GENETICALLY MODIFIED PIGS FOR XENOTRANSPLANTATION OF VASCULARIZED XENOGRAFTS AND DERIVATIVES THEREOF
The present invention provides certain donor animals, tissues and cells that are particularly useful for xenotransplantation therapies. In particular, the invention includes porcine animals, as well as tissue and cells derived from these, which lack any expression of functional alpha 1,3 galactosyltransferase (αGT) and express one or more additional transgenes which make these animals suitable donors for xenotransplantation of vascularized xenografts and derivatives thereof. Methods of treatment and using organs, tissues and cells derived from such animals are also provided.
1 . A method of treating an organ dysfunction in a subject in need thereof comprising administering to the subject an organ, a tissue, or a cell from a transgenic animal, wherein the transgenic animal comprises genetic modifications that result in:
(a) lack of any expression of a functional alpha 1,3 galactosyltransferase (GTKO); and
(b) incorporation into the genome and expression of:
a cytoprotective transgene that is ubiquitously expressed or is under the control of a constitutive promoter, wherein the cytoprotective transgene is selected from the group consisting of HO-1, FAT-1, A20, and soluble tumor necrosis factor-alpha (TNF-alpha) receptor;
an immunosuppressant transgene selected from the group consisting of cluster of differentiation 47 (CD47), and Class II transactivator-DN (CIITA-DN); and
one anticoagulant transgene under the control of an endothelial cell-specific promoter, wherein the one anticoagulant transgene is endothelial protein C receptor (EPCR) or thrombomodulin (TBM);
wherein the EPCR or the TBM is expressed in an aorta, a lung, a kidney, a heart, a liver, an intestine, a stomach, or a spleen; and
wherein the transgenic animal is a transgenic ungulate.
2 . The method of claim 1 , wherein at least two transgenes are under the control of:
(a) an endothelial cell-specific promoter; or
(b) ICAM-2 promoter, Tie-2 promoter, or pecam-2 promoter.
3 . The method of claim 1 , wherein the transgenic animal comprises two cytoprotective transgenes, and wherein the cytoprotective transgenes are HO-1 and A20.
4 . The method of claim 1 , wherein the constitutive promoter or an ubiquitous promoter is a CAG promoter, a CMV promoter, a SV40 promoter, a gamma-actin promoter, a GAPDH promoter, a H 2 K promoter, a ubiquitin promoter, or a rosa promoter.
5 . The method of claim 1 , wherein the transgenic animal expresses two anticoagulant transgenes, and wherein the two anticoagulant transgenes are TBM and EPCR.
6 . The method of claim 4 , further comprising one additional transgene, wherein the one additional transgene is a complement inhibitor transgene, and wherein the complement inhibitor transgene is selected from the group consisting of CD46, DAF (CD55), CD59, and CR1.
7 . The method of claim 6 , wherein the constitutive promoter or an ubiquitous promoter is a CAG promoter.
8 . The method of claim 6 , wherein the anticoagulant transgene is thrombomodulin and:
(a) the cytoprotective transgene is HO-1;
(b) the immunosuppressant transgene is CD47; and
(c) the complement inhibitor transgene is CD46 or CD55.
9 . The method of claim 6 , wherein the anticoagulant transgene is EPCR and
(a) the complement inhibitor transgene is CD46 or CD59;
(b) the cytoprotective transgene is HO-1; and
(c) the immunosuppressant transgene is CD47.
10 . The method of claim 6 , wherein the transgenic animal comprises a vector comprising a ubiquitous promoter, a CD46 transgene, a CD55 transgene, and a CD47 transgene nucleic acid.
11 . The method of claim 10 , wherein the ubiquitous promoter is a CAG promoter.
12 . The method of claim 1 , wherein the subject in need thereof has a disease selected from the group consisting of heart disease, atherosclerosis, coronary artery disease, peripheral vascular disease, aortic aneurysm, chronic kidney disease, diabetes, type 1 diabetes, acute macular degeneration, diabetes-induced retinopathy, kidney failure, liver failure, eye disease, pulmonary dysfunction, pancreatic dysfunction, retinal disease, and corneal disease.
13 . The method of claim 1 , wherein the organ dysfunction comprises atherosclerosis, a coronary artery disease, a peripheral vascular disease, or an aortic aneurysm.
14 . The method of claim 1 , wherein:
(a) the organ is selected from the group consisting of heart, lung, liver, pancreas, intestine, spleen, and kidney;
(b) the tissue is selected from the group consisting of a vascular tissue, a retinal tissue, and a corneal tissue;
(c) the cell is selected from the group consisting of endothelial cells, corneal endothelial cells, retinal endothelial cells, and hepatic cell;
(d) the transgenic animal comprises a nucleic acid sequence of SEQ ID NO: 7, and SEQ ID NO: 10, or a complementary sequence thereof; or
(e) the subject is a human.
15 . The method of claim 14 , wherein the vascular tissue is a vascular graft.
16 . The method of claim 1 , wherein administering comprises a bioartificial liver device, an extracorporeal liver perfusion, or a hepatic cell transplantation.
17 . The method of claim 16 , wherein:
(a) the bioartificial liver device is an extracorporeal artificial liver device; or
(b) the bioartificial liver device, the extracorporeal liver perfusion, or the hepatic cell transplantation comprises a liver tissue or a liver cell from the transgenic animal.
18 . The method of claim 1 further comprising administering an immunosuppressant regimen to the subject.
19 . A method of treating an organ dysfunction in a subject in need thereof comprising administering to the subject an organ, a tissue, or a cell from a transgenic porcine animal, wherein the transgenic porcine animal comprises genetic modifications that result in:
(a) lack of expression of functional alpha 1,3 galactosyltransferase; and
(b) incorporation into the genome and expression of
(i) two complement inhibitor transgenes, wherein the two complement inhibitors are CD46 and DAF; CD46 and CD59; or DAF and CD59, and wherein the complement inhibitors are ubiquitously expressed and are under the control of a constitutive promoter; and
(ii) two anticoagulant transgenes under the control of an endothelial cell-specific promoter, wherein the two anticoagulant transgenes are endothelial protein C receptor (EPCR), and thrombomodulin, and wherein the EPCR and the thrombomodulin are expressed in an aorta, a lung, a kidney, a heart, a liver, an intestine, a stomach, or a spleen; and
(iii) a CD47 transgene under the control of a constitutive promoter.
20 . The method of claim 19 , wherein the transgenic animal comprises:
(a) a nucleic acid sequence of SEQ ID NO: 7, SEQ ID NO: 10, or a complementary sequence thereof, or
(b) a vector comprising a ubiquitous promoter, a CD46 transgene, a CD55 transgene, and a CD47 transgene nucleic acids.
21 . A method of treating an organ dysfunction in a subject in need thereof comprising administering to the subject an organ, a tissue, or a cell from a transgenic porcine animal, wherein the transgenic porcine animal comprises genetic modifications that result in:
(a) lack of expression of functional alpha 1,3 galactosyltransferase; and
(b) incorporation into the genome and expression of:
(i) one complement inhibitor transgene, wherein the complement inhibitor is selected from the group consisting of CD46 and DAF, and wherein the complement inhibitor is ubiquitously expressed and is under the control of a constitutive promoter;
(ii) a CD47 transgene under the control of a constitutive promoter; (iii) a HO-1 transgene; and (iv) one anticoagulant transgene under the control of an endothelial cell-specific promoter, wherein the anticoagulant transgene is selected from the group consisting of endothelial protein C receptor (EPCR) and thrombomodulin, and wherein the anticoagulant transgene is expressed in an aorta, a lung, a kidney, a heart, a liver, an intestine, a stomach, or a spleen.
22 . The method of claim 21 , wherein the transgenic animal comprises:
(a) a nucleic acid sequence of SEQ ID NO: 7, SEQ ID NO: 10, or a complementary sequence thereof; or
(b) a vector comprising a ubiquitous promoter, a CD46 transgene, a CD55 transgene, and a CD47 transgene nucleic acids.