Immunologically compatible cells, tissues, organs, and methods for transplantation for silencing, humanization, and personalization with minimized collateral genomic disruptions

A method of producing a porcine donor protein, cell, tissue, or organ for xenotransplantation from a genetically reprogrammed porcine donor comprising a reprogrammed genome, wherein cells of said porcine donor tissue or organ are genetically reprogrammed to be characterized by a recipient-specific surface phenotype comprising:

• a) obtaining a biological sample containing DNA from a prospective human transplant recipient;
• b) performing whole genome sequencing of the biological sample to obtain a human capture reference sequence;
• c) comparing the human capture reference sequence with the wild-type genome of the porcine donor at loci (i)-(v):
• (i) exon regions encoding SLA-3;
• (ii) exon regions encoding SLA-6, SLA-7, and SLA-8;
• (iii) exon regions encoding SLA-DQ;
• (iv) one or more exons encoding Beta-2-Microglobulin (B2M);
• (v) exon regions of SLA-MIC-2 gene, PD-L1, CTLA-4, EPCR, TBM, and TFPI;
• d) creating synthetic nucleotide sequences of 3 to 350 base pairs in length for one or more of said loci (i)-(v), wherein said synthetic nucleotide sequences are orthologous to the human capture reference sequence at loci corresponding to porcine donor loci (i)-(vi);
• e) obtaining a porcine fetal fibroblast cell, a porcine zygote, a porcine mesenchymal stem cell (MSC), or a porcine germline cell;
• f) genetically altering said cell in e) to lack functional alpha-1,3 galactosyltransferase (GalT), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and beta-1,4-N-acetylgalactosaminyltransferase (B4GALNT2);
• g) genetically reprogramming said cell in e) or f) using clustered regularly interspaced short palindromic repeats (CRISPR) or multiplex gene editing to perform site-directed mutagenic substitutions of nucleotides by replacing nucleotide sequences in (i)-(v) with said synthetic nucleotide sequences;
• wherein the reprogrammed porcine donor genome comprises endogenous exon and/or intron regions of the wild-type porcine donors' 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 are disrupted, silenced or otherwise not functionally expressed on (95%) of extracellular surfaces achieved through specific combinations of precise, site-directed mutagenic substitutions or modifications;
• wherein the reprogrammed porcine donor genome comprises endogenous exon and/or intron regions of the wild-type porcine donor's B2M, PD-L1, CTLA-4, EPCR, TBM, TFPI, and/or MIC-2, and any combination thereof, that are reprogrammed through specific combinations of precise, site-directed mutagenic substitutions or modifications with synthetic nucleotides from orthologous exons of a known human B2M, PD-L1, CTLA-4, EPCR, TBM, TFPI, and/or MIC-2 from the human captured reference sequence, designed from the human captured reference sequence and which minimizes collateral genomic disruptions and has 5% or less net gain or net loss of total numbers of nucleotides and avoids genomic organizational disruption and that render the donor animal's cells, tissues, and organs tolerogenic when transplanted into a human without sacrificing the natural immune function of the B2M, PD-L1, CTLA-4, EPCR, TBM, TFPI, and/or MIC-2 proteins;
• wherein the reprogrammed porcine donor genome comprises endogenous exon and/or intron regions of the wild-type porcine donor'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 are reprogrammed through specific combinations of precise, site-directed mutagenic substitutions or modifications with synthetic 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 from the human captured reference sequence, designed from the human captured reference sequence and which minimizes collateral genomic disruptions and ideally results in no net gain or loss of total numbers of nucleotides and avoids genomic organizational disruption and that render the donor animal's cells, tissues, and organs tolerogenic when transplanted into a human without sacrificing the natural immune function of the SLA-1, SLA-2, SLA-3, SLA-6, SLA-7, SLA-8, SLA-DRA, SLA-DRB, SLA-DQA, and/or SLA-DQB proteins;
• h) generating an embryo from the genetically reprogrammed cell in g);
• i) transferring the embryo into a surrogate pig and growing the transferred embryo in the surrogate pig, wherein said surrogate pig gives birth to said genetically reprogrammed porcine donor;
• and
• j) harvesting the porcine donor protein, cell, tissue, or organ from the genetically reprogrammed porcine donor.