option for many serious conditions, including kidney, heart, lung, liver and other organ disease. However, it is also well known that there are not enough suita ble organs availa ble for transplant to meet current or expected clinical demands for organ transplants.
[0006] Xenotransplantation, the transplant of organs, tissue or cells from one animal into another animal of a different species, such as the transplantation of a pig organ into a human recipient, has the potential to eliminate the shortage of organs ava ila ble for transplant, potentially helping hundreds of thousands of people worldwide. For instance, suita ble organs for transplant from non-human donors, such as from a pig, could help keep seriously ill patients alive, either permanently or temporarily, until a suita ble human organ is availa ble for transplant.
[0007] While many mammalian animals may be suitable candidates for xenotransplantation, much of the current focus is on the pig. Using pig organs, tissue or cells for xenotransplantaion offers many advantages over other non-human mammalian donors. For instance, pigs are easily obtainable, they are inexpensive to breed and maintain, and, most importantly, many pig organs are similar to humans in size, shape and function.
[0008] However, xenotransplantation using standard, unmodified pig tissue into a human (or other primate) is accompanied by severe rejection of the transplanted tissue. The rejection may be a hyperacute rejection, an acute rejection or a chronic rejection. The hyperacute response to the pig antibodies present on the transplanted tissue is so strong that the transplant is typically damaged by the human immune system within minutes or hours of transplant into the human recipient.
[0009] Pig cells express al,3 galactosyltransferase (aGal) and cytidine monophosphate-N- acetylneuraminic acid hydroxylase (CMAH), which are not found in human cells. The aGal enzyme produces the aGal epitope. CMAH converts the sialic acid N-acetylneuraminic acid (Neu5Ac) to N- glycolylneuraminic acid (Neu5Gc). Accordingly, when pig tissue is transplanted into a human, these epitopes elicit an antibody-mediated rejection from the human patient immediately following implantation. The antibodies are present in the patient's blood prior to implantation of the tissue, resulting in the intense and immediate rejection of the implanted tissue.
[0010] Many strategies have been employed to address the rejection caused by aGal and CMAH, including removing the genes encoding aGal or CMAH to prevent expression of the enzymes, modifying the genes encoding aGal or CMAH to reduce or limit expression of the enzymes or otherwise limiting the ability of the enzymes to trigger a rejection response. For instance, U.S Patent 7,547,816 to Day et al. describes a knockout pig with decreased expression of aGal as compared to wild-type pigs. U.S. Patents 7, 166,278 and 8,034,330 to Zhu et al. describe methods for making porcine organs for transplantation that are less likely to be subject to hyperacute rejection. However, progress in this field is critically dependent upon the development of the genetically modified pigs.
[0011] Unfortunately, developing homozygous knockout pigs is a slow process, requiring as long as three years using homologous recombination in fetal fibroblasts followed by somatic cell nuclear transfer (SCNT), and then breeding of heterozygous knockout animals. The development of new knockout pigs for xenotransplantation has been hampered by the lack of pluripotent stem cells, relying instead on the fetal fibroblast as the cell upon which genetic engineering was carried out. For instance, the production of the first live pigs lacking any functional expression of aGal (GTKO) was first reported in
2003. U.S. Patent 7,795,493 to Phelps et al. describes a method for the production of a pig that lacks any expression of functional aGal.
[0012] Unfortunately, while the GTKO pig may have eliminated anti-aGal antibodies as a barrier to xenotransplantation, studies using GTKO cardiac and renal xenografts in baboons show that the GTKO organs still trigger an immunogenic response, resulting in rejection or damage to the transplanted organ. Baboons transplanted with GTKO kidneys and treated with two different immunosuppressive regimens died within 16 days of surgery. Chen et al. concluded "genetic depletion of Gal antigens does not provide a major benefit in xenograft survival" (Chen et al., 2005, Nature Med. 11(12):1295-1298. Basnet et al examined the cytotoxic response of human serum to CMAH-/- mouse cells. Basnet et al. concluded "the anti-NeuGc Ab-mediated immune response may be significantly involved in graft loss in xenogeneic cell transplantation, but not in organ transplantation" (Basnet et al., 2010, Xenotransplantation, 17(6):440-448).
[0013] Thus, there is a need in the art for an improved, simple, replica ble, efficient and standardized method of producing double knockout (aGAL and CMAH) pigs having no aGAL and CMAH expression (GT/CMAH-KO) as a source of human transplant material for organs, tissue and cells for human transplant recipients.
BRIEF SUMMARY
[0014] This disclosure relates generally to methods of making porcine organs, tissues or cells for transplantation into a human that do not express aGal and CMAH.
[0015] The present disclosure provides, in one embodiment, a knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)- galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and when tissue from said pig is transplanted into a human, hyperacute rejection is decreased as compared to when tissue from a wild-type pig is transplanted into a human.
[0016] In another embodiment, the specification provides porcine organs, tissue or cells for transplantation into a human having reduced expression of aGal and CMAH on the porcine organs, tissue or cells.
[0017] In another embodiment, the specification provides a method for modifying a porcine organs, tissue or cells for transplantation into a human, the method comprising removing or reducing expression of aGal and CMAH on the porcine organs, tissue or cells. The porcine organs, tissue, or cells may be
selected from the group consisting of red blood cells, skin, heart, livers, kidneys, lung, pancreas, thyroid, small bowel, and components thereof.
[0018] In another embodiment, the specification provides a method for making porcine organs, tissue or cells for transplantation into a human, the method comprising reducing expression of aGal and CMAH on the porcine organs, tissue or cells. The porcine organs, tissue, or cells may be selected from the group consisting of red blood cells, skin, heart, liver, kidneys, lung, pancreas, small bowel, and components thereof.
[0019] In another embodiment, the specification provides a knockout pig comprising disrupted a(l,3)- galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and wherein when tissue from a knockout pig is transplanted into a human, thrombocytopenia is decreased as compared to when tissue from a wild-type pig is transplanted into a human.
[0020] In another embodiment, the specification provides a knockout pig comprising disrupted a(l,3)- galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and wherein a liver from said pig exhibits reduced uptake of human platelets when said liver is exposed to said human platelets.
[0021] In another embodiment, the specification provides a method of increasing the duration of the period between when a human subject is identified as a subject in need of human liver transplant and when said human liver transplant occurs, said method comprising providing a liver from a knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type swine and surgically attaching said liver from said knockout pig to said human su bject in a therapeutically effective manner. The liver from the knockout pig may be internal or external to the human subject, and may be directly or indirectly attached to the human subject.
[0022] In another embodiment, the specification provides a method of preparing organs, tissues, or cells for xenotransplantation into human patients with reduced rejection, the method comprising providing a transgenic pig as a source of transplant material wherein the transplant material is selected from the group consisting of organs, tissues, or cells, and wherein the pig masks or reduces the expression of at least two xenoreactive antigens on the transplant material. At least two xenoreactive antigens may be aGal and Neu5Gc.
[0023] In another embodiment, the specification provides a knockout pig comprising disrupted a(l,3)- galactosyltransferase and CMAH genes, wherein the disruption of said a(l,3)-galactosyltransferase gene is selected from the group of disruptions comprising a 3 base pair deletion adjacent to a G to A substitution, a single base pair deletion, a single base pair insertion, a six base pair deletion, a two base pair insertion, a ten base pair deletion, a seven base pair deletion, and an eight base pair substitution for a five base pair sequence; wherein the disruption of said CMAH gene is selected from the group of disruptions comprising a four base pair insertion, a two base pair deletion, a single base pair insertion, an eight base pair deletion, a five base pair deletion, a three base pair deletion, a two base pair substitution for a single base pair, and a twenty base pair deletion; and wherein expression of functional a(l,3)-galactosyltransferase and CMAH in said knockout pig is decreased as compared to a wild-type pig and when tissue from said knockout pig is transplanted into a human, a hyperacute rejection related symptom is improved as compared to when tissue from a wild-type pig is transplanted into a human.
[0024] In another embodiment, the specification provides a method of improving symptoms of hyperacute rejection in a patient comprising transplanting porcine organs, tissue or cells having reduced expression of aGal and CMAH on the porcine organs, tissue or cells into a human, wherein the symptoms of hyperacute rejection are improved as compared to tissue from a wild-type swine when transplanted into a human.
[0025] In another embodiment, the disclosure provides a cell culture reagent derived from a knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein the disruption of said a(l,3)-galactosyltransferase gene is selected from the group of disruptions comprising a 3 base pair deletion adjacent to a G to A substitution, a single base pair deletion, a single base pair insertion, a six base pair deletion, a two base pair insertion, a ten base pair deletion, a seven base pair deletion, and an eight base pair substitution for a five base pair sequence; wherein the disruption of said CMAH gene is selected from the group of disruptions comprising a four base pair insertion, a two base pair deletion, a single base pair insertion, an eight base pair deletion, a five base pair deletion, a three base pair deletion, a two base pair substitution for a single base pair, and a twenty base pair deletion; and wherein expression of functional a(l,3)-galactosyltransferase and CMAH in said knockout pig is decreased as compared to a wild-type pig. A cell culture reagent may be selected from the group of cell culture reagents comprising cell culture media, cell culture serum, a cell culture additive and an isolated cell capable of proliferation.
[0026] In a further embodiment, the invention provides a method of producing a glycoprotein of interest comprising the step of incubating an isolated cell capable of expressing the glycoprotein of interest with a cell culture reagent derived from a knockout pig comprising disrupted a(l,3)- galactosyltransferase and CMAH genes, wherein the amount of Neu5Gc or aGal epitopes on the glycoprotein of interest is lower than the amount of Neu5Gc or aGal epitopes on the glycoprotein of interest when an isolated cell capable of expressing said glycoprotein of interest is incubated with a cell culture reagent derived from a wild-type pig. The glycoprotein of interest may be selected from the group comprising an antibody, growth factor, cytokine, hormone and clotting factor. In an aspect of the disclosure, the disruption of the a(l,3)-galactosyltransferase gene is selected from the group of disruptions comprising a 3 base pair deletion adjacent to a G to A substitution, a single base pair deletion, a single base pair insertion, a six base pair deletion, a two base pair insertion, a ten base pair deletion, a seven base pair deletion, and an eight base pair substitution for a five base pair sequence; the disruption of said CMAH gene is selected from the group of disruptions comprising a four base pair insertion, a two base pair deletion, a single base pair insertion, an eight base pair deletion, a five base pair deletion, a three base pair deletion, a two base pair substitution for a single base pair, and a twenty base pair deletion; and expression of the functional a(l,3) galactosyltransferase and CMAH in the knockout pig from which the cell culture reagent is derived is decreased as compared to a wild-type pig.
[0027] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will be apparent, the invention is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. Accordingly, the detailed descriptions are to be regarded as illustrative in nature and not restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The disclosure will be better understood and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings.
[0029] Figure 1 depicts a schematic of a protocol used to develop the double knock-out CMAH/GGTA1- pigs. Step a) shows the delivery of CMAH ZFNs to liver derived cells. Step b) illustrates identified CMAH mutant cells by screening individual clones. Step c) illustrates the delivery of GGTA1 ZFNs to CMAH KO cells. Step d) illustrates the counterselection of Gal negative cells. Step e) illustrates CMAH and GGTA1
double KO cells. Step f) illustrates somatic cell nuclear transfer (SCNT) resulting in CMAH and GGTAl double KO cells.
[0030] Figure 2 provides genotype and phenotype analysis of double-KO fetuses. Panel A presents a photographic image of harvested double-KO fetuses. Panel B provides representative electropherograms of the mutations found in the CMAH gene in the double knockout fetuses. Mutations are underlined while the DNA binding sites of the ZFN are italicized. Panel C provides representative electropherograms of the mutations found in the GGTAl gene in the double knockout fetuses. Mutations are underlined while the DNA binding sites of the ZFN are italicized. Panel D presents data obtained from flow cytometric analysis of red blood cells (RBC) obtained from six month old wild-type piglets (WT), six-month-old GGTA1-KO (GGTA1-KO) piglets, double-KO fetuses and adult humans showing cells stained with an antibody recognizing Neu5GC. Panel E presents data obtained from flow cytometric analysis of red blood cells (RBC) obtained from six month old wild-type piglets (WT), six-month-old GGTA1-KO (GGTA1-KO) piglets, double-KO fetuses and adult humans showing cells stained with fluorescently labeled IB4 lectin to measure the level of the Gal epitopes. Unstained RBC were used as negative controls for IB4 lectin staining and an isotype matched control was used for Neu5Gc staining. Some negative control histograms are difficult to see because of significant overlap with the experimental group.
[0031] Figure 3A presents a photograph of viable double-KO piglets. Panel B provides sequence information regarding the wild-type (WT) sequences for the CMAH and GGTAl target regions. The alterations that occur in the double-KO piglets in either the CMAH or GGTAl target region are underlined, while the binding sites are italicized.
[0032] Figure 4 presents results from a series of experiments analyzing carbohydrate expression in genetically modified pigs. Panels A and B provide confocal micrographs of tissues from wild type (WT), single (GGTA1-KO) and CMAH-/-/GGTA1-/- (dou ble-KO) pigs. DAPI staining of nuclei may be visible. Heart, kidney and liver tissues were stained with anti-Neu5Gc antibody in the micrographs of panel A. Limited staining of Neu5Gc occurs in tissues from double-KO pigs. Heart, kidney and liver tissues were stained with IB4 lectin in the micrographs of panel B. Limited IB4 binding occurs in tissues from GGTA1- KO and double-KO pigs. Panel C presents results obtained from flow cytometry analysis of peripheral blood mononuclear cells (PBMC). Traces were obtained from cells labeled with anti-Neu5Gc antibody (left column) and IB4 lectin (right column). Unstained PBMC were the negative controls for IB4 lectin; an
isotype negative control was used in the anti-Neu5Gc staining. Negative controls are shown (*) but are difficult to see in some panels because of significant overlap with the experimental group.
[0033] Figure 5 presents results from a series of experiments examining human antibody recognition of PBMC from GGTA1-KO and double-KO pigs. Panels A and B show IgG (A) and IgM (B) histograms of a representative sample (Subject 5) of randomly chosen normal human serum against GGTA1-KO or double-KO cells. Bar graphs C and D show the mean fluorescent intensities (MFI) of IgM) or IgG binding to peripheral blood monocytes (PBMCs). The results of testing ten unique human subjects are shown. Panels E and F illustrate example curves (Subject 3) for antibody-mediated complement-dependent cytotoxicity of normal human serum against GGTA1-KO and double KOPBMCs. Percent cytotoxicity for each serum tested (2.0% final concentration) is shown.
[0034] Figure 6 shows flow cytometry used to analyze human antibody binding to GT-KO and GT/CMAH-KO porcine fetal fibroblasts in a dose dependent manner. GT/CMAH-KO cells bind less human IgM as compared to GT-KO porcine fetal fibroblasts.
[0035] Figure 7 provides panels pertaining to the generation of GGTA1/CMAH double knockout porcine LDCs through a C ISP /Cas9 technique. LDCs that survived IB4 lectin counter-selection were expanded and stained with IB4-FITC. Figure 7A provides results of flow cytometry analysis of the a-Gal epitope on LDCs that survived IB4 lectin counter-selection. Also shown are unlabeled LDC and labeled, non- transfected LDC. In the particular example shown, 91.7% of the selected cells were free of the a-Gal epitope. Figure 7B provides an electropherogram of mutations found in Crispr-targeted GGTA1 and CMAH regions. The wild-type sequence of a portion of each targeted region is shown above the sequencing results. The top electropherogram is a portion of the GGTA1 gene; the bottom electropherogram is a portion of the CMAH gene.
[0036] Figure 8 depicts flow cytometry results of labeled liver derived cells (LDCs) simultaneously transfected with CrispR CMAH and a-Gal targeting plasmids (CRISPR LDC); labeled, non-transfected LDC; and unlabeled LDC. CMAH expression in the cells was evaluated by assessing the anti-Neu5Gc antibody binding to Neu5Gc epitopes on the cell surface. CMAH produces Neu5Gc. As shown in Panel B (Unselected) a small percentage of the bulk transfected cell population shows a CMAH deficiency. As shown in Panel A, after IB4 lectin counter-selection of the bulk cell population, a significantly higher percentage of the IB4 nonbinding cells (IB4 lectin selected) were CMAH deficient cells. In one experiment, 41.6% of the IB4 counter-selected cells were CMAH deficient while 6.7% of unselected bulk transfected cells were CMAH deficient.
[0037] Figure 9 provides panels pertaining to generation of CMAH/aGal DKO fetuses from CMAH- CrispR and aGal-CrispR transfected LDC. Figure 9A provides a photograph of 10 fetuses harvested at day 32 of gestation after SCNT of Crispr-transfected LDC that survived IB4 counterselection and implantation of resulting embryos in recipient sows. DNA sequencing analysis of the GGTA1 and CMAH target regions was performed on each fetus. For GGTA1, seven fetuses contained biallelic mutations, one fetus contained a monoallelic mutation, and two fetuses were wild-type. For CMAH, five fetuses contained biallelic mutations, one fetus contained a monoallelic mutation, and four fetuses were wild-type. Figure 9B provides the results of DNA sequencing analysis of the targeted GGTA1 and CMAH regions of five DKO fetuses with mutations in both alleles of both CMAH and GGTA1. If the two alleles have different mutations in a particular fetus, the sequence of both alleles is shown. If the two alleles of a fetus have the same mutation, the sequence is only shown once. The net change in nucleotide number is indicated to the right of the sequences. The wild-type sequence of the relevant portions of the GGTA1 and CMAH genes is shown above the fetal sequences. Fetal fibroblasts were derived from fetus 7. Panel C provides data from flow cytometry analysis of aGal and Neu5Gc on double knockout (DKO) and wild-type (WT) fetal fibroblasts. Unlabeled DKO and WT cells were also analyzed. The upper trace shows aGal results; the lower trace shows Neu5Gc results.
[0038] Figure 10 provides panels of flow cytometry analysis of Neu5Gc on CMAH/GAL double knockout fibroblast cells grown in media supplemented with either fetal bovine serum (FBS) or CMAH/aGAL double knockout derived serum (CMAH/Gal KO). Figure 10A illustrates data from an isotype control. Figure 10B provides Neu5Gc data. Cells grown in the CMAH deficient serum show lower anti-Neu5Gc antibody binding than cells grown in FBS.
[0039] Figure 11 provides panels of flow cytometry analysis of Neu5Gc on double knockout (CMAH/Gal KO) fibroblasts and single aGal knockout (GGTA KO) renal cells grown with a variety of cell culture reagents. The upper panel provides flow cytometry results obtained from isotype control analysis of a CMAH/aGal DKO cell line (CMAH/Gal KO Fibroblast cell line) and aGall single knockout kidney cells (GGTA KO renal cells). The lower panel provides flow cytometry results of Neu5Gc on a CMAH/aGal DKO cell line (CMAH/Gal KO Fibroblast cell line) grown in culture media supplemented with CMAH/aGal derived serum and a GGTA-1 kidney cell line grown in culture media supplemented with FBS.
[0040] Figure 12 provides a graph of data obtained from perfusion experiments involving porcine livers perfused with human platelets. The post-perfusion initiation time in minutes is indicated on the x-axis (Time); the percent of human platelets remaining in the perfusion solution is indicated on the y-axis
(Percent Platelets Remaining in Perfusion). Data obtained from experiments with single aGal knockout pig livers (GGTA17-, n=2) and CMAH/aGal DKO livers (CMAH7~/GGTA17 ~ , n=3) are provided.
[0041] Figure 13 provides individual curves for antibody-mediated, complement-dependent cytotoxicity of 10 randomly selected baboon serum samples against single (GGTA1-KO) and GGTA1/CMAH double (double-KO) peripheral blood monocytes (PBMCs). Results from a human serum sample against single (GGTA1-KO) and double-KO PBMC are also shown on each graph. Unexpectedly cytotoxicity of the baboon serum to the CMAH/aGAL double knockout PBMC differed widely from cytotoxicity of human serum to CMAH/aGal PBMCs. Cytotoxicity of baboon serum with CMAH/aGal double knockout PBMCs consistently increased as compared to cytotoxicity of baboon serum with aGal single knockout PBMCs. The dashed horizontal line indicates 50% killing. The %CTX for each sample was plotted against the log of each serum dilution and the sigmoid curve was analyzed by non-linear regression.
[0042] Figure 14 provides histograms showing results of flow cytometry analysis of IgM (Figure 14) and IgG (Figure 14B) antibody recognition. Histograms show the results of 5 randomly selected human sera (1, 2, 3, 4 and 5) incu bated with GGTA1-KO pig, double-KO pig, baboon, chimpanzee and human PBMCs. Secondary only control antibody staining is shown in the bottom graph in each column. In four of the five samples shown, IgG antibody recognition for CMAH/GAL DKO PBMC and human PBMC yield almost overlaying traces. In the same four samples shown, the IgM traces for the CMAH/GAL DKO PBMC are shifted closer to the traces for the human PBMC than the baboon, chimpanzee and single knockout (GGTA1-KO) traces.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The present invention provides pigs and porcine organs, tissues or cells for transplantation into a human that do not express aGal and CMAH and methods of making the same. In one embodiment, the invention provides a knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and when tissue from said pig is transplanted into a human, hyperacute rejection is decreased as compared to when tissue from a wild-type pig is transplanted into a human.
I. In General.
[0044] In the specification and in the claims, the terms "including" and "comprising" are open-ended terms and should be interpreted to mean "including, but not limited to. . . . " These terms encompass the more restrictive terms "consisting essentially of" and "consisting of."
[0045] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", "characterized by" and "having" can be used interchangeably.
[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes including describing and disclosing the chemicals, instruments, statistical analyses and methodologies which are reported in the publications which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
II. The Invention.
[0047] The present invention provides transgenic animals suitable for use in xenotransplantation and methods of producing mammals suitable for use in xenotransplantation. Specifically, the present invention describes the production of homozygous double knockout pigs lacking any functional expression of alpha 1,3 galactosyltransferase (aGAL) and CMAH. In one embodiment, the production of pigs with copies of the aGal and CMAH genes knocked out prior to somatic cell nuclear transfer (SCNT) is described. The time to create a two gene homozygous knockout took less than 10 months, significantly reducing the time required to create new pigs to speed up the progress of xenotransplantation research.
[0048] The term "knockout mammal" refers to a transgenic mammal wherein a given gene has been altered, removed or disrupted. By a "double knockout" we mean a transgenic mammal wherein two genes have been altered, removed or disrupted. It is to be emphasized that the term is intended to include all progeny generations. Thus, the founder animal and all Fl, F2, F3, and so on, progeny thereof are included. In principle, knockout animals may have one or both copies of the gene sequence of interest disrupted. In the latter case, in which a homozygous disruption is present, the mutation is termed a "null" mutation. In the case where only one copy of the nucleic acid sequence of interest is
disrupted, the knockout animal is termed a "heterozygous knockout animal". The knockout animals of the invention are typically homozygous for disruptions of both genes being targeted.
[0049] The term "chimera," "mosaic" or "chimeric mammal" refers to a transgenic mammal with a knockout in some of its genome-containing cells.
[0050] The term "heterozygote" or "heterozygotic mammal" refers to a transgenic mammal with a disruption on one of a chromosome pair in all of its genome containing cells.
[0051] The term "homozygote" or "homozygotic mammal" refers to a transgenic mammal with a disruption on both members of a chromosome pair in all of its genome-containing cells.
[0052] A "non-human mammal" of the invention includes mammals such as rodents, sheep, dogs, ovine such as lamb, bovine such as beef cattle and milk cows, and swine such as pigs and hogs. Although the invention uses a typical non-human animal (e.g., porcine), other mammals can similarly be genetically modified using the methods and compositions of the invention.
[0053] A "mutation" is a detecta ble change in the genetic material in the animal, which is transmitted to the animal's progeny. A mutation is usually a change in one or more deoxyribonucleotides, such as, for example, adding, deleting, inverting, or substituting nucleotides.
[0054] By "pig" we mean any pig known to the art, including a wild pig, a domestic pig, mini pigs, a Sus scrofa pig, a Sus scrofa domesticus pig, as well as inbred pigs. Without limitation, the pig can be selected from the group consisting of, for example, Landrace, Yorkshire, Hampshire, Duroc, Chinese Meishan, Chester White, Berkshire Goettingen, Landrace/York/Chester White, Yucatan, Bama Xiang Zhu, Wuzhishan, Xi Shuang Banna, and Pietrain pigs. Porcine organs, tissue or cells are organs, tissue or cells from a pig.
[0055] Transgenic Animals. The present invention provides a transgenic animal lacking any expression of functional aGal and CMAH genes. The animal can be any mammal suitable for xenotransplantation. In a specific embodiment, the animal is a pig. "CMAH/aGAL double knockouts", "CMAH/aGAL DKO", "CMAH/aGal", "CMAH/aGal DKO", "CMAH7~/GAL.7~", "aGal/CMAH DKOs", "aGAL/CMAH double knockouts", "GGTA1/CMAH DKO", "GT1/CMAH DKO", "GGTA17~/CMAH7~", "GGT17~/CMAH7", "CMAH/GGTA DKO", "GT/CMAH-KO", "GGTA1/CMAH KO", "DKO (aGal/CMAH)", "DKO (aGAL & CMAH)", "CMAH-/aGal-", "aGal-/CMAH-", "CMAH-/aGAL-" and variants thereof refer to animals, cells, or tissues that lack expression of functional alpha 1,3 galactosyltransferase and cytidine monophosphate-N- acetylneuraminic acid hydroxylase.
[0056] Transgenic Material. In another embodiment, the invention provides organs, tissue and/or cells from animals lacking any expression of functional aGal and CMAH for use as xenografts. The tissues from animals lacking any functional expression of the aGal and CMAH gene can be obtained from a prenatal, neonatal, immature, or fully mature animal, such as a porcine, bovine or ovine. The organ may be used as a temporary or permanent organ replacement for a patient in need of an organ transplant. Any porcine organ can be used, including but not limited to the brain, heart, lungs, eye, stomach, pancreas, kidneys, liver, intestines, uterus, bladder, skin, hair, nails, ears, glands, nose, mouth, lips, spleen, gums, teeth, tongue, salivary glands, tonsils, pharynx, esophagus, large intestine, small intestine, rectum, anus, thyroid gland, thymus gland, bones, cartilage, tendons, ligaments, suprarenal capsule, skeletal muscles, smooth muscles, blood vessels, blood, spinal cord, trachea, ureters, urethra, hypothalamus, pituitary, pylorus, adrenal glands, ovaries, oviducts, vagina, mammary glands, testes, seminal vesicles, penis, lymph, lymph nodes and lymph vessels.
[0057] In another embodiment, the invention provides non-human tissues that are useful for xenotransplantation. In one embodiment, the non-human tissue is porcine tissue. Any porcine tissue can be used, including but not limited to epithelium, connective tissue, blood, bone, cartilage, muscle, nerve, adenoid, adipose, areolar, bone, brown adipose, cancellous, muscle, cartilaginous, cavernous, chondroid, chromaffin, dartoic, elastic, epithelial, fatty, fibrohyaline, fibrous, Gamgee, gelatinous, granulation, gut-associated lymphoid, skeletal muscle, Haller's vascular, indifferent, interstitial, investing, islet, lymphatic, lymphoid, mesenchymal, mesonephric, multilocular adipose, mucous connective, myeloid, nasion soft, nephrogenic, nodal, osteoid, osseous, osteogenic, retiform, periapical, reticular, rubber, smooth muscle, hard hemopoietic, and subcutaneous tissue.
[0058] The invention also provides cells and cell lines from porcine animals that lack expression of functional aGal and CMAH. In one embodiment, these cells or cell lines can be used for xenotransplantation. Cells from any porcine tissue or organ can be used, including, but not limited to: epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, Islets of Langerhans cells, pancreatic insulin secreting cells, pancreatic alpha-2 cells, pancreatic beta cells, pancreatic alpha-1 cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, liver stellate cells, aortic smooth muscle cells, cardiac
myocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells, parotid cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, heart cells, pacemaker cells, spleen cells, antigen presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testicular cells, germ cells, egg cells, leydig cells, peritubular cells, Sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, dopamiergic cells, squamous epithelial cells, osteocytes, osteoblasts, osteoclasts, dopaminergic cells, embryonic stem cells, fibroblasts and fetal fibroblasts.
[0059] Nonviable derivatives include tissues stripped of viable cells by enzymatic or chemical treatment these tissue derivatives can be further processed through crosslinking or other chemical treatments prior to use in transplantation. In a preferred embodiment, the derivatives include extracellular matrix derived from a variety of tissues, including skin, bone, urinary, bladder or organ submucosal tissues. In addition, tendons, joints, and bones stripped of viable tissue to including but not limited to heart valves and other nonviable tissues as medical devices are provided. In an embodiment, serum or medium suitable for cell culture and isolated from a knockout pig of the invention are provided. Components of porcine knockout organs, tissues or cells are also provided. Components may also be modified through any means known in the art including but not limited to crosslinking and aldehyde crosslinking. Components may vary depending on the larger organ or tissue from which the component is obtained. Skin components may include but are not limited to stripped skin, collagen, epithelial cells, fibroblasts and dermis. Bone components may include but are not limited to collagen and extracellular matrix. Heart components may include but are not limited to valves and valve tissue.
[0060] Expression of a gene product is decreased when total expression of the gene product is decreased, a gene product of an altered size is produced or when the gene product exhibits an altered functionality. Thus if a gene expresses a wild-type amount of product but the product has an altered enzymatic activity, altered size, altered cellular localization pattern, altered receptor-ligand binding or other altered activity, expression of that gene product is considered decreased. Expression may be analyzed by any means known in the art including, but not limited to, T-PC , Western blots, Northern blots, microarray analysis, immunoprecipitation, radiological assays, polypeptide purification, spectrophotometric analysis, Coomassie staining of acrylamide gels, ELISAs, 2-D gel electrophoresis, in
situ hybridization, chemiluminescence, silver staining, enzymatic assays, ponceau S staining, multiplex T-PC , immunohistochemical assays, radioimmunoassay, colorimetric assays, immunoradiometric assays, positron emission tomography, fluorometric assays, fluorescence activated cell sorter staining of permeablized cells, radioimunnosorbent assays, real-time PCR, hybridization assays, sandwich immunoassays, flow cytometry, SAGE, differential amplification or electronic analysis. Expression may be analyzed directly or indirectly. Indirect expression analysis may include but is not limited to, analyzing levels of a product catalyzed by an enzyme to evaluate expression of the enzyme. See for example, Ausubel et al, eds (2013) Current Protocols in Molecular Biology, Wiley-lnterscience, New York, N.Y. and Coligan et al (2013) Current Protocols in Protein Science, Wiley-lnterscience New York, NY. Gene expression assays for porcine ASGR1 are commercially available (Applied Biosystems™, Carlsbad CA).
[0061] "As compared to" is intended encompass comparing something to a similar but different thing, such as comparing a data point obtained from an experiment with a knockout pig to a data point obtained from a similar experiment with a wildtype pig. The word "comparing" is intended to encompass examining character, qualities, values, quantities, or ratios in order to discover resemblances or differences between that which is being compared. Comparing may reveal a significant difference in that which is being compared. By "significant difference" is intended a statistically significant difference in results obtained for multiple groups such as the results for material from a knockout pig and material from a wild-type pig. Generally statistical significance is assessed by a statistical significance test such as but not limited to the student's t-test, Chi-square, one-tailed t-test, two-tailed t-test, ANOVA, Dunett's post hoc test, Fisher's test and z-test. A significant difference between two results may be results with a p<0.1, p<0.05, p<0.04, p<0.03, p<0.02, p<0.01 or greater.
[0062] The word "isolated" is intended to encompass an entity that is physically separated from another entity or group. An isolated cell is physically separated from another group of cells. Examples of a group of cells include, but are not limited to, a developing cell mass, a cell culture, a cell line, a tissue, and an animal. The word "isolating" is intended to encompass physically separating an entity from another entity or group. Examples include physically separating a cell from other cells, physically separating a cell component from the remainder of the cell and physically separating tissue or organ from an animal. An isolated cell or cell component is separated by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, up to 100% of other naturally occurring cells or cell components. Methods for isolating one or more cells from another group of cells are known in the art. See for example Freshney (ED) Culture of Animal Cells: a manual of
basic techniques (3 Ed.) 1994, Wiley-Liss; Spector et al (Eds)(1998) Cells: a Laboratory Manual (vol.1) Cold Spring Harbor Laboratory Press and Darling et al (1994) Animal Cells: culture and media John Wiley & Sons. Methods of isolating a tissue or an organ from an animal are known in the art and vary depending on the tissue or organ to be isolated and the desired method of transplanting the tissue or organ.
[0063] A "skin related product" encompasses products isolated from skin and products intended for use with skin. Skin related products isolated from skin or other tissues may be modified before use with skin. Skin related products include but are not limited to replacement dressings, burn coverings, dermal products, replacement dermis, dermal fibroblasts,collagen, chondroitin, connective tissue, keratinocytes, cell-free xenodermis,cell-free pig dermis,composite skin su bstitutes and epidermis and temporary wound coverings. See for example Matou-Kovd et al (1994) Ann Med Burn Club 7:143, herein incorporated by reference in its entirety.
[0064] "Xenotransplantation" encompasses any procedure that involves the transplantation, implantation or infusion of cells, tissues or organs into a recipient subject from a different species. Xenotransplantation in which the recipient is a human is particularly envisioned. Thus xenotransplantation includes but is not limited to vascularized xenotransplant, partially vascularized xenotransplant, unvascularized xenotransplant, xenodressings, xenobandages, and xenostructures.
[0065] In another embodiment, the invention provides a method of improving a hyperacute rejection related symptom in a patient comprising transplanting porcine organs, tissue or cells having reduced expression of aGal and Neu5Gc on the porcine organs, tissue or cells into a human, wherein the hyperacute rejection related symptoms are improved as compared to when tissue from a wild-type swine is transplanted into a human. By "improving", "bettering", "ameliorating", "enhancing", and "helping" is intended advancing or making progress in what is desirable. It is also envisioned that improving a hyperacute rejection related symptom may encompass a decrease, lessening, or diminishing of an undesirable symptom. By "hyperacute rejection" we mean rejection of the transplanted material or tissue occurring or beginning within the first 24 hours post-transplant involving one or more mechanisms of rejection. Hyperacute rejection encompasses but is not limited to "acute humoral rejection" and "antibody mediated rejection".
[0066] "Hyperacute rejection related symptom" is intended to encompass any symptom known to the field as related to or caused by hyperacute rejection. It is recognized that hyperacute rejection related symptoms may vary depending upon the type of organ, tissue or cell that was transplanted. Hyperacute
rejection related symptoms may include, but are not limited to, thrombotic occlusion, hemorrhage of the graft vasculature, neutrophil influx, ischemia, mottling, cyanosis, edema, organ failure, reduced organ function, necrosis, glomerular capillary thrombosis, lack of function, hemolysis, fever, clotting, decreased bile production, asthenia, hypotension, oliguria, coagulopathy, elevated serum aminotransferase levels, elevated alkaline phosphatase levels, jaundice, lethargy, acidosis and hyperbilirubenemia and thrombocytopenia.
[0067] Thrombocytopenia is a quantity of platelets below the normal range of 140,000 to 440,000/μΙ. Thrombocytopenia related symptoms include, but are not limited to, internal hemorrhage, intracranial bleeding, hematuria, hematemesis, bleeding gums, abdominal distension, melena, prolonged menstruation, epistaxis, ecchymosis, petechiae or purpura. Uptake of human platelets by pig livers contributes to the development of thrombocytopenia in xenograft recipients.
[0068] Platelets, also known as thrombocytes, are enucleate fragments of megakaryocytes involved in blood coagulation, hemostasis and blood thrombus formation. Human platelets are routinely isolated through a variety of methods including, but not limited to, platelet apheresis, plateletpheresis and ultracentrifugation.
[0069] The phrase "platelet uptake" is intended to encompass the incorporation of a platelet into a liver or liver cell. While not being limited by mechanism, such uptake may occur through a phagocytic process. Platelet uptake may be monitored by any platelet uptake monitoring assay known in the art. Platelet uptake monitoring assays include, but are not limited to immunological methods, western blots, immunoblotting, microscopy, confocal microscopy, transmission electron microscopy and phagosome isolation. It is recognized that the appropriate platelet uptake monitoring assay may depend upon the type of label used. Platelet uptake may be measured as a percentage of total platelets absorbed, percentage of total platelets not absorbed, a ratio of absorbed to unabsorbed platelets, percentage of cells absorbing at least one platelet, percentage of cells not absorbing a platelet, or number of platelets absorbed per cell. It is recognized that platelet uptake by more than one cell type may contribute to the total platelet uptake of the liver. Total platelet uptake by an animal liver may include platelet uptake by liver sinusoidal endothelial cells, platelet uptake by Kuppffer cells, platelet uptake by LSECs and Kupffer cells and platelet uptake by additional cell types. It is recognized that platelet uptake by different cell types may contribute similar or disparate fractions of the total platelet uptake by a liver. Thus an alteration, inhibition, reduction, decrease, or lowering of platelet uptake by a liver comprises an alteration, inhibition, reduction, decrease, or lowering of platelet uptake by one or more liver cell types.
[0070] The word "providing" is intended to encompass preparing, procuring, getting ready, making ready, supplying or furnishing. It is recognized that methods of providing a cell may differ from methods of providing a subject and that methods of providing a liver may differ from methods of providing a pig.
[0071] In an embodiment, a cell culture reagent derived from a transgenic pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes is provided. Cell culture reagents are reagents utilized for tissue culture, in vitro tissue culture, microfluidic tissue culture, cell culture or other means of growing isolated cells or cell lines. Cell culture reagents may include but are not limited to cell culture media, cell culture serum, a cell culture additive, a feeder cell, and an isolated cell capable of proliferation. By an "isolated cell capable of proliferation" is intended a cell isolated or partially isolated from other cell types or other cells wherein the cell is capable of proliferating, dividing, or multiplying into at least one additional clonal cell.
[0072] Cells grown in culture may synthesize or meta bolically incorporate antigenic epitopes into glycoproteins secreted by the cultured cell. The antigenic epitopes may result in increased binding by human antibodies and decreased efficacy of the glycoprotein. See Ghaderi et al, 2010 Nature Biotechnology 28(8):863-867, herein incorporated by reference in its entirety. Growing the producing cell in a cell culture reagent with less aGal or Neu5Gc may reduce the amount of aGal, Neu5Gc, or both aGal and Neu5Gc epitopes on the glycoprotein of interest. Glycoproteins of interest may include any glycoprotein, particularly glycoproteins intended for use in human su bjects such as but not limited to, an antibody, growth factor, cytokine, hormone, or clotting factor.
[0073] In summary, xenoantigens aGal and Neu5Gc were eliminated in transgenic pigs by genetic modification. Double knockout pigs (GT/CMAH-KO) were produced within 5-10 months or less.
[0074] In embodiments of the present invention, tissues are provided in which both the aGal and CMAH genes are rendered inactive, such that the resultant aGal and CMAH products can no longer generate alpha 1,3-galactosyl epitopes or Neu5Gc on the cell surface. In an alternative embodiment, the aGal and CMAH genes can be inactivated in such a way that no transcription of the gene occurs.
[0075] In yet another aspect, the present invention provides a method for producing viable pigs lacking any functional expression of aGal and CMAH. In one embodiment, the pigs are produced as described below. Methods of making transgenic pigs, and the challenges thereto, are discussed in Galli et al. 2010 Xenotransplantation, 17(6) p. 397-410, incorporated by reference herein for all purposes. The methods and cell cultures of the invention are further detailed below.
EXAMPLES
[0076] The following Examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims.
Example 1. Design of Zinc Finger Nucleases (ZFN).
[0077] A pair of ZFN were designed to bind and cleave the sequence of porcine CMAH exon 9 (SEQ ID NO: 1-AAACTCCTGAACTACAAGGCTCGGCTGGTGAAGGA) beginning at position 1,341 of Ensemble transcript ENSSSCT00000001195. Another pair of ZFN were designed to bind and cleave the region of GGTA1 exon 8 (SEQ ID NO: 2-GTCATCTTTTACATCATGGTGGATGATATCTCCAGGATGCC) beginning at position 1165 of Ensemble transcript ENSSSCT00000006069. The ZFN activities were validated in yeast (Sigma-Aldrich, St. Louis, MO). Additional detail regarding the ZFN of the present invention can be found in Li et al., Journal of Surgical Research, (2012)E1-E7 Epub 2012 July 3, which is incorporated by reference herein for all purposes.
Example 2. Cell Culture and Transfection of Porcine Liver Derived Cells.
[0078] Porcine adult liver derived cells (LDC) were isolated with modifications from the method described in Li et al, 2010 Cell Reprogram. 12:599. Isolated LDC were cultured in a combination stem cell media (SCM) (a-MEM :EGM-MV 3:1) (Invitrogen/Lonza, Switzerland) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 10% horse serum (Invitrogen, Carlsbad, CA), 12mM HEPES, and 1% Pen/Strep (Invitrogen) as described in Li et al. 2012 JSR Epub 2012 July 3. A commercial porcine strain (Landrace/York/Chester White) was used as the source of the LDC. Neon transfection system was used according to the manufacturer's instruction (Invitrogen).
[0079] Briefly, LDC were harvested by trypsin digestion, washed with calcium and magnesium free Dulbecco's phosphate buffered saline (DPBS) (Invitrogen) and centrifuged. 10s cells were suspended in 120 μΙ of resuspension buffer (Invitrogen) containing 2 μgs of each CMAH ZFN plasmid. pEGFP-N 1 (Clonetech, Mountain View, CA) was used as a control to measure transfection efficiency as well as ZFN activity. Cells were electroporated at 1300 V, 30 msec for 1 pulse.
[0080] Then cells were transferred in SCM without antibiotics and plated onto collagen I coated plates. Cells were cultured with 5% C02 and 10% 02 at 30°C for 3 days and 37°C for 2 days.
Example 3. Surveyor Mutation Detection Assay (CEL I assay).
[0081] ZFN-induced mutation was detected using the Surveyor Mutation Detection kit (Transgenomic, Omaha, NE). At day 5 post-transfection, genomic DNA from ZFN-treated and control plasmid pEGFP-Nl treated cells was extracted and PCR was performed using primers ZFN-CMAH-F (SEQ ID NO: 3-5' GGACCTGCTTTATCTTGCTCGC 3'), ZFN-CMAH-R (SEQ ID NO: 4-5' CC ATACTTGTCTG CTGGGTGGG 3'). Pwo SuperYield DNA Polymerase, dNTPack (Roche, Indianapolis, IN) was used and the PCR conditions were as follows: 94°C, 2 minutes; 94°C, 15 seconds, 55°C, 30 seconds and 68°C, 50 seconds for 15 cycles; 94°C, 15 seconds, 5 5°C, 30 seconds and 68°C, 50 seconds with additional 5 seconds for each cycle, for 25 cycles and a final extension step of 68°C for 5 minutes.
[0082] PCR product was denatured and annealed using the following program on a MyCycler (Bio-Rad): 95°C, 10 minutes; 95°C to 85°C, -2°C /second; 85°C to 25°C, -0.1°C /second. 200-400 ng of PCR product was digested with 1 μΙ of Nuclease S and 1 μΙ of enhancer (Transgenomic, Omaha, NE) at 42°C for 40 minutes. The product was separated on a 10% polyacrylamide gel and stained with SYBR Safe to assess ZFN-induced mutations.
Example 4. Screening CMAH Mutant Cells.
[0083] ZFN-treated cells were plated at 1 cell/well in ten 96-well plates coated with collagen I (BD, Franklin Lakes New Jersey). After 14 days, single cell clones became evident. Cells were expanded to 48- well plate. Some cells were harvested for mutation screening. PCR was performed as described above herein. PCR products were resolved on a 0.8% agarose gel and purified by QIAquick Gel Extraction Kit (Qiagen). Primer CMAH-S1 (SEQ ID NO: 5-5' CCAAACCCTGTCATTCCAG 3') was used to sequence the ZFN targeted CMAH region. A clone with an identical mutation in both CMAH gene copies was identified.
[0084] Disruption of GGTA 1 gene on CMAH mutant cells and counter selection of a-Gal- cells 10s of CMAH mutant cells were transfected with 2 μgs of each GGTAl ZFN plasmid as described below herein. aGal- cells were isolated by the counter selection method described below herein.
Example 5. Counter Selection of aGal- Cells.
[0085] CMAH deficient LDC were treated with zinc finger plasmids targeting porcine 1,3 aGal. Alternatively LDC were transfected with Crispr CMAH sgRNA expression plasmids and GGTAl sgRNA expression plasmids and grown for 48 hours as described elsewhere herein. The cells were harvested, washed and counted. A total of 2.3 x 10s cells were incubated with 5 μg of biotin-conjugated Griffonia
simplicifolia isolectin IB4 (IB4 lectin from Enzo Life Science, Farmingdale NY) in 600 μΙ of PBS supplemented with 0.1% BSA and 2 mM ethylenediaminetetracetic acid (pH 7.4) on ice for 30 minutes. After 30 minutes, the cells were washed once and incubated with 50 μΙ Dynabeads Biotin Binder (Invitrogen) at 4°C with rotation for 30 minutes. A magnet was applied to deplete Dynabeads-bound a- Gal+ cells. The IB4 lectin is specific for the Gal epitope; cells that did not bind the Dynabeads remained in the supernatant. CMAH cells that did not bind IB4 and the Dynabeads were identified as Gal-/- cells. Crispr CMAH and GGTAl treated cells that did not bind IB4 and the Dynabeads were utilized in SCNT as described below herein.
Example 6. TALEN Constructs.
[0086] TALEN constructs are designed to bind and cleave the sequence of porcine CMAH at a suitable site. Additional Talen constructs are designed to bind and cleave GGTAl at a suitable site.
Example 7. Production of Double Knockout (otGal and CMAH) Pigs.
[0087] Somatic Cell Nuclear Transfer (SCNT) was performed using in vitro matured oocytes (DeSoto Biosciences Inc., St. Seymour TN and Minitube of America, Mount Horeb, Wl). Cumulus cells were removed from the oocytes by pipetting in 0.1% hyaluronidase. Oocytes with normal morphology and a visible polar body were selected and incubated in manipulation media (calcium-free NCSU-23 with 5% fetal bovine serum (FBS) containing 5 μg/ml bisbenzimide and 7.5 μg/mL cytochalasin B for 15 minutes. Following this incubation period, oocytes were enucleated by removing the first polar body and metaphase II plate. Single cells of CMAH deficient LDC that survived IB4 counterselection were injected into each enucleated oocyte. Alternatively Crispr transfected cells that survived IB4 counterselection were injected into each enucleated oocyte. Electrical fusion was induced with a BTX cell electroporator (Harvard Apparatus, Holliston, MA). Enucleated oocytes injected with a cell (couples) were exposed to two DC pulses of 140 V for 50 μ≤ in 280 mM mannitol, 0.001 mM CaCI2 and 0.05 mM MgCI2. After one hour, reconstructed oocytes were activated by two DC pulses of 120 V for 60 μ≤ in 280 mM mannitol, 0. 1 mM CaCI2 and 0.05 mM MgCI2. After activation, oocytes were placed in NCSU-23 medium with 0.4% bovine serum albumin (BSA) and incubated at 38.5°C, 5% C02 in a humidified atmosphere for less than one hour. Within an hour after activation, oocytes were transferred into a recipient pig. Recipient pigs were synchronized occidental pigs on their first day of estrus. Pregnancies were verified by ultrasound at day 25 or day 26 after embryo transfer. Results of SCNT using double knockout cells are summarized in Table 1. Results of SCNT using Crispr-transfected IB4 counterselected cells are summarized in Table 2.
Table 1: Results of somatic cell nuclear transfer using double-KO cells.
[0088] Dou ble-KO cells (cl22) were used as donor cells in SCNT and transferred into 2 recipients, 01 and 02. Fetal fibroblasts from fetus num ber 1 of recipient 02 were re-cloned and transferred to animals 03, 04, 05, and 06. 477 embryos were delivered to four gilts, resulting in one pregnancy and the birth of 5 piglets. The four via ble piglets appeared healthy as they were nursing since birth and needed no feeding support.
[0089] In at least one experiment, SCNT of the selected cells deficient for CMAH and GGTAl resulted in a pregnancy that was terminated at day 30 of gestation. Five normal fetuses and one resorbed fetus were harvested, photogra phed and shown in Figure 1A. Red blood cells from the dou ble knockout fetuses were analyzed by flow cytometry as described below herein.
[0090] Fibroblasts were grown from fetus number one. The fibroblasts were utilized in SCNT to generate 477 em bryos which were transferred into four recipient gilts. The SCNT resulted in one pregnancy and the birth of four live piglets and one still born piglet. The four via ble piglets needed no feeding support and appeared healthy. Three of the aGal/CMAH dou ble knockout pigs are shown in Figure 3A. The above described disruptions of the CMAH and GGTAl genes were validated in the four via ble pigs from the second pregnancy as described below herein. Results of somatic cell nuclear transfer using dou ble-KO cells are su mmarized in Ta ble 1 a bove.
[0091] Crispr-transfected I B4 counterselected cells were used as donor cells in SCNT and transferred into 2 recipients, Juneau and Honda. Eighty-eight em bryos were transferred into Ju neau; pregnancy did not occur in Juneau. Over 100 em bryos were transferred into Honda; pregnancy occurred. Ten healthy
fetuses were collected at day 32 of gestation (Figure 9A). DNA sequencing results showed that five out of the ten fetuses were GGTA1 and CMAH double knockouts. For GGTA1, seven fetuses were biallelic mutations, one fetus was a monoallelic mutation and two fetuses were wild-type. For CMAH, five fetuses were biallelic mutations, one fetus was a monoallelic mutation and four fetuses were wild-type. Among the five double knockout fetuses, two homozygous mutations were found in fetus 7 at the GGTA1 locus and in fetus 2 at the CMAH locus. The remaining mutations were heterozygous biallelic mutations. Results are summarized in Table 2 below and sequence data are presented in Figure 9B. Fetal fibroblasts were harvested from one dou ble knockout fetus. The fetal fibroblasts were utilized in SCNT to generate embryos which were transferred into recipient sows. SCNT of the double knockout fetal fibroblasts resulted in at least one pregnancy.
[0092] Table 2 Results of Somatic Cell Nuclear Transfer Using Crispr-Transfected Cells
[0093] Somatic Cell Nuclear Transfer. All the animals used in this study were approved by Institutional Biosafety Committee (IBC) and Institutional Animal Care and Use Committee (IACUC). In Table 2, * indicates pregnant animals/total recipients and ** indicates fetuses/embryos transferred.
Example 8. DNA Sequencing Analysis of Zn-finger Targeted CMAH and GGTA1 Regions.
[0094] Genomic DNA from double knockout Zn finger cloned fetuses and the four viable piglets was extracted using DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA). PCR amplification of the CMAH region was performed as described above herein.
[0095] Primer CMAH-S1 (SEQ ID NO: 5-5' CCAAACCCTGTCATTCCAG 3') and GGTA1-F primer (SEQ ID NO: 6-5' CTAGAAATCCCAGAGGTTAC 3') were used to sequence the targeted CMAH region and GGTAl region, respectively.
[0096] The GGTAl region was amplified by PCR using the GGTA1-F primer (SEQ ID NO: 6 (5' CTAGAAATCCCAGAGGTTAC 3')) and GGTA1-R primer (SEQ ID NO:7 (5'TCCTTGTCCTGGAGGATTCC3')). Pwo Master (Roche, Indianapolis IN) was used and PCR conditions were as follows: 94°C, 2 min; 94°C, 15 s, 57°C, 30 s, and 72°C, 30s for 40 cycles; and a final extension step of 72°C for 5 min. A total of 200-400 ng of PCR product was denatured and annealed using the following program on a Mycycler (Bio-Rad): 95°C, 10 min; 95° to 85°C, -2°C/s; 85°C to 25°C, -0.1°C/s. One microliter of enhancer and 1 μΙ of Nuclease S (Transgenomic Omaha NE) was added to each reaction and incubated at 42°C for 40 minutes. The product was separated on a 10% polyacrylamide gel and stained with SYBR Safe (Invitrogen USA Eugene OR).
[0097] Results from an exemplary DNA sequence analysis are shown in Figure 2B. DNA sequence analysis confirmed homozygous alterations of the GGTAl and CMAH genes in at least one fetus. The CMAH gene sequence is altered by a four base pair insertion. The inserted sequence is "GGAA". The GGTAl gene sequence is altered by a 3 base pair deletion adjacent to a G to A substitution. The sequences of the targeted CMAH and GGTAl regions of the genomic DNA of four viable piglets are shown in Figure 3B.
[0098] Genomic DNA from IB4 counter-selected cells and Crispr cloned fetuses was extracted using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma Aldrich, St. Louis MO).
[0099] The GGTAl region was amplified by PCR using the GGTA1-F seq primer (SEQ ID NO: 8 (5' CCTTAGTATCCTTCCC AACCC AG AC-3' ) ) and GGTA1-R seq primer (SEQ ID NO: 9 (5' GCTTTCTTTACGGTGTCAGTGAATCC-3')). The CMAH region was amplified by PCR using the CMAH-F seq primer (SEQ ID NO: 10 (5'- CTTGGAGGTGATTTGAGTTGGG-3')) and the CMAH-R seq primer (SEQ ID NO: 11 (5'- CATTTTCTTCGGAGTTGAGGGC-3')). Pwo Superyield DNA polymerase (Roche, Indianapolis IN) was used and PCR conditions for GGTAl were as follows: 94°C, 2 min; 94°C, 15 s, 54°C, 30 s, and 72°C, 45s for 15 cycles; 94°C, 15 s, 54°C, 30 s, and 72°C, 45s + 5 seconds/cycle for 25 cycles; and a final extension step of 72°C for 5 min. The PCR conditions for CMAH were as follows: 94°C, 2 min; 94°C, 15 s, 56°C, 30 s, and 72°C, 45s for 15 cycles; 94°C, 15 s, 56°C, 30 s, and 72°C, 45s + 5 seconds/cycle for 25 cycles; and a final extension step of 72°C for 5 min. The products were separated on a 1% agarose gel, purified by the
GenElute Gel Extraction kit (Sigma-Aldrich, St. Louis MO) and sequenced by the Sanger method (DNA Sequencing Core Facility, Indiana University School of Medicine).
[00100] Primer CMAH-F seq (SEQ ID NO: 10) and GGTA1-F seq primer (SEQ ID NO: 8) were used to sequence the targeted CMAH region and GGTAl region, respectively.
[00101] Results from exemplary DNA sequence analyses are shown in Figure 9B. DNA sequence analysis confirmed bia I lei ic alterations of the GGTAl and CMAH genes in five fetuses. In fetus 1 and 5, one GGTAl allele has a single base pair deletion (deletes a C), the other allele has a single nucleotide insertion (T). In fetus 2, one GGTAl allele has a six base pair deletion, the other allele has a two nucleotide insertion (AT). In fetus 6, one GGTAl allele has a ten base pair deletion, the other allele has a seven base pair deletion. In fetus 7, both GGTAl alleles have an eight nucleotide insertion that substitute for a five nucleotide sequence. In fetus 1, one CMAH allele has a double base pair deletion (deletes TG), the other allele has a single nucleotide insertion (inserts T). In fetus 2, both CMAH alleles have an 8 base pair deletion. In fetus 5, one CMAH allele has a 5 nucleotide deletion, the other has a 3 nucleotide deletion. In fetus 6, one CMAH allele has a single base pair deletion (T), the other has single base pair deletion and a colocalized two base pair insertion. In fetus 7, one CMAH allele has a 5 base pair deletion, the other has a 20 base pair deletion. Sequences of the targeted CMAH and GGTAl regions of the genomic DNA of five fetuses are shown in Figure 9B.
Example 9. Flow Cytometry of Red Blood Cells (RBCs).
[00102] Fetal livers were removed from double knockout fetuses and incubated in RPM I1640 for
24 hours at 37°C. RBCs were collected from cells released into the media after incubation of the fetal livers. RBCs were also obtained from adult human donors, six month old wild-type pigs and GGTA- knockout pigs (GGTA-KO pigs (fetal or 6 month old)). Porcine and human peripheral blood monocytes (PBMCs) were prepared using Ficoll-Paque Plus from whole blood collected in anticoagulant citrate dextrose (ACD).
[00103] Cells were stained with IB4 lectin Alexa Fluor 647 (Invitrogen, Grand Island, NY) and anti-
Neu5Gc antibody (Sialix, Vista CA). A negative control antibody for comparison with anti-Neu5Gc antibody was also used (Sialix, Vista CA). Cells were incubated with IB4 lectin for 20 minutes at 4°C. The cells were washed with blocking agent (Sialix, Vista, Ca) diluted in HBSS. Gal epitopes bind IB4 lectin. Cells were then stained with anti-Neu5Gc antibody for one hour at 4°C followed by Donkey anti-chicken DyLight 649 (Jackson ImmunoResearch Laboratories Inc, West Grove PA) for 40-60 minutes at 4°C. Cells
stained with anti-Neu5Gc antibody were washed before and after secondary antibody with Sialix blocking agent diluted in PBS. In various experiments unstained BC or PBMC were used as negative controls for IB4 lectin staining. An Accuri C6 flow cytometer and CFlow Software (Accuri, Ann Arbor, Ml) were used for analysis.
[00104] FACS analysis validated the absence of functional GGTA1 and CMAH genes eliminated the a-Gal and Neu5Gc modifications on the RBCs isolated from the cloned fetuses. Results obtained from one such experiment are shown in Figure 2C.
Example 10. Confocal microscopy analysis.
[00105] One of the four double knockout (Double-KO, DKO, CMAH-/aGAL-) piglets with mutations generated by Zn-finger targeting was euthanized. Liver, heart and kidney tissues were obtained from the double-ko pig. Liver, heart and kidney tissues were also obtained from wild-type (WT) and GGTA1 knockout (GGTA-ko) pigs. Frozen sections of each tissue were prepared. Mounted tissues were blocked in Odyssey blocking buffer (Ll-Cor Biosciences, Lincoln NE) in HBSS for one hour. The slides were then fixed in 4% paraformaldehyde for 10 minutes. Tissues were stained with IB4 lectin Alexa Fluor 647 (Invitrogen, Grand Island NY) to visualize the presence of the Gal epitope. To visualize the Neu5Gc epitope, tissues were stained with a chicken anti-Neu5Gc antibody or with a control antibody (Sialix, Vista, CA) for one hour. Tissues were washed three times with HBSS. Donkey anti- chicken DyLight 649 (Jackson ImmunoResearch Laboratories Inc., West Grove PA) secondary antibody was incubated with the tissue for approximately one hour. Tissues were washed three times with 0.1% HBSS Tween. To stain the nucleus, DAPI stain (Invitrogen, Grand Island NY) was added to all the slides for 1 minute followed by two 0.1% HBSS Tween washes. Tissues were mounted in ProLong Gold (Invitrogen, Grand Island NY). Confocal microscopy was performed using an Olympus FV1000.
[00106] Confocal microscopy indicated the presence of a-Gal and Neu5Gc in liver, heart and kidney tissues obtained from a wild-type pig. GGTA1-KO pigs displayed only Neu5Gc in liver, heart and kidney tissues. Confocal microscopy indicated the absence of a-Gal and Neu5Gc in liver, heart and kidney in tissues obtained from a double knockout piglet (exemplary fields are shown in Figure 4A and 4B).
Example 11. Crossmatch of Human Sera with GGTA1-KO and double-KO PBMCs.
[00107] Porcine whole blood from GGTA1-KO and dou ble-KO pigs was collected in ACD. Porcine
PBMCs were prepared from the whole blood using Ficoll-Paque Plus. Cell viability was assessed microscopically with Trypan Blue. Sera were obtained from ten healthy human volunteers. Twenty-five percent heat-inactivated serum was prepared. Approximately 2 X 106/ml GGTA-KO and double-KO PBMCs were incubated with each human serum sample for 2 hours at 4°C. After incubation of the serum and PBMCs, the PBMCs were washed three times in 0.5% PBS Sialix Blocking agent. PBMCs were stained with DyLight 649-conjugated Donkey anti-human IgM or DyLight 488 Donkey anti-human IgG (Jackson Immunoresearch Laboratories Inc, West Grove PA) for 1 hour at 4°C. PBMCs were washed three times using 0.5% PBS Sialix blocking agent. Analyses were performed using an Accuri C6 flow cytometer and BD CFlow Plus Software (Accuri, Ann Arbor Ml). Overlays were produced using Kaluza version 1.2 software from Beckman Coulter (Brea, CA). A representative histogram is shown in Figure 5A. MFI's obtained from flow cytometry crossmatch analysis of 10 unique human subjects are also shown in Figure 5A.
[00108] Lower Mean fluorescent Intensities (MFI) of double-KO PBMCs indicate less IgM bound to double-KO cells than to GGTA1-KO cells in 9 of 10 samples. In an experiment, the median MFI observed for human IgM binding to double-KO and GGTA-KO PBMCs were 4,411 and 15,059 respectively (n=10, p=0.0039). Lower MFI of double-KO PBMCs indicate less IgG bound to double-KO cells than to GGTA1-KO cells in 10 of 10 samples. In an experiment, the median MFI observed for human IgG binding to double-KO and GGTA-KO PBMC were 714 and 2306 respectively (n=10, p=0.002). (Figure 5A).
Example 12. Antibody-mediated Complement-Dependent Cytotoxicity.
[00109] Antibody-mediated complement dependent cytotoxicity assays are known in the art. A modified method of Diaz et al (Diaz et al, 2004, Transplant Immunology 13(4):313-317) was performed. Human serum was obtained from ten healthy volunteers (Figures 5 and 6). Twenty-five percent heat- inactivated human serum was prepared. The heat-inactivated human sera were serially diluted and 100 μΙ of each concentration were placed in a 96 well v-bottom assay plate. Sera were mixed with a 100 μΙ aliquot of PBMC obtained from either GGTA1-KO or double-KO pigs. The final concentration of PBMC in each well was 5 X 106/ml; in some experiments 1 x 106/ml PBMC were used. The serum concentrations varied from 50%, 17%, 6%, 2%, 0.6%, 0.2% and 0.07%. The mixtures were incubated for 30 minutes at 4°C. After 30 minutes, the plates were centrifuged for 4 minutes at 400 x g. The plates were decanted
and washed with HBSS. Rabbit complement (150 μΙ of a 1:15 dilution) was added to each well and incubated for 30 minutes at 37°C. PBMC were labeled with a fluorescein diacetate (FDA) stock solution, prepared fresh daily in HBSS (1 μ§/ιηΙ-) from a 1 mg/ml stock solution in acetone and with propidium iodide (PI), prepared at 50 μ§/ιηΙ in phosphate buffered saline (PBS). After incubation in complement, the samples were transferred by pipette to tubes containing 250 μΙ of HBSS and 10 μΙ of FDA/PI for analysis using an Accuri C6 flow cytometer.
[00110] The percentage of dead cells (PI+/FDA-), damaged cells (PI+/FDA+) and live cells (Pl-
/FDA+) was determined. Double negative events (PI-/FDA-) were excluded from calculations. The percentage of cytotoxicity in cells not exposed to serum was considered spontaneous killing (% CTXspont). Values for cytotoxicity (%CTX) are shown after correction for spontaneous cytotoxicity (%CTXspont), using the following formula: %CTX = (%CTXeXp-%CTXSpont)/(100-%CTXSpOnt) x 100, where %CTXexp is the percentage of dead cells under the experimental condition.
[00111] Data from a series of antibody-mediated complement dependent cytotoxicity assays are shown in Figure 5B. At 2% concentration, most of the subjects' sera lysed greater than 90% of GGTAl- KO PBMCs but less than 50% of the double-KO PBMCs. The median percent cytotoxicity of GGTAl-KO and double-KO PBMCs in this series was 98% and 29% respectively, at 2% human serum (n=10, p=0.002). The data shown indicate these human sera were less cytotoxic to double-KO cells than to GGTAl-KO cells. While not being bound by mechanism, the data suggest in the absence of the Gal epitope, a significant portion of IgM and IgG binding is to Neu5Gc.
Example 13. Statistical Analysis.
[00112] Flow cytometric crossmatch results were reported as medians of the M FI values for each human serum crossmatched with GGTAl-KO and double-KO PBMCs for both IgM and IgG. The Wilcoxon matched pairs signed rank test was used to analyze the data using Prism 5 for Windows (GraphPad Software Inc, La Jolla Ca).
[00113] Antibody-mediated, complement dependent cytotoxicity data was also analyzed as above. The log of each serum dilution was plotted against the %CTX for each sample and the sigmoid curve was analyzed by non-linear regression to estimate the serum LD50. The %CTX of GGTAl-KO and double-KO PBMCs at each serum dilution for each sample was analyzed using the Wilcoxon matched pairs signed rank test.
Example 14. Ex vivo Perfusion of Human Platelet through a Double KO Liver.
[00114] A double knockout CMAH/aGal pig is anesthetized and intubated. A midline abdominal incision is made. The liver is removed and placed in a perfusion device under normothermic conditions. Humidity, temperature and air flow are maintained in the perfusion device. Human platelets obtained from healthy volunteer subjects are circulated through the double knockout liver. Platelet levels in the pre-perfusion and post-perfusion samples are evaluated. Pre and post-perfusion evaluation of the pig liver is performed.
Example 15. Evaluation of Response to a Double Knockout (ctGal and CMAH) Xenograft.
[00115] A porcine liver obtained from double knockout (aGal-/CMAH-) pigs is surgically transplanted into a recently deceased human cadaver using the piggyback method. After the surgery, biological samples are obtained from the human cadaver. Clinical indicia of graft rejection are monitored.
Example 16. Evaluation of a Response to a Double Knockout (ctGal and CMAH) Xenograft.
[00116] Porcine kidneys are obtained from double knockout (GGTA1/CMAH double-KO) pigs. A highly sensitized human subject is administered compounds to manage preexisting and de novo donor- specific antibodies. Porcine double-KO kidneys are surgically transplanted into the subject. Clinical indicia of graft rejection are monitored.
Example 17. Construction of single guide RNAs (sgRNAs) Expression Vector
[00117] Targeting sites for the CMAH and GGTA1 genes were identified. Forward and reverse oligonucleotides for each targeting site were obtained. The GGTA1 forward sgRNA sequence was CACCGAGAAAATAATGAATGTCAA (SEQ ID NO: 12); the GGTA1 reverse sgRNA sequence was AAACTTGACATTCATTATTTTCTC (SEQ ID NO: 13). The CMAH forward sgRNA sequence was CACCGAGTAAGGTACGTGATCTGT (SEQ ID NO: 14); the CMAH reverse sgRNA sequence was AAACACAGATCACGTACCTTACT (SEQ ID NO: 15). The oligonucleotide pairs were annealed together to generate short double strand DNA fragments with Bbsl compatible overhangs. The pX330 bicistronic expression vector expressing Cas9 and sgRNA of interest (Addgene, www.addgene.org/crispr-cas) was linearized with Bbsl. pX330 expresses a mammalian codon optimized Cas9. The linearized pX330
expression vector and the double strand DNA fragments for each sg NA (GGTA1 and CMAH) were incubated with ligase.
[00118] Liver derived cells (LDCs) were cultured as described in Li et al (2013) J. Surg Res
181:e39-45, herein incorporated by reference in its entirety. LDC were cotransfected with pX330- CMAHsgRNA and pX300-GGTAlsgRNA expression plasmids using the Neon transfection system (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were harvested and used for isolectin B4 counter-selection.
Example 18. Flow cytometry analysis
[00119] Isolectin B4 (IB4) counter-selected LDC, cloned fetal fibroblasts from DKO fetuses, wild- type LDC and wild-type fetal fibroblasts were stained with IB4 conjugated with FITC (Enzo Life Science, Farmingdale NY) to assess the aGal epitope level. To evaluate the Neu5Gc level, IB4 counter-selected LDC cloned fetal fibroblasts from DKO fetuses, wild-type LDC and wild-type fetal fibroblasts were stained with antiNeu5Gc antibody (Sialix, Vista CA) followed by donkey anti-chicken DyLight 649 (Jackson ImmunoResearch Laboratories Inc., West Grove PA). A negative control antibody for comparison with anti-Neu5Gc antibody was also used (Sialix, Vista CA). An Accuri C6 flow cytometer (Accuri, Ann Arbor Ml) and FlowJo software (Tree Star, Inc. Ashland OR) were used for analysis. Representative flow cytometry results of aGal from IB4 counter selected cells are shown in Figure 7A. Representative flow cytometry results of Neu5Gc on IB4 counter selected cells are shown in Figure 8A. Representative flow cytometry results of the aGal and Neu5Gc on fetal fibroblasts derived from fetus 7 are shown in Figure 9C.
Example 19. Ex vivo Perfusion of Human Platelets through a Double KO Liver
[00120] Double knockout CMAH/aGAL and single aGal knockout pigs were anesthetized and intubated. A midline abdominal incision was made in each pig. Livers were removed and placed in a perfusion device under normothermic conditions. Humidity, temperature and air flow were maintained in the perfusion device. Human platelets obtained from healthy volunteer subjects were circulated through the double knockout liver. Platelet levels in the pre-perfusion and post-perfusion samples were evaluated after circulation through three double knockout (CMAH ^/GGTAl7 ) livers. Pre and post- perfusion evaluation of the pig livers were performed. GGTA17" livers (n=2) were obtained, and the livers
were perfused under similar conditions. Data from one experimental series are summarized in Figure 12.
Example 20. Neu5Gc Levels in Cells Cultured with a DKO (aGal/CMAH) Piq Cell Culture Reagent.
[00121] Fibroblasts were cultured from a CMAH/aGal Double knockout pig. Because the DKO fibroblasts lack CMAH, they do not produce Neu5Gc. DKO serum was isolated from the blood of CMAH/aGAL pigs. The DKO fibroblasts were cultured in cell culture media supplemented with either DKO serum or bovine serum (FBS) for six weeks. After six weeks, cells were evaluated by flow cytometry with anti-Neu5Gc antibodies. Results from one such experiment are presented in Figure 10.
Example 21. Concordant Analysis of DKO Pig, Baboon and Chimpanzee Material.
[00122] Antibody-mediated complement dependent cytotoxicity assays are known in the art. A modified method of Diaz et al (Diaz et al, 2004, Transplant Immunology 13(4):313-317) was performed. Serum samples were obtained from 10 randomly selected baboons. Twenty-five percent heat- inactivated baboon serum was prepared. The heat-inactivated baboon sera were serially diluted and 100 μΙ of each concentration were placed in a 96 well v-bottom assay plate. Sera were mixed with a 100 μΙ aliquot of PBMC obtained from either GGTA1-KO or double-KO (GGTA1/CMAH DKO) pigs. The final concentration of PBMC in each well was 5 X 106/ml; in some experiments 1 x 10106/ml PBMC were used.
[00123] The mixtures were incubated for 30 minutes at 4°C. After 30 minutes, the plates were centrifuged for 4 minutes at 400 x g. The plates were decanted and washed with HBSS. Rabbit complement (150 μΙ of a 1:15 dilution) was added to each well and incubated for 30 minutes at 37°C. PBMC were labeled with a fluorescein diacetate (FDA) stock solution, prepared fresh daily in HBSS (1 μg/mL) from a 1 mg/ml stock solution in acetone and with propidium iodide (PI), prepared at 50 μg/ml in phosphate buffered saline (PBS). After incubation in complement, the samples were transferred by pipette to tu bes containing 250 μΙ of HBSS and 10 μΙ of FDA/PI for analysis using an Accuri C6 flow cytometer.
[00124] The %CTX for each sample was plotted against the log of each serum dilution and the sigmoid curve was analyzed by non-linear regression. Data from one such set of experiments are presented in Figure 13.
[00125] Serum was obtained from five randomly selected human donors. Twenty-five percent heat-inactivated serum was prepared. Human, baboon and chimpanzee PBMCs were obtained. PBMCs
were obtained from single aGal knockout pigs (GGTA1-KO) and double CMAH/aGal (double-KO) pigs. Approximately 2 X 106/ml PBMCs were incubated with each human serum sample for 2 hours at 4°C. After incu bation of the serum and PBMCs, the PBMCs were washed three times in 0.5% PBS Sialix Blocking agent. PBMCs were stained with DyLight 649-conjugated Donkey anti-human IgM or DyLight 488 Donkey anti-human IgG (Jackson Immunoresearch Laboratories Inc, West Grove PA) for 1 hour at 4°C. PBMCs were washed three times using 0.5% PBS Sialix blocking agent. Analyses were performed using an Accuri C6 flow cytometer and BD CFlow Plus Software (Accuri, Ann Arbor Ml). Overlays were produced using Kaluza version 1.2 software from Beckman Coulter (Brea, CA). Secondary only control antibody staining was also evaluated. Histograms from one such experiment are presented in Figure 14.
[00126] The invention is not limited to the embodiments set forth herein for illustration, but includes everything that is within the scope of the claims. Furthermore, all references cited herein are hereby incorporated by reference in their entirety and for all purposes as if fully set forth herein.
Claims
1. A knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and when tissue from said pig is transplanted into a human, hyperacute rejection is decreased as compared to when tissue from a wild-type pig is transplanted into a human.
2. Porcine organs, tissue or cells for transplantation into a human having reduced expression of aGal and Neu5Gc on the porcine organs, tissue or cells.
3. A method for modifying a porcine organs, tissue or cells for transplantation into a human, the method comprising removing or reducing expression of aGal and Neu5Gc on the porcine organs, tissue or cells.
4. The method of claim 3 wherein the porcine organs, tissue, or cells are selected from the group consisting of skin, heart, liver, kidneys, lung, pancreas, thyroid, small bowel, and components thereof.
5. A method for making porcine organs, tissue or cells for transplantation into a human, the method comprising reducing expression of aGal and CMAH on the porcine organs, tissue or cells.
6. The method of claim 5 wherein said porcine organs, tissue, or cells are selected from the group consisting of skin, heart, liver, kidneys, lung, pancreas, small bowel, and components thereof.
7. A knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and wherein when tissue from said knockout pig is transplanted into a human, thrombocytopenia is decreased as compared to when tissue from a wild-type pig is transplanted into a human.
8. A knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig and wherein a liver from said pig exhibits reduced uptake of human platelets when said liver is exposed to said human platelets.
9. A method of increasing the duration of the period between when a human subject is identified as a subject in need of human liver transplant and when said human liver transplant occurs, said method comprising providing a liver from a knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in the knockout pig is decreased as compared to a wild-type pig, and surgically attaching said liver from said knockout pig to said human subject in a therapeutically effective manner.
10. The method of claim 9, wherein said liver from said knockout pig is internal to said human subject.
11. The method of claim 9, wherein said liver from said knockout pig is external to said human subject.
12. The method of claim 9, wherein said liver is directly or indirectly attached to said subject.
13. A method of preparing organs, tissues, or cells for xenotransplantation into human patients with reduced rejection, the method comprising providing a transgenic pig as a source of transplant material wherein the transplant material is selected from the group consisting of organs, tissues, or cells, and wherein the pig masks or reduces the expression of at least two xenoreactive antigens on the transplant material.
14. The method of claim 13, wherein the at least two xenoreactive antigens are aGal and Neu5Gc.
15. A knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein the disruption of said a(l,3)-galactosyltransferase gene is selected from the group of disruptions comprising a 3 base pair deletion adjacent to a G to A substitution, a single base pair deletion, a single base pair insertion, a six base pair deletion, a two base pair insertion, a ten base pair deletion, a seven base pair deletion, and an eight base pair substitution for a five base pair sequence; wherein the disruption of said CMAH gene is selected from the group of disruptions comprising a four base pair insertion, a two base pair deletion, a single base pair insertion, an eight base pair deletion, a five base pair deletion, a three base pair deletion, a two base pair substitution for a single base pair, and a twenty base pair deletion; and wherein expression of functional a(l,3)-galactosyltransferase and CMAH in said knockout pig is decreased as compared to a wild-type pig and when tissue from said knockout pig is transplanted into a human, a hyperacute rejection related symptom is improved as compared to when tissue from a wild- type pig is transplanted into a human.
16. A method of improving a hyperacute rejection related symptom in a patient comprising transplanting porcine organs, tissue or cells having reduced expression of aGal and CMAH into a human, wherein said hyperacute rejection related symptom is improved as compared to when tissue from a wild-type swine is transplanted into a human.
17. A cell culture reagent derived from a knockout pig comprising disrupted a(l,3)- galactosyltransferase and CMAH genes, wherein expression of functional a(l,3)-galactosyltransferase and CMAH in said knockout pig is decreased as compared to a wild-type pig, wherein said cell culture reagent is selected from the group comprising cell culture media, cell culture serum, cell culture additive and an isolated cell capable of proliferation.
18. A method of producing a glycoprotein of interest, said method comprising the step of incubating an isolated cell capable of expressing said glycoprotein of interest with a cell culture reagent derived from a knockout pig comprising disrupted a(l,3)-galactosyltransferase and CMAH genes, wherein the amount of Neu5Gc or alphaGal epitopes on said glycoprotein of interest is lower than the amount of Neu5Gc or alphaGal epitopes on said glycoprotein of interest when said isolated cell capable of expressing said glycoprotein of interest is incu bated with a cell culture reagent derived from a wild-type Pig-
19. The method of claim 18 wherein said glycoprotein of interest is a glycoprotein selected from the group comprising an antibody, growth factor, cytokine, hormone and clotting factor.
20. The method of claim 18, wherein the disruption of said a(l,3)-galactosyltransferase gene is selected from the group of disruptions comprising a 3 base pair deletion adjacent to a G to A substitution, a single base pair deletion, a single base pair insertion, a six base pair deletion, a two base pair insertion, a ten base pair deletion, a seven base pair deletion, and an eight base pair substitution for a five base pair sequence; wherein the disruption of said CMAH gene is selected from the group of disruptions comprising a four base pair insertion, a two base pair deletion, a single base pair insertion, an eight base pair deletion, a five base pair deletion, a three base pair deletion, a two base pair substitution for a single base pair, and a twenty base pair deletion; and wherein expression of functional a(l,3)-galactosyltransferase and CMAH in said knockout pig is decreased as compared to a wild-type pig.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/436,963 US9888674B2 (en) | 2012-10-24 | 2013-10-23 | Double knockout (GT/CMAH-KO) pigs, organs and tissues |
US15/848,870 US10667500B2 (en) | 2012-10-24 | 2017-12-20 | Double knockout (GT/CMAH-KO) pigs, organs and tissues |
US16/855,905 US11666039B2 (en) | 2012-10-24 | 2020-04-22 | Double knockout (GT/CMAH-KO) pigs, organs and tissues |
US18/101,810 US20240196872A1 (en) | 2012-10-24 | 2023-01-26 | Double knockout (gt/cmah-ko) pigs, organs and tissues |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201261717845P | 2012-10-24 | 2012-10-24 | |
US61/717,845 | 2012-10-24 | ||
US13/804,365 | 2013-03-14 | ||
US13/804,365 US20140115728A1 (en) | 2012-10-24 | 2013-03-14 | Double knockout (gt/cmah-ko) pigs, organs and tissues |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/804,365 Continuation US20140115728A1 (en) | 2012-10-24 | 2013-03-14 | Double knockout (gt/cmah-ko) pigs, organs and tissues |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/436,963 A-371-Of-International US9888674B2 (en) | 2012-10-24 | 2013-10-23 | Double knockout (GT/CMAH-KO) pigs, organs and tissues |
US15/848,870 Division US10667500B2 (en) | 2012-10-24 | 2017-12-20 | Double knockout (GT/CMAH-KO) pigs, organs and tissues |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2014066505A1 true WO2014066505A1 (en) | 2014-05-01 |
Family
ID=50486651
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2013/066387 WO2014066505A1 (en) | 2012-10-24 | 2013-10-23 | Double knockout (gt/cmah-ko) pigs, organs and tissues |
Country Status (2)
Country | Link |
---|---|
US (5) | US20140115728A1 (en) |
WO (1) | WO2014066505A1 (en) |
Cited By (37)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9068179B1 (en) | 2013-12-12 | 2015-06-30 | President And Fellows Of Harvard College | Methods for correcting presenilin point mutations |
US9163284B2 (en) | 2013-08-09 | 2015-10-20 | President And Fellows Of Harvard College | Methods for identifying a target site of a Cas9 nuclease |
US9228207B2 (en) | 2013-09-06 | 2016-01-05 | President And Fellows Of Harvard College | Switchable gRNAs comprising aptamers |
WO2016059161A1 (en) * | 2014-10-15 | 2016-04-21 | Xenothera | Composition with reduced immunogenicity |
US9322006B2 (en) | 2011-07-22 | 2016-04-26 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US9322037B2 (en) | 2013-09-06 | 2016-04-26 | President And Fellows Of Harvard College | Cas9-FokI fusion proteins and uses thereof |
WO2016065046A1 (en) | 2014-10-22 | 2016-04-28 | Indiana University Research & Technology Corporation | Triple transgenic pigs suitable for xenograft |
US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US9834791B2 (en) | 2013-11-07 | 2017-12-05 | Editas Medicine, Inc. | CRISPR-related methods and compositions with governing gRNAS |
US9888673B2 (en) | 2014-12-10 | 2018-02-13 | Regents Of The University Of Minnesota | Genetically modified cells, tissues, and organs for treating disease |
JP2018526015A (en) * | 2015-09-09 | 2018-09-13 | レビビコア, インコーポレイテッド | Multi-transgenic pigs for xenotransplantation |
US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
WO2020041384A1 (en) | 2018-08-20 | 2020-02-27 | The Broad Institute, Inc. | 3-phenyl-2-cyano-azetidine derivatives, inhibitors of rna-guided nuclease activity |
WO2020041387A1 (en) | 2018-08-20 | 2020-02-27 | The Brigham And Women's Hospital, Inc. | Degradation domain modifications for spatio-temporal control of rna-guided nucleases |
WO2020068304A2 (en) | 2018-08-20 | 2020-04-02 | The Broad Institute, Inc. | Inhibitors of rna-guided nuclease target binding and uses thereof |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
WO2020236967A1 (en) | 2019-05-20 | 2020-11-26 | The Broad Institute, Inc. | Random crispr-cas deletion mutant |
WO2021041922A1 (en) | 2019-08-30 | 2021-03-04 | The Broad Institute, Inc. | Crispr-associated mu transposase systems |
WO2021050974A1 (en) | 2019-09-12 | 2021-03-18 | The Broad Institute, Inc. | Engineered adeno-associated virus capsids |
US11180751B2 (en) | 2015-06-18 | 2021-11-23 | The Broad Institute, Inc. | CRISPR enzymes and systems |
US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
WO2022087089A1 (en) | 2020-10-21 | 2022-04-28 | Tissue Testing Technologies Llc | Minimizing immunogenicity of decellularized tissues |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11479752B2 (en) | 2017-10-20 | 2022-10-25 | Indiana University Research And Technology Corporation | Scaffold-free 3D bioprinting of porcine cells |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140115728A1 (en) | 2012-10-24 | 2014-04-24 | A. Joseph Tector | Double knockout (gt/cmah-ko) pigs, organs and tissues |
BR112017018932A2 (en) | 2015-03-03 | 2018-07-31 | Univ Minnesota | etv2 and uses thereof |
EP3313176A4 (en) * | 2015-06-26 | 2019-06-26 | Indiana University Research&technology Corporation | Transgenic pigs with genetic modifications of sla |
RU2018103093A (en) | 2015-06-30 | 2019-07-31 | Реджентс Оф Зэ Юниверсити Оф Миннесота | HUMANIZED skeletal muscle |
JP2018523999A (en) | 2015-06-30 | 2018-08-30 | リージェンツ オブ ザ ユニバーシティ オブ ミネソタ | Humanized myocardium |
EP3468356A4 (en) * | 2016-06-14 | 2020-02-26 | Regents Of The University Of Minnesota | Genetically modified cells, tissues, and organs for treating disease |
KR102363891B1 (en) * | 2017-04-28 | 2022-02-17 | 서울대학교산학협력단 | Transgenic pigs expressing both shTNFRI-Fc and HA-HO-1 without Gal epitope and N-glycolylneuraminic acid epitope and the Use of thereof |
AU2019356014A1 (en) | 2018-10-05 | 2021-05-20 | Alexis Bio, Inc. | Xenotransplantation products and methods |
US10883084B2 (en) | 2018-10-05 | 2021-01-05 | Xenotherapeutics, Inc. | Personalized cells, tissues, and organs for transplantation from a humanized, bespoke, designated-pathogen free, (non-human) donor and methods and products relating to same |
EP3863400A4 (en) * | 2018-10-10 | 2022-07-13 | Revivicor Inc. | Compositions and methods for preventing allergies |
CN113425906A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Cartilage material and preparation method and application thereof |
US20230203176A1 (en) | 2021-09-17 | 2023-06-29 | Novartis Ag | Methods For Prevention Of Graft Rejection In Xenotransplantation |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002088351A1 (en) * | 2001-04-30 | 2002-11-07 | Rbc Biotechnology, Inc. | Modified organs and cells for xenotransplantation |
WO2004028243A2 (en) * | 2002-08-21 | 2004-04-08 | Revivicor, Inc. | Porcine animals lacking any expression of functional alpha 1,3 galactosyltransferase |
US7547816B2 (en) | 2001-12-21 | 2009-06-16 | The Curators Of The University Of Missouri | α(1,3)-galactosyltransferase knockout swine, tissues and organs |
Family Cites Families (67)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5352240A (en) | 1989-05-31 | 1994-10-04 | Promedica International, Inc. | Human heart valve replacement with porcine pulmonary valve |
US5705732A (en) | 1989-06-12 | 1998-01-06 | Oklahoma Medical Research Foundation | Universal donor cells |
WO1991016449A1 (en) | 1990-04-16 | 1991-10-31 | The Trustees Of The University Of Pennsylvania | Saccharide compositions, methods and apparatus for their synthesis |
US5846715A (en) | 1990-05-11 | 1998-12-08 | The Austin Research Institute | CD46 variants |
WO1994021799A1 (en) | 1993-03-16 | 1994-09-29 | Austin Research Institute | USE OF PORCINE GAL α(1,3) GALACTOSYL TRANSFERASE IN XENOGRAFT THERAPIES |
US6331658B1 (en) | 1993-04-20 | 2001-12-18 | Integris Baptist Medical Center, Inc. | Genetically engineered mammals for use as organ donors |
US5560911A (en) | 1993-10-12 | 1996-10-01 | Oklahoma Medical Research Foundation | Method of inhibiting acute complement mediated cytotoxicity with anti-idiotypic antibodies |
US5849991A (en) | 1994-01-27 | 1998-12-15 | Bresatch Limited | Mice homozygous for an inactivated α 1,3-galactosyl transferase gene |
WO1995024494A1 (en) | 1994-03-09 | 1995-09-14 | Abbott Laboratories | Humanized milk |
US6204431B1 (en) | 1994-03-09 | 2001-03-20 | Abbott Laboratories | Transgenic non-human mammals expressing heterologous glycosyltransferase DNA sequences produce oligosaccharides and glycoproteins in their milk |
US6204053B1 (en) | 1994-11-08 | 2001-03-20 | Diacrin, Inc. | Porcine cortical cells and their use in treatment of neurological deficits due to neurodegenerative diseases |
US6294383B1 (en) | 1994-11-08 | 2001-09-25 | The Mclean Hospital Corporation | Porcine neural cells and their use in treatment of neurological deficits due to neurodegenerative diseases |
US5922577A (en) | 1995-04-11 | 1999-07-13 | Cytel Corporation | Enzymatic synthesis of glycosidic linkages |
US5728554A (en) | 1995-04-11 | 1998-03-17 | Cytel Corporation | Enzymatic synthesis of glycosidic linkages |
GB9517780D0 (en) | 1995-08-31 | 1995-11-01 | Roslin Inst Edinburgh | Biological manipulation |
US6166288A (en) | 1995-09-27 | 2000-12-26 | Nextran Inc. | Method of producing transgenic animals for xenotransplantation expressing both an enzyme masking or reducing the level of the gal epitope and a complement inhibitor |
WO1998005768A1 (en) | 1996-08-02 | 1998-02-12 | The Austin Research Institute | Improved nucleic acids encoding a chimeric glycosyltransferase |
AUPO182396A0 (en) | 1996-08-23 | 1996-09-12 | Austin Research Institute, The | Improved nucleic acids for reducing carbohydrate epitopes |
US6423316B1 (en) | 1997-03-26 | 2002-07-23 | Imperial College Innovative Limited | Anticoagulant fusion protein anchored to cell membrane |
GB9809280D0 (en) | 1998-04-30 | 1998-07-01 | Rpms Technology Ltd | Immunosupression |
WO2000004191A1 (en) | 1998-07-17 | 2000-01-27 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods and devices for detection of xenogeneic graft persistence and infectious agents |
US6444205B2 (en) | 1998-09-30 | 2002-09-03 | Diacrin, Inc. | Transplantation of neural cells for the treatment of chronic pain or spasticity |
GB2365445A (en) | 1998-10-12 | 2002-02-20 | Geron Bio Med Ltd | Porcine occcytes with improved developmental competence |
US6258998B1 (en) | 1998-11-24 | 2001-07-10 | Infigen, Inc. | Method of cloning porcine animals |
US7531715B1 (en) | 1999-01-13 | 2009-05-12 | Ppl Therapeutics (Scotland) | Double nuclear transfer method and results thereof |
US20020012660A1 (en) | 1999-03-04 | 2002-01-31 | Alan Colman | Method of preparing a somatic cells for nuclear transfer |
EP1158999A2 (en) | 1999-03-05 | 2001-12-05 | Diacrin, Inc. | Methods for improving graft acceptance in a recipient by administration of a cytokine profile altering agent |
US6245890B1 (en) | 1999-03-26 | 2001-06-12 | New York Blood Center, Inc. | Porcine protein and uses thereof |
WO2001030992A2 (en) | 1999-10-22 | 2001-05-03 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | α1-3 GALACTOSYLTRANSFERASE GENE AND PROMOTER |
CA2925231A1 (en) | 1999-10-26 | 2001-05-03 | Stichting Dienst Landbouwkundig Onderzoek | Mammalian-type glycosylation in transgenic plants expressing galactosyltransferase and sialyl transferase |
US20020081654A1 (en) | 2000-04-07 | 2002-06-27 | Sandrin Mauro Sergio | Targeting hydrolase enzymes |
WO2002015681A1 (en) | 2000-08-25 | 2002-02-28 | Nippon Meat Packers, Inc. | Transgenic mammals |
MXPA03008209A (en) | 2001-03-12 | 2004-11-12 | Progenetics Llc | Production of high levels of transgenic factor viii with engineered stability, and its therapeutic uses. |
JP2005510202A (en) | 2001-03-28 | 2005-04-21 | ネクストラン インコーポレイテッド | Elimination of endogenous porcine retroviruses |
US7214660B2 (en) | 2001-10-10 | 2007-05-08 | Neose Technologies, Inc. | Erythropoietin: remodeling and glycoconjugation of erythropoietin |
US20030124023A1 (en) | 2001-12-21 | 2003-07-03 | Wilson Burgess | Method of sterilizing heart valves |
AU2003265415A1 (en) | 2002-08-14 | 2004-03-03 | Immerge Biotherapeutics, Inc. | Alpha(1,3)-GALACTOSYLTRANSFERASE NULL CELLS, METHODS OF SELECTING AND Alpha(1,3)-GALACTOSYLTRANSFERASE NULL SWINE PRODUCED THEREFROM |
US8106251B2 (en) | 2002-08-21 | 2012-01-31 | Revivicor, Inc. | Tissue products derived from porcine animals lacking any expression of functional alpha 1,3 galactosyltransferase |
US7626075B2 (en) | 2002-09-19 | 2009-12-01 | Ximerex, Inc. | Growth of foreign cells in fetal animals facilitated by conditional and selective destruction of native host cells |
CA2528500A1 (en) | 2003-06-06 | 2004-12-16 | University Of Pittsburgh | Porcine cmp-n-acetylneuraminic acid hydroxylase gene |
WO2005010485A2 (en) | 2003-07-15 | 2005-02-03 | The Regents Of The University Of California | Methods for detecting and analyzing n-glycolylneuraminic acid (neu5gc) in biological materials |
CA2533259C (en) | 2003-07-21 | 2014-01-28 | Lifecell Corporation | Acellular tissue matrices made from galactose .alpha.-1,3-galactose-deficient tissue |
EP1685148A2 (en) | 2003-11-05 | 2006-08-02 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | PORCINE ISOGLOBOSIDE 3 SYNTHASE PROTEIN, cDNA, GENOMIC ORGANIZATION, AND REGULATORY REGION |
US20090043383A1 (en) | 2004-03-29 | 2009-02-12 | Mayo Foundation For Medical Education And Research | Genetically modified heart valve xenografts |
AU2005243169A1 (en) | 2004-05-07 | 2005-11-24 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Porcine Forssman synthetase protein, cDNA, genomic organization, and regulatory region |
US7582741B2 (en) | 2004-07-26 | 2009-09-01 | University Of Massachusetts | Conditional disruption of dicer1 in cell lines and non-human mammals |
NZ539491A (en) | 2005-04-15 | 2008-04-30 | Living Cell Products Pty Ltd | Swine population and uses thereof |
US20080166805A1 (en) | 2005-06-08 | 2008-07-10 | The Regents Of The University Of California | Culturing human cells and tissues in an N-glycolylneuraminic acid-free environment |
US20070033666A1 (en) | 2005-06-24 | 2007-02-08 | Harris Reuben S | Using cytosine deaminases to diminish retroelement transfer from pigs to humans |
PT1904636E (en) | 2005-07-12 | 2010-09-14 | Greenovation Biotech Gmbh | Improvements in or relating to protein production |
CA2617930A1 (en) | 2005-08-09 | 2007-03-29 | Revivicor, Inc. | Transgenic ungulates expressing ctla4-ig and uses thereof |
GB0606231D0 (en) | 2006-03-29 | 2006-05-10 | Univ Leeds | Improvements relating to decellurisation of tissue matrices for bladder implantation |
US20100105140A1 (en) | 2008-07-16 | 2010-04-29 | Fahrenkrug Scott C | Plaice dna transposon system |
WO2010008562A2 (en) | 2008-07-16 | 2010-01-21 | Recombinetics | Methods and materials for producing transgenic animals |
US20120045816A1 (en) | 2008-09-09 | 2012-02-23 | Sialix, Inc. | Novel Glycosylated Polypeptides |
CA2736488A1 (en) | 2008-09-09 | 2010-03-18 | The Regents Of The University Of California | Elimination of a contaminating non-human sialic acid by metabolic competition |
US8709400B2 (en) | 2009-07-27 | 2014-04-29 | Washington University | Inducement of organogenetic tolerance for pancreatic xenotransplant |
ES2550202T3 (en) | 2009-08-03 | 2015-11-05 | Recombinetics, Inc. | Methods and compositions for targeted gene modification |
KR20120050485A (en) | 2009-08-14 | 2012-05-18 | 레비비코르 인코포레이션 | Multi-transgenic pigs for diabetes treatment |
WO2011139488A2 (en) | 2010-05-06 | 2011-11-10 | Mayo Foundation For Medical Education And Research | Methods and materials for reducing cardiac xenograft rejection |
US8945868B2 (en) | 2010-07-21 | 2015-02-03 | Sangamo Biosciences, Inc. | Methods and compositions for modification of a HLA locus |
JP2014520533A (en) | 2011-06-30 | 2014-08-25 | シグマ−アルドリッチ・カンパニー・リミテッド・ライアビリティ・カンパニー | Cells deficient in CMP-N-acetylneuraminic acid hydroxylase and / or glycoprotein alpha-1,3-galactosyltransferase |
WO2013019625A2 (en) | 2011-07-29 | 2013-02-07 | The University Of Wyoming | Methods of using a bacterial glcnac-6-p 2'-epimerase to promote sialylation of glycoconjugates |
WO2013151649A1 (en) | 2012-04-04 | 2013-10-10 | Sialix Inc | Glycan-interacting compounds |
WO2013169929A1 (en) | 2012-05-08 | 2013-11-14 | The General Hospital Corporation | Reducing immunogenicity of xenogeneic transplant tissues |
US20140115728A1 (en) | 2012-10-24 | 2014-04-24 | A. Joseph Tector | Double knockout (gt/cmah-ko) pigs, organs and tissues |
CN107106607A (en) | 2014-10-22 | 2017-08-29 | 印第安纳大学研究与技术公司 | Suitable for triple transgene pigs of heterograft |
-
2013
- 2013-03-14 US US13/804,365 patent/US20140115728A1/en not_active Abandoned
- 2013-10-23 US US14/436,963 patent/US9888674B2/en active Active
- 2013-10-23 WO PCT/US2013/066387 patent/WO2014066505A1/en active Application Filing
-
2017
- 2017-12-20 US US15/848,870 patent/US10667500B2/en active Active
-
2020
- 2020-04-22 US US16/855,905 patent/US11666039B2/en active Active
-
2023
- 2023-01-26 US US18/101,810 patent/US20240196872A1/en active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002088351A1 (en) * | 2001-04-30 | 2002-11-07 | Rbc Biotechnology, Inc. | Modified organs and cells for xenotransplantation |
US7166278B2 (en) | 2001-04-30 | 2007-01-23 | Rbc Biotechnology, Inc. | Modified organs and cells for xenotransplantation |
US8034330B2 (en) | 2001-04-30 | 2011-10-11 | Rbc Biotechnology, Inc. | Modified organs and cells for xenotransplantation |
US7547816B2 (en) | 2001-12-21 | 2009-06-16 | The Curators Of The University Of Missouri | α(1,3)-galactosyltransferase knockout swine, tissues and organs |
WO2004028243A2 (en) * | 2002-08-21 | 2004-04-08 | Revivicor, Inc. | Porcine animals lacking any expression of functional alpha 1,3 galactosyltransferase |
US7795493B2 (en) | 2002-08-21 | 2010-09-14 | Revivicor, Inc. | Porcine animals lacking any expression of functional alpha 1, 3 galactosyltransferase |
Non-Patent Citations (21)
Title |
---|
"Cells: a Laboratory Manual", vol. 1, 1998, COLD SPRING HARBOR LABORATORY PRESS |
"Current Protocols in Molecular Biology", 2013, WILEY-INTERSCIENCE |
ANDREW J. LUTZ ET AL: "Double knockout pigs deficient in N-glycolylneuraminic acid and Galactose [alpha]-1,3-Galactose reduce the humoral barrier to xenotransplantation", XENOTRANSPLANTATION, vol. 20, no. 1, 5 January 2013 (2013-01-05), pages 27 - 35, XP055069928, ISSN: 0908-665X, DOI: 10.1111/xen.12019 * |
BASNET ET AL., XENOTRANSPLANTATION, vol. 17, no. 6, 2010, pages 440 - 448 |
BASNET NABIN B ET AL: "Deficiency of N-glycolylneuraminic acid and Gal alpha 1-3Gal beta 1-4GlcNAc epitopes in xenogeneic cells attenuates cytotoxicity of human natural antibodies", XENOTRANSPLANTATION, vol. 17, no. 6, November 2010 (2010-11-01), pages 440 - 448, XP002717542, ISSN: 0908-665X * |
CESARE GALLI ET AL: "Genetic engineering including superseding microinjection: new ways to make GM pigs", XENOTRANSPLANTATION, vol. 17, no. 6, 1 November 2010 (2010-11-01), pages 397 - 410, XP055007654, ISSN: 0908-665X, DOI: 10.1111/j.1399-3089.2010.00590.x * |
CHEN ET AL., NATURE MED., vol. 11, no. 12, 2005, pages 1295 - 1298 |
CHEN GANG ET AL: "Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys", NATURE MEDICINE, vol. 11, no. 12, December 2005 (2005-12-01), pages 1295 - 1298, XP002717545, ISSN: 1078-8956 * |
COLIGAN ET AL.: "Current Protocols in Protein Science", 2013, WILEY-INTERSCIENCE |
DARLING ET AL.: "Animal Cells: culture and media", 1994, JOHN WILEY & SONS |
DIAZ ET AL., TRANSPLANT IMMUNOLOGY, vol. 13, no. 4, 2004, pages 313 - 317 |
FRESHNEY: "ED) Culture of Animal Cells: a manual of basic techniques", WILEY-LISS |
GALLI ET AL., XENOTRANSPLANTATION, vol. 17, no. 6, 2010, pages 397 - 410 |
GHADERI, NATURE BIOTECHNOLOGY, vol. 28, no. 8, 2010, pages 863 - 867 |
LI ET AL., CELL REPROGRAM., vol. 12, 2010, pages 599 |
LI ET AL., J. SURG RES, vol. 181, 2013, pages E39 - 45 |
LI ET AL., JOURNAL OF SURGICAL RESEARCH, 3 July 2012 (2012-07-03), pages E1 - E7 |
LI ET AL., JSR, 3 July 2012 (2012-07-03) |
LI PING ET AL: "Biallelic knockout of the alpha-1,3 galactosyltransferase gene in porcine liver-derived cells using zinc finger nucleases", JOURNAL OF SURGICAL RESEARCH, vol. 181, no. 1, 3 July 2012 (2012-07-03), pages E39 - E45, XP002717543 * |
MATOU-KOVD, ANN MED BURN CLUB, vol. 7, 1994, pages 143 |
PARK JONG-YI ET AL: "Alpha 1,3-galactosyltransferase deficiency in pigs increases sialyltransferase activities that potentially raise non-gal xenoantigenicity.", JOURNAL OF BIOMEDICINE & BIOTECHNOLOGY, vol. 2011, 560850, 2011, pages 8PP, XP002717544, ISSN: 1110-7251 * |
Cited By (82)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9322006B2 (en) | 2011-07-22 | 2016-04-26 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US12006520B2 (en) | 2011-07-22 | 2024-06-11 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US9163284B2 (en) | 2013-08-09 | 2015-10-20 | President And Fellows Of Harvard College | Methods for identifying a target site of a Cas9 nuclease |
US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US10227581B2 (en) | 2013-08-22 | 2019-03-12 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US9322037B2 (en) | 2013-09-06 | 2016-04-26 | President And Fellows Of Harvard College | Cas9-FokI fusion proteins and uses thereof |
US9228207B2 (en) | 2013-09-06 | 2016-01-05 | President And Fellows Of Harvard College | Switchable gRNAs comprising aptamers |
US9737604B2 (en) | 2013-09-06 | 2017-08-22 | President And Fellows Of Harvard College | Use of cationic lipids to deliver CAS9 |
US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
US9388430B2 (en) | 2013-09-06 | 2016-07-12 | President And Fellows Of Harvard College | Cas9-recombinase fusion proteins and uses thereof |
US9340800B2 (en) | 2013-09-06 | 2016-05-17 | President And Fellows Of Harvard College | Extended DNA-sensing GRNAS |
US9340799B2 (en) | 2013-09-06 | 2016-05-17 | President And Fellows Of Harvard College | MRNA-sensing switchable gRNAs |
US10190137B2 (en) | 2013-11-07 | 2019-01-29 | Editas Medicine, Inc. | CRISPR-related methods and compositions with governing gRNAS |
US9834791B2 (en) | 2013-11-07 | 2017-12-05 | Editas Medicine, Inc. | CRISPR-related methods and compositions with governing gRNAS |
US10640788B2 (en) | 2013-11-07 | 2020-05-05 | Editas Medicine, Inc. | CRISPR-related methods and compositions with governing gRNAs |
US11390887B2 (en) | 2013-11-07 | 2022-07-19 | Editas Medicine, Inc. | CRISPR-related methods and compositions with governing gRNAS |
US9068179B1 (en) | 2013-12-12 | 2015-06-30 | President And Fellows Of Harvard College | Methods for correcting presenilin point mutations |
US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
EP3613764A1 (en) * | 2014-10-15 | 2020-02-26 | Xenothera | Composition with reduced immunogenicity |
WO2016059161A1 (en) * | 2014-10-15 | 2016-04-21 | Xenothera | Composition with reduced immunogenicity |
US11725044B2 (en) | 2014-10-15 | 2023-08-15 | Xenothera | Method for producing polyclonal antibodies with improved complement-dependent cytotoxicity |
CN107001449A (en) * | 2014-10-15 | 2017-08-01 | 芝诺锡拉公司 | The composition of immunogenicity with reduction |
EP3220925A4 (en) * | 2014-10-22 | 2018-07-18 | Indiana University Research&technology Corporation | Triple transgenic pigs suitable for xenograft |
EP4129308A1 (en) * | 2014-10-22 | 2023-02-08 | Indiana University Research & Technology Corporation | Triple transgenic pigs suitable for xenograft |
WO2016065046A1 (en) | 2014-10-22 | 2016-04-28 | Indiana University Research & Technology Corporation | Triple transgenic pigs suitable for xenograft |
US11234418B2 (en) | 2014-12-10 | 2022-02-01 | Regents Of The University Of Minnesota | Genetically modified cells, tissues, and organs for treating disease |
US9888673B2 (en) | 2014-12-10 | 2018-02-13 | Regents Of The University Of Minnesota | Genetically modified cells, tissues, and organs for treating disease |
US10278372B2 (en) | 2014-12-10 | 2019-05-07 | Regents Of The University Of Minnesota | Genetically modified cells, tissues, and organs for treating disease |
US10993419B2 (en) | 2014-12-10 | 2021-05-04 | Regents Of The University Of Minnesota | Genetically modified cells, tissues, and organs for treating disease |
US11180751B2 (en) | 2015-06-18 | 2021-11-23 | The Broad Institute, Inc. | CRISPR enzymes and systems |
JP2018526015A (en) * | 2015-09-09 | 2018-09-13 | レビビコア, インコーポレイテッド | Multi-transgenic pigs for xenotransplantation |
JP2021101741A (en) * | 2015-09-09 | 2021-07-15 | レビビコア, インコーポレイテッド | Multi-transgenic pig for xenotransplantation |
EP3347458A4 (en) * | 2015-09-09 | 2019-08-07 | Revivicor Inc. | Multi-transgenic pig for xenotransplantation |
JP7050665B2 (en) | 2015-09-09 | 2022-04-08 | レビビコア, インコーポレイテッド | Multi-transgenic pigs for xenotransplantation |
US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US11999947B2 (en) | 2016-08-03 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
US11479752B2 (en) | 2017-10-20 | 2022-10-25 | Indiana University Research And Technology Corporation | Scaffold-free 3D bioprinting of porcine cells |
WO2020041387A1 (en) | 2018-08-20 | 2020-02-27 | The Brigham And Women's Hospital, Inc. | Degradation domain modifications for spatio-temporal control of rna-guided nucleases |
WO2020041384A1 (en) | 2018-08-20 | 2020-02-27 | The Broad Institute, Inc. | 3-phenyl-2-cyano-azetidine derivatives, inhibitors of rna-guided nuclease activity |
WO2020068304A2 (en) | 2018-08-20 | 2020-04-02 | The Broad Institute, Inc. | Inhibitors of rna-guided nuclease target binding and uses thereof |
US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
WO2020236967A1 (en) | 2019-05-20 | 2020-11-26 | The Broad Institute, Inc. | Random crispr-cas deletion mutant |
WO2021041922A1 (en) | 2019-08-30 | 2021-03-04 | The Broad Institute, Inc. | Crispr-associated mu transposase systems |
WO2021050974A1 (en) | 2019-09-12 | 2021-03-18 | The Broad Institute, Inc. | Engineered adeno-associated virus capsids |
US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
WO2022087089A1 (en) | 2020-10-21 | 2022-04-28 | Tissue Testing Technologies Llc | Minimizing immunogenicity of decellularized tissues |
Also Published As
Publication number | Publication date |
---|---|
US20140115728A1 (en) | 2014-04-24 |
US10667500B2 (en) | 2020-06-02 |
US11666039B2 (en) | 2023-06-06 |
US20240196872A1 (en) | 2024-06-20 |
US20210076647A1 (en) | 2021-03-18 |
US9888674B2 (en) | 2018-02-13 |
US20150264900A1 (en) | 2015-09-24 |
US20180153146A1 (en) | 2018-06-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11666039B2 (en) | Double knockout (GT/CMAH-KO) pigs, organs and tissues | |
AU2017285224B2 (en) | Genetically modified cells, tissues, and organs for treating disease | |
EP3220925B1 (en) | Triple transgenic pigs suitable for xenograft | |
JP2021126129A (en) | Genetically modified cells, tissues and organs for treating diseases | |
JP5329502B2 (en) | Pig animals lacking any expression of functional alpha-1,3-galactosyltransferase | |
US20180184630A1 (en) | Transgenic pigs with genetic modifications of sla | |
US20170251646A1 (en) | Transgenic pigs lacking one or more cellular transport genes | |
Joanna et al. | The production of UL16-binding protein 1 targeted pigs using CRISPR technology | |
US9420770B2 (en) | Methods of modulating thrombocytopenia and modified transgenic pigs | |
JPWO2015155904A1 (en) | Gene knockout pig | |
Hossein et al. | Cloning missy: obtaining multiple offspring of a specific canine genotype by somatic cell nuclear transfer | |
JP5771240B2 (en) | Immunodeficient pig | |
LE ANH | Studies on genome editing techniques using CRISPR/Cas9 in porcine embryos | |
WO2023076897A1 (en) | Viable galactosyltransferase knock-out sheep and related methods | |
Ishikawa et al. | Insertion of transgene in sex chromosomes in generating transgenic mice |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13786121 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14436963 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 13786121 Country of ref document: EP Kind code of ref document: A1 |