DOUBLE KNOCKOUT (GT/CMAH-KO) PIGS, ORGANS AND TISSUES
The invention provides double knockout transgenic pigs (GT/CMAH-KO pigs) lacking expression of any functional αGAL 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.
1 .- 20 . (canceled)
21 . A knockout pig comprising a disrupted α(1,3)-galactosyltransferase gene and a disrupted cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene and having decreased expression of functional α(1,3)-galactosyltransferase and functional CMAH as compared to a wild-type pig,
wherein the decreased expression results in a reduced amount of N-glycolylneuraminic acid (Neu5Gc) epitopes and a reduced amount of αGal epitopes when compared to amounts of the epitopes on cells derived from a wild-type pig.
22 . The knockout pig of claim 21 , wherein cells of the knockout pig lack functional expression of α(1,3)-galactosyltransferase protein and lack functional expression of CMAH protein.
23 . The knockout pig of claim 21 , wherein the knockout pig lacks Neu5Gc epitopes and lacks αGal epitopes on glycoproteins of interest.
24 . The knockout pig of claim 23 , wherein the glycoprotein of interest is selected from the group consisting of an antibody, a growth factor, a cytokine, a hormone, and a clotting factor.
25 . The knockout pig of claim 23 , wherein cells from the knockout pig lack Neu5Gc epitopes and lack αGal epitopes on glycoproteins of interest on their cell surfaces.
26 . The knockout pig of claim 21 , wherein the reduced amount of Neu5Gc epitopes and the reduced amount of αGal epitopes decreases the likelihood of antibody-mediated rejection when an organ or tissue from the knockout pig is transplanted into a human as compared to when the organ or tissue from a wild-type pig is transplanted into a human.
27 . The knockout pig of claim 26 , wherein the reduced amount of Neu5Gc epitopes and the reduced amount of αGal epitopes decreases the likelihood of hyperacute rejection when an organ or tissue from the knockout pig is transplanted into a human as compared to when the organ or tissue from a wild-type pig is transplanted into a human.
28 . The knockout pig of claim 26 , wherein antibodies directed to the Neu5Gc epitopes or to the αGal epitopes are present in the human's blood prior to transplantation of the organ or tissue.
29 . The knockout pig of claim 26 , wherein the tissue is a brain, heart, lung, eye, stomach, pancreas, kidney, liver, uterus, bladder, skin, spleen, tongue, pharynx, esophagus, large intestine, small intestine, rectum, anus, thyroid gland, thymus gland, bone, cartilage, tendon, ligament, suprarenal capsule, skeletal muscle, smooth muscle, blood vessel, blood, spinal cord, trachea, ureter, urethra, hypothalamus, pituitary, pylorus, adrenal gland, ovary, oviduct, vagina, mammary gland, testes, seminal vesicle, penis, lymph, lymph node, or lymph vessel tissue.
30 . The knockout pig of claim 29 , wherein the tissue is kidney, heart, liver, or lung tissue.
31 . The knockout pig of claim 26 , wherein the organ is a heart, lung, eye, stomach, pancreas, kidney, liver, uterus, bladder, skin, spleen, tongue, pharynx, esophagus, large intestine, small intestine, rectum, anus, thyroid gland, thymus gland, bone, cartilage, tendon, ligament, suprarenal capsule, skeletal muscle, smooth muscle, blood vessel, blood, spinal cord, trachea, ureter, urethra, hypothalamus, pituitary, pylorus, adrenal gland, ovary, oviduct, vagina, mammary gland, testes, seminal vesicle, penis, lymph, lymph node, or lymph vessel.
32 . The knockout pig of claim 31 , wherein the organ is a kidney, heart, liver, or lung.
33 . The knockout pig of claim 21 , wherein the disruption of said α(1,3)-galactosyltransferase gene is selected from the group consisting of 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 and wherein expression of functional αGal in said pig is decreased as compared to a wild-type pig.
34 . The knockout pig of claim 21 , wherein the disruption of said CMAH gene is selected from the group consisting of a four base pair insertion, a two base pair deletion, a single 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 CMAH in said pig is decreased as compared to a wild-type pig.
35 . The knockout pig of claim 33 , wherein the disruption of said CMAH gene is selected from the group consisting of a four base pair insertion, a two base pair deletion, a single 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 CMAH in said pig is decreased as compared to a wild-type pig.
36 . An organ or tissue obtained from the knockout pig of claim 21 .
37 . The organ or tissue of claim 36 , wherein the organ is a kidney, heart, liver, or lung.
38 . The organ or tissue of claim 36 , wherein the tissue is a brain, heart, lung, eye, stomach, pancreas, kidney, liver, uterus, bladder, skin, spleen, tongue, pharynx, esophagus, large intestine, small intestine, rectum, anus, thyroid gland, thymus gland, bone, cartilage, tendon, ligament, suprarenal capsule, skeletal muscle, smooth muscle, blood vessel, blood, spinal cord, trachea, ureter, urethra, hypothalamus, pituitary, pylorus, adrenal gland, ovary, oviduct, vagina, mammary gland, testes, seminal vesicle, penis, lymph, lymph node, or lymph vessel tissue.
39 . A cell obtained from or a cell line derived from the knockout pig of claim 21 .
40 . The cell or a cell line of claim 39 , wherein the cell is an epithelial cell, fibroblast cell, neural cell, keratinocyte, hematopoietic cell, melanocyte, chondrocyte, B lymphocyte, T lymphocyte, macrophage, monocyte, mononuclear cell, cardiac muscle cell, other muscle cell, granulosa cell, cumulus cell, epidermal cell, endothelial cell, Islets of Langerhans cell, pancreatic insulin secreting cell, pancreatic alpha-2 cell, pancreatic beta cell, pancreatic alpha-1 cell, blood cell, blood precursor cell, bone cell, bone precursor cell, neuronal stem cell, primordial stem cell, hepatocyte, keratinocyte, umbilical vein endothelial cell, aortic endothelial cell, microvascular endothelial cell, fibroblast, liver stellate cell, aortic smooth muscle cell, cardiac myocyte, neuron, Kupffer cell, smooth muscle cell, Schwann cell, and epithelial cell, erythrocyte, platelet, neutrophil, lymphocyte, monocyte, eosinophil, basophil, adipocyte, chondrocyte, pancreatic islet cell, thyroid cell, parathyroid cell, parotid cell, tumor cell, glial cell, astrocyte, red blood cell, white blood cell, macrophage, epithelial cell, somatic cell, pituitary cell, adrenal cell, hair cell, bladder cell, kidney cell, retinal cell, rod cell, cone cell, heart cell, pacemaker cell, spleen cell, antigen presenting cell, memory cell, T cell, B cell, plasma cell, muscle cell, ovarian cell, uterine cell, prostate cell, vaginal epithelial cell, sperm cell, testicular cell, germ cell, egg cell, Leydig cell, peritubular cell, Sertoli cell, lutein cell, cervical cell, endometrial cell, mammary cell, follicle cell, mucous cell, ciliated cell, nonkeratinized epithelial cell, keratinized epithelial cell, lung cell, goblet cell, columnar epithelial cell, dopaminergic cell, squamous epithelial cell, osteocyte, osteoblast, osteoclast, dopaminergic cell, embryonic stem cell, fibroblast, or fetal fibroblast.