Granted Patent B2

US 12,297,458 · App. 18/046,678 · Granted May 13, 2025

Inventors: Paul W. Holzer (Enfield, NH); Jon Adkins (Londonderry, NH); Rodney L. Monroy (North Fort Myers, FL); Elizabeth J. Chang (Pittsford, NY)

Assignee: XENOTHERAPEUTICS CORPORATION

C12N5/0697A01K67/0278A61K35/12C12N15/102C12N15/113C12Q1/14A01K2207/15A01K2227/108A01K2267/025C12N2310/20C12N2800/80

Abstract

Claims (37)

1. A genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ comprising live cells that vascularize after xenotransplantation, wherein the genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ has been obtained from a non-wild type, biologically engineered porcine comprising a nuclear genome that has been reprogrammed to replace a plurality of nucleotides in a plurality of exon regions of a major histocompatibility complex of a wild-type porcine with a plurality of synthesized nucleotides from a human captured reference sequence, and

wherein cells of said genetically reprogrammed porcine do not present one or more surface glycan epitopes selected from alpha-Gal, Neu5Gc, and SD a ,

wherein genes encoding alpha-1,3 galactosyltransferase, cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and β1,4-N-acetylgalactosaminyltransferase are altered such that the genetically reprogrammed porcine lacks functional expression of surface glycan epitopes encoded by said genes,

wherein the genetically reprogrammed porcine's genome comprises endogenous exon and/or intron regions of the wild-type porcine's Major Histocompatibility Complex corresponding to exon regions of SLA-1, SLA-2, SLA-3, SLA-6, SLA-7, SLA-8, SLA-DRA, SLA-DRB, SLA-DQA, and/or SLA-DQB, and any combination thereof, that have been substituted with nucleotides from orthologous exons of a known human HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DRA, HLA-DRB, HLA-DQA, and/or HLA-DQB sequence of the human captured reference sequence,

wherein intron regions of the wild-type porcine's genome are not reprogrammed, and

wherein the reprogrammed genome comprises at least one of A-D:

A) wherein the reprogrammed porcine nuclear genome comprises site-directed mutagenic substitutions of nucleotides at exon regions of the wild-type porcine's β2-microglobulin with nucleotides from orthologous exons of a known human β2-microglobulin from the human captured reference sequence;

B) wherein the reprogrammed porcine nuclear genome comprises a polynucleotide that encodes a polypeptide that is a humanized beta 2 microglobulin (hB2M) polypeptide sequence that is at least 95% identical to the amino acid sequence of beta 2 microglobulin glycoprotein expressed by the human captured reference genome;

C) wherein the reprogrammed porcine nuclear genome has been reprogrammed such that, at the porcine's endogenous β2-microglobulin locus, the nuclear genome has been reprogrammed to comprise a nucleotide sequence encoding β2-microglobulin polypeptide of the human recipient;

D) wherein the genetically reprogrammed porcine's genome comprises endogenous exon and/or intron regions of the wild-type porcine's B2M, PD-L1, CTLA-4, EPCR, TBM, TFPI, and/or MIC-2, and any combination thereof, that have been substituted with nucleotides from orthologous exons of a known human B2M, PD-L1, CTLA-4, EPCR, TBM, TFPI, and MIC-2 sequence,

wherein the reprogrammed porcine nuclear genome has been reprogrammed such that the genetically reprogrammed porcine lacks functional expression of the wild-type porcine's endogenous β2-microglobulin polypeptides, and

wherein said genetically reprogrammed porcine is free of at least the following pathogens:

(i) Ascaris species, cryptosporidium species, Echinococcus , Strongyloids sterocolis , and Toxoplasma gondii in fecal matter;

(ii) Leptospira species, Mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies, transmissible gastroenteritis virus (TGE)/Porcine Respiratory Coronavirus, and Toxoplasma Gondii by determining antibody titers;

(iii) Porcine Influenza;

(iv) the following bacterial pathogens as determined by bacterial culture: Bordetella bronchisceptica, Coagulase-positive staphylococci, Coagulase-negative staphylococci, Livestock-associated methicillin resistant Staphylococcus aureus (LA MRSA), Microphyton and Trichophyton spp.;

(v) Porcine cytomegalovirus; and

(vi) Brucella suis.

2. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the nucleic acid sequence that is substituting the endogenous exon and/or intron regions of the wild-type porcine gene with orthologous exons of a known human gene is 10 to 350 base pairs in length.

3. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the wild-type porcine genome comprises reprogrammed nucleotides at SLA-MIC-2 gene and at exon regions encoding SLA-3, SLA-6, SLA-7, SLA-8, SLA-DQ, CTLA-4, PD-L1, EPCR, TBM, TFPI, and beta-2-microglobulin using the human capture reference sequence, wherein the human cell, tissue, or organ lacks functional expression of porcine beta-2-microglobulin, SLA-1, SLA-2, and SLA-DR.

4. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 3 , wherein the wild-type porcine genome comprises reprogrammed nucleotides at one or more of a CTLA-4 promoter and a PD-L1 promoter, wherein the one or more of the CTLA-4 promoter and the PD-L1 promoter are reprogrammed to increase expression of one or both of reprogrammed CTLA-4 and reprogrammed PD-L1 compared to the wild-type porcine's endogenous expression of CTLA-4 and PD-L1.

5. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein a total number of the synthesized nucleotides is equal to a total number of the replaced nucleotides, such that there is no net loss or net gain in number of nucleotides after reprogramming the genome of the wild-type porcine with the synthesized nucleotides.

6. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the nuclear genome is reprogrammed using scarless exchange of the exon regions, wherein the nuclear genome is reprogrammed without introduction of any net insertions, deletions, truncations, or other genetic alterations that would cause a disruption of protein expression via frame shift, nonsense, and missense mutations.

7. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein site-directed mutagenic substitutions are made in porcine fetal fibroblast cell, a porcine zygote, a porcine Induced Pluripotent Stem Cells (IPSC), or a porcine germ-line cells.

8. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein site-directed mutagenic substitutions are made in germ-line cells used to produce the non-human animal.

9. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the human captured reference sequence is a human patient capture sequence, a human population-specific human capture sequence, or an allele-group-specific human capture sequence.

10. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the reprogrammed genome comprises site-directed mutagenic substitutions of nucleotides at exon regions of the wild-type porcine's MHC class I chain-related 2 (MIC-2).

11. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the reprogrammed genome lacks functional expression of SLA-1, SLA-2, SLA-DR, or a combination thereof.

12. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the reprogrammed genome comprises site-directed mutagenic substitutions of nucleotides at exon regions of the wild-type porcine's SLA-DQA from an orthologous exon region of a HLA-DQA1 captured reference sequence.

13. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the reprogrammed genome comprises site-directed mutagenic substitutions of nucleotides at exon regions of the wild-type porcine's SLA-DQB from an orthologous exon region of a HLA-DQB1 captured reference sequence.

14. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the reprogrammed genome comprises site-directed mutagenic substitutions of nucleotides at exon regions of the wild-type porcine's SLA-DRA and SLA-DRB1 with nucleotides from orthologous exon regions of HLA-DRA1 and HLA-DRB1 of the human captured reference sequence, or wherein the reprogrammed genome lacks functional expression of SLA-DRA and SLA-DRB1.

15. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 14 , wherein the site-directed mutagenic substitutions of nucleotides are at codons that are not conserved between the wild-type porcine's nuclear genome and the known human sequence.

16. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the reprogrammed genome comprises site-directed mutagenic substitutions of nucleotides at exon regions of the wild-type porcine's SLA-DQA and SLA-DQB1 with nucleotides from orthologous exon regions of HLA-DQA1 and HLA-DQB1 of the human captured reference sequence.

17. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the reprogrammed genome comprises site-directed mutagenic substitutions of nucleotides at exon regions of SLA-3, SLA-6, SLA-7, SLA-8, and MIC-2.

18. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the reprogrammed genome comprises site-directed mutagenic substitutions of nucleotides at exon regions of SLA-DQ and MIC-2.

19. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein the reprogrammed genome comprises site-directed mutagenic substitutions of nucleotides at SLA-3, SLA-6, SLA-7, SLA-8, SLA-DQ, and MIC-2.

20. The genetically reprogrammed, biologically active and metabolically active cell, tissue, and/or an organ of claim 1 , wherein said nuclear genome is reprogrammed to be homozygous at the reprogrammed exon regions and wherein cells of said genetically reprogrammed porcine have extracellular, phenotypic surface expression of polypeptides that are tolerogenic when the cells are transplanted into the human recipient.

Assignments (1)

Continuity (17)

References Cited (400)