Cells, tissues, organs, and animals having one or more modified genes for enhanced xenograft survival and tolerance
The present disclosure relates to cells, tissues, organs, and/or animals having one or more modified genes for enhanced xenograft survival and/or tolerance. In addition, the present disclosure relates to methods of making and using the cells, tissues, organs, and/or animals having one or more of the modified genes.
1 . A genetically modified porcine cell comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises:
(a) a first polycistronic cassette comprising: (i) an endothelial protein C receptor (EPCR) transgene and (ii) a thrombomodulin (THBD) transgene;
(b) a second polycistronic cassette comprising: (i) a tumor necrosis factor a-induced protein 3 (A20) transgene and (ii) a heme oxygenase-1 (HO1) transgene; and
(c) a third polycistronic cassette comprising: (i) a cluster of differentiation 46 (CD46) transgene, (ii) a CD55 transgene, and (iii) a CD47 transgene,
wherein the nucleic acid molecule is integrated at a single genomic locus.
2 . The genetically modified porcine cell of claim 1 , wherein:
(a) the EPCR transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 92, 93, or 181;
(b) the THBD transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 97-102, 166, or 265-266;
(c) the A20 transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 64-65, 104, 182, or 188;
(d) the HO1 transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 63, 109, 165, 183, or 189;
(e) the CD46 transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 71-76, 185, 200, or 253-258;
(f) the CD55 transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 84, 85, 107, or 184; and
(g) the CD47 transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 77-83, 180, or 259.
3 . The genetically modified porcine cell of claim 2 , wherein:
(a) the EPCR transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 92, 93, or 181;
(b) the THBD transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 97-102, 166, or 265-266;
(c) the A20 transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 64-65, 104, 182, or 188;
(d) the HO1 transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 63, 109, 165, 183, or 189;
(e) the CD46 transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 71-76, 185, 200, or 253-258;
(f) the CD55 transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 84, 85, 107, or 184; and
(g) the CD47 transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 77-83, 180, or 259.
4 . The genetically modified porcine cell of claim 3 , wherein:
(a) the EPCR transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 92, 93, or 181;
(b) the THBD transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 97-102, 166, or 265-266;
(c) the A20 transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 64-65, 104, 182, or 188;
(d) the HO1 transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 63, 109, 165, 183, or 189;
(e) the CD46 transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 71-76, 185, 200, or 253-258;
(f) the CD55 transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 84, 85, 107, or 184; and
(g) the CD47 transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 77-83, 180, or 259.
5 . The genetically modified porcine cell of claim 4 , wherein:
(a) the EPCR transgene comprises any one of SEQ ID NOS: 92, 93, or 181;
(b) the THBD transgene comprises any one of SEQ ID NOS: 97-102, 166, or 265-266;
(c) the A20 transgene comprises any one of SEQ ID NOS: 64-65, 104, 182, or 188;
(d) the HO1 transgene comprises any one of SEQ ID NOS: 63, 109, 165, 183, or 189;
(e) the CD46 transgene comprises any one of SEQ ID NOS: 71-76, 185, 200, or 253-258;
(f) the CD55 transgene comprises any one of SEQ ID NOS: 84, 85, 107, or 184; and
(g) the CD47 transgene comprises any one of SEQ ID NOS: 77-83, 180, or 259.
6 . The genetically modified porcine cell of claim 1 , wherein the nucleic acid molecule comprises a sequence having at least 80% identity to SEQ ID NO: 30.
7 . The genetically modified porcine cell of claim 6 , wherein the nucleic acid molecule comprises a sequence having at least 90% identity to SEQ ID NO: 30.
8 . The genetically modified porcine cell of claim 7 , wherein the nucleic acid molecule comprises a sequence having at least 95% identity to SEQ ID NO: 30.
9 . The genetically modified porcine cell of claim 8 , wherein the nucleic acid molecule comprises a sequence having at least 98% identity to SEQ ID NO: 30.
10 . The genetically modified porcine cell of claim 9 , wherein the nucleic acid molecule comprises a sequence having at least 99% identity to SEQ ID NO: 30.
11 . The genetically modified porcine cell of claim 10 , wherein the nucleic acid molecule comprises SEQ ID NO: 30.
12 . A genetically modified porcine cell comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises:
(a) a first polycistronic cassette comprising: (i) a tissue factor pathway inhibitor (TFPI) transgene, (ii) an endothelial protein C receptor (EPCR) transgene, and (iii) a thrombomodulin (THBD) transgene;
(b) a second polycistronic cassette comprising: (i) (A) a beta 2 microglobulin (B2M) transgene and a human leukocyte antigen E (HLA-E) transgene, or (B) a transgene encoding a B2M-HLA-E fusion; and (ii) a cluster of differentiation 47 (CD47) transgene; and
(c) a third polycistronic cassette comprising: (i) a CD46 transgene and (ii) a CD55 transgene,
wherein the nucleic acid molecule is integrated at a single genomic locus.
13 . The genetically modified porcine cell of claim 12 , wherein the second polycistronic cassette comprises the transgene encoding the B2M-HLA-E fusion, and wherein:
(a) the TFPI transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 94-96, 103, or 187;
(b) the EPCR transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 92, 93, or 181;
(c) the THBD transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 97-102, 166, or 265-266;
(d) the transgene encoding the B2M-HLA-E fusion comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 62, 66, or 105;
(e) the CD47 transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOs: SEQ ID NOS: 77-83, 180, or 259;
(f) the CD46 transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 71-76, 185, 200, or 253-258; and
(g) the CD55 transgene comprises a nucleic acid sequence having at least 80% identity to any one of SEQ ID NOS: 84, 85, 107, or 184.
14 . The genetically modified porcine cell of claim 13 , wherein:
(a) the TFPI transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 94-96, 103, or 187;
(b) the EPCR transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 92, 93, or 181;
(c) the THBD transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 97-102, 166, or 265-266;
(d) the transgene encoding the B2M-HLA-E fusion comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 62, 66, or 105;
(e) the CD47 transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: SEQ ID NOs: 77-83, 180, or 259;
(f) the CD46 transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 71-76, 185, 200, or 253-258; and
(g) the CD55 transgene comprises a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOS: 84, 85, 107, or 184.
15 . The genetically modified porcine cell of claim 14 , wherein:
(a) the TFPI transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 94-96, 103, or 187;
(b) the EPCR transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 92, 93, or 181;
(c) the THBD transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 97-102, 166, or 265-266;
(d) the transgene encoding the B2M-HLA-E fusion comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 62, 66, or 105;
(e) the CD47 transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: SEQ ID NOs: 77-83, 180, or 259;
(f) the CD46 transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 71-76, 185, 200, or 253-258; and
(g) the CD55 transgene comprises a nucleic acid sequence having at least 95% identity to any one of SEQ ID NOS: 84, 85, 107, or 184.
16 . The genetically modified porcine cell of claim 15 , wherein:
(a) the TFPI transgene comprises any one of SEQ ID NOS: 94-96, 103, or 187;
(b) the EPCR transgene comprises any one of SEQ ID NOS: 92, 93, or 181;
(c) the THBD transgene comprises any one of SEQ ID NOS: 97-102, 166, or 265-266;
(d) the transgene encoding the B2M-HLA-E fusion comprises any one of SEQ ID NOS: 62, 66, or 105;
(e) the CD47 transgene comprises any one of SEQ ID NOS: SEQ ID NOs: 77-83, 180, or 259;
(f) the CD46 transgene comprises any one of SEQ ID NOS: 71-76, 185, 200, or 253-258; and
(g) the CD55 transgene comprises any one of SEQ ID NOS: 84, 85, 107, or 184.
17 . The genetically modified porcine cell of claim 12 , wherein the nucleic acid molecule comprises a sequence having at least 80% identity to SEQ ID NO: 31.
18 . The genetically modified porcine cell of claim 17 , wherein the nucleic acid molecule comprises a sequence having at least 90% identity to SEQ ID NO: 31.
19 . The genetically modified porcine cell of claim 18 , wherein the nucleic acid molecule comprises a sequence having at least 95% identity to SEQ ID NO: 31.
20 . The genetically modified porcine cell of claim 19 , wherein the nucleic acid molecule comprises a sequence having at least 98% identity to SEQ ID NO: 31.
21 . The genetically modified porcine cell of claim 20 , wherein the nucleic acid molecule comprises a sequence having at least 99% identity to SEQ ID NO: 31.
22 . The genetically modified porcine cell of claim 21 , wherein the nucleic acid molecule comprises SEQ ID NO: 31.
23 . The genetically modified porcine cell of claim 2 or 13 , wherein:
(a) the first polycistronic cassette comprises a first promoter, wherein the first promoter comprises a sequence having at least 80% identity to any one of SEQ ID NOS: 126-145, 167-168, 178-179, 231-238, or 250;
(b) the second polycistronic cassette comprises a second promoter, wherein the second promoter comprises a sequence having at least 80% identity to any one of SEQ ID NOS: 126-145, 167-168, 178-179, 231-238, or 250; and
(c) the third polycistronic cassette comprises a third promoter, wherein the third promoter comprises a sequence having at least 80% identity to any one of SEQ ID NOS: 126-145, 167-168, 178-179, 231-238, or 250.
24 . The genetically modified porcine cell of claim 23 , wherein:
(a) the first promoter comprises any one of SEQ ID NOS: 126-145, 167-168, 178-179, 231-238, or 250;
(b) the second promoter comprises any one of SEQ ID NOS: 126-145, 167-168, 178-179, 231-238, or 250; and
(c) the third promoter comprises any one of SEQ ID NOS: 126-145, 167-168, 178-179, 231-238, or 250.
25 . The genetically modified porcine cell of claim 2 or 13 , wherein:
(a) the first polycistronic cassette comprises a first poly A sequence having at least 80% identity to any one of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192, or 239-240;
(b) the second polycistronic cassette comprises a second poly A sequence having at least 80% identity to any one of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192, or 239-240; and
(c) the third polycistronic cassette comprises a third poly A sequence having at least 80% identity to any one of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192, or 239-240.
26 . The genetically modified porcine cell of claim 25 , wherein:
(a) the first poly A sequence comprises any one of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192, or 239-240;
(b) the second poly A sequence comprises any one of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192, or 239-240; and
(c) the third poly A sequence comprises any one of SEQ ID NOS: 112-125, 154-156, 159-162, 190-192, or 239-240.
27 . The genetically modified porcine cell of claim 2 or 13 , wherein the CD46 transgene or the THBD transgene comprises a splice site mutation.
28 . The genetically modified porcine cell of claim 2 or 13 , wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding a 2A peptide.
29 . The genetically modified porcine cell of claim 28 , wherein the 2A peptide is selected from the group consisting of E2A, T2A, F2A, and P2A.
30 . The genetically modified porcine cell of claim 2 or 13 , wherein the nucleic acid molecule further comprises an IRES sequence.
31 . The genetically modified porcine cell of claim 1 or 12 , wherein the nucleic acid molecule is integrated into a genomic safe harbor locus in a genome of the genetically modified porcine cell.
32 . The genetically modified porcine cell of claim 31 , wherein the genomic safe harbor locus is Adeno-Associated Virus Integration Site 1 (AAVS1), CCR5, or Fos5.
33 . The genetically modified porcine cell of claim 1 or 12 , wherein at least one polycistronic cassette of the nucleic acid molecule is a bicistronic cassette.
34 . The genetically modified porcine cell of claim 1 or 12 , wherein the genetically modified porcine cell lacks expression of an endogenous glycoprotein alpha-1,3-galactosyltransferase (GGTA1) gene, an endogenous β-1,4-N-acetyl-galactosaminyltransferase 2 (β4GALNT2) gene, and an endogenous cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene.
35 . The genetically modified porcine cell of claim 1 or 12 , wherein each transgene is a human transgene.
36 . A genetically modified organ comprising the genetically modified porcine cell of claim 1 or 12 .
37 . The genetically modified organ of claim 36 , wherein the genetically modified organ is a liver, a heart, or a kidney.
38 . A genetically modified pig comprising the genetically modified porcine cell of claim 1 or 12 .
39 . The genetically modified pig of claim 38 , wherein the genetically modified pig is a genetically modified Yucatan pig or a genetically modified Yorkshire pig.