WO2020228043A1 - Cellules, tissus, organes et/ou animaux ayant un ou plusieurs gènes modifiés pour une meilleure survie et/ou tolérance aux xénogreffes - Google Patents

Cellules, tissus, organes et/ou animaux ayant un ou plusieurs gènes modifiés pour une meilleure survie et/ou tolérance aux xénogreffes Download PDF

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WO2020228043A1
WO2020228043A1 PCT/CN2019/087314 CN2019087314W WO2020228043A1 WO 2020228043 A1 WO2020228043 A1 WO 2020228043A1 CN 2019087314 W CN2019087314 W CN 2019087314W WO 2020228043 A1 WO2020228043 A1 WO 2020228043A1
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Prior art keywords
transgenes
tissue
organ
animal
vector
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PCT/CN2019/087314
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English (en)
Inventor
Luhan Yang
Yangbin Gao
Marc Guell
Yinan KAN
Wenning Qin
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Egenesis, Inc.
Hangzhou Qihan Biotechnology Co., Ltd.
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Application filed by Egenesis, Inc., Hangzhou Qihan Biotechnology Co., Ltd. filed Critical Egenesis, Inc.
Priority to PCT/CN2019/087314 priority Critical patent/WO2020228043A1/fr
Priority to MX2021013914A priority patent/MX2021013914A/es
Priority to KR1020217041209A priority patent/KR20220033468A/ko
Priority to AU2020274150A priority patent/AU2020274150A1/en
Priority to JP2021568652A priority patent/JP2022532783A/ja
Priority to US17/611,838 priority patent/US20220267805A1/en
Priority to CN202080050306.1A priority patent/CN115176020A/zh
Priority to BR112021023029A priority patent/BR112021023029A2/pt
Priority to SG11202112675SA priority patent/SG11202112675SA/en
Priority to PCT/CN2020/090440 priority patent/WO2020228810A1/fr
Priority to CA3139928A priority patent/CA3139928A1/fr
Priority to EP20806445.1A priority patent/EP3969596A4/fr
Publication of WO2020228043A1 publication Critical patent/WO2020228043A1/fr
Priority to IL288049A priority patent/IL288049A/en

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Definitions

  • porcine organs trigger rejection by the human immune system in a number of forms, including (i) hyperacute rejection, (ii) acute humoral rejection consisting of disordered thromboregulation and type II endothelial cell (EC) activation with leukocyte recruitment, (iii) thrombotic microangiopathy consisting of intravascular thrombosis with platelet consumption and EC activation, fibrin deposition, and thrombosis due to lack of thromboregulation, and (iv) chronic vasculopathy.
  • hyperacute rejection consisting of disordered thromboregulation and type II endothelial cell (EC) activation with leukocyte recruitment
  • thrombotic microangiopathy consisting of intravascular thrombosis with platelet consumption and EC activation, fibrin deposition, and thrombosis due to lack of thromboregulation
  • chronic vasculopathy including (i) hyperacute rejection, (ii) acute humoral rejection consisting of disordered thromboregulation and type II endotheli
  • porcine cells, tissues, organs, and/or porcine animals having a novel combination of gene modifications for use in xenotransplantation and for developing associated methods.
  • the present disclosure provides cells, tissues, organs, and animals comprising genetic modifications that result in enhanced immunological compatibility, as well as vectors and methods for use in generating these cells, tissues, organs, and animals, and the use of these cells, tissues, organs, and animals in xenotransplantation.
  • the genetic modifications giving rise to enhanced immunological compatibility include one or more complement response genes (interchangeably referred to herein as complement toxicity genes) , coagulation response genes (interchangeably referred to herein as coagulation genes) , inflammatory response genes (interchangeably referred to herein as apoptosis/inflammation genes) , immune response genes (interchangeably referred to herein as cellular toxicity genes) , and/or immunomodulator genes.
  • the present disclosure provides isolated cells, tissues, organs, and animals comprising a plurality of transgenes of at least two types selected from the group consisting of inflammatory response transgenes, immune response transgenes, immunomodulator transgenes, or combinations thereof.
  • the present disclosure provides for an isolated cell, tissue, organ, or animal comprising a plurality of transgenes, wherein the plurality of transgenes comprises at least one inflammatory response transgene, at least one immune response transgene, and at least one immunomodulator transgene.
  • the plurality of transgenes comprises at least three transgenes selected from the group consisting of inflammatory response transgenes, immune response transgenes, immunomodulator transgenes, or combinations thereof.
  • the inflammatory response transgenes are selected from the group consisting of tumor necrosis factor ⁇ -induced protein 3 (A20) , heme oxygenase (HO-1 or HMOX1) , Cluster of Differentiation 47 (CD47) , and combinations thereof.
  • the immune response transgenes are selected from the group consisting of human leukocyte antigen-E (HLA- E) , beta-2 microglobulin (B2M) , and combinations thereof.
  • the immunomodulator transgene is selected from the group consisting of programmed death ligand 1 (PD-L1) , Fas ligand (FasL) , and combinations thereof.
  • the plurality of transgenes further comprises at least one coagulation response transgene.
  • the coagulation response transgenes are selected from the group consisting of Cluster of Differentiation 39 (CD39) , thrombomodulin (THBD, TBM, or TM) , tissue factor pathway inhibitor (TFPI) , and combinations thereof.
  • the plurality of transgenes further comprises at least one complement response transgene.
  • the complement response transgene is selected from the group consisting of human membrane cofactor protein (hCD46 or simply CD46) ; human complement decay accelerating factor (hCD55 or simply CD55) , human MAC-inhibitor factor (hCD59 or simply CD59) , and combinations thereof.
  • human membrane cofactor protein hCD46 or simply CD46
  • human complement decay accelerating factor hCD55 or simply CD55
  • human MAC-inhibitor factor hCD59 or simply CD59
  • the present disclosure provides isolated cells, tissues, organs, and animals comprising one or more transgenes, each independently selected from the group consisting of complement response transgenes (e.g., CD46, CD55, CD59) ; coagulation response transgenes (e.g., CD39, THBD or TBM, TFPI) ; inflammatory response transgenes (e.g., A20, HO-1, CD47) ; immune response transgenes (e.g., HLA-E, B2M) ; and/or immunomodulator transgenes (e.g., PD-L1, FasL) .
  • complement response transgenes e.g., CD46, CD55, CD59
  • coagulation response transgenes e.g., CD39, THBD or TBM, TFPI
  • inflammatory response transgenes e.g., A20, HO-1, CD47
  • immune response transgenes e.g., HLA-E
  • the cells, tissues, organs, or animals may further comprise one or more additional transgenes from other gene categories.
  • the isolated cells, tissues, organs, and animals provided herein comprise one or more complement response transgenes selected from the group consisting of hCD46, hCD55, and hCD59.
  • expression of one or more of the complement response transgenes is driven by a ubiquitous promoter.
  • the isolated cells, tissues, organs, and animals provided herein comprise one or more coagulation response transgenes selected from the group consisting of CD39, THBD, and TFPI.
  • expression of one or more of the coagulation response transgenes is driven by a tissue-specific promoter.
  • the tissue-specific promoter is an endothelial-specific promoter, and in certain of these embodiments, the endothelial-specific promoter is a low expression endothelial-specific promoter.
  • the isolated cells, tissues, organs, and animals provided herein comprise one or more inflammatory response transgenes selected from the group consisting of A20, HO-1, and CD47.
  • expression of one or more of the inflammatory response transgenes is driven by a ubiquitous promoter, a tissue-specific promoter such as an endothelial-specific promoter, or a combination thereof.
  • the isolated cells, tissues, organs, and animals provided herein comprise one or more immune response transgenes selected from the group consisting of HLA-E and B2M.
  • expression of one or more of the immune response transgenes is driven by a ubiquitous promoter.
  • the isolated cells, tissues, organs, and animals provided herein comprise one or more immunomodulator transgenes, including but not limited to PD-L1, FasL, or both.
  • the isolated cells, tissues, organs, and animals provided herein comprise at least 6, 7, 8, 9, 10, 11, or 12 transgenes selected from the group consisting of complement response, coagulation response, inflammatory response, immune genes, and immunomodulator transgenes.
  • the cells, tissues, organs, or animals may comprise at least one transgene from each category. In other embodiments, certain categories of transgenes may be excluded.
  • the complement response, coagulation response, inflammatory response, immune response, and/or immunomodulator transgenes may all be expressed at detectable and/or clinically effective levels simultaneously. In other embodiments, only specific subsets of transgenes may be expressed at clinically effective levels at certain timepoints or in response to certain signals. In these embodiments, expression of one or more of the transgenes may drop below detectable and/or clinically effective levels at certain timepoints.
  • the cells, tissues, organs, and animals provided herein comprise the transgenes CD46, CD55, CD59, HLA-E, B2M, CD47, CD39, THBD, TFPI, A20, PD-L1, and HO-1.
  • the isolated cells, tissues, organs, and animals disclosed herein further comprise one or more modifications to a complement response gene, coagulation response genes, inflammatory response genes, immune response genes, and/or immunomodulator genes.
  • a complement response gene coagulation response genes
  • inflammatory response genes inflammatory response genes
  • immune response genes immune response genes
  • immunomodulator genes include one or more modifications to a complement response gene, coagulation response genes, inflammatory response genes, immune response genes, and/or immunomodulator genes.
  • the cell, tissue, organ, or animal may comprise an alteration of the von Willebrand factor (vWF) gene, including in some instances alterations that result in humanization of the gene.
  • vWF von Willebrand factor
  • the cells, tissues, organs, and animals disclosed herein further comprise one or more modifications to other categories of genes. These modifications may include, for example, deletion or excision of all or part of the gene (i.e., knockout) , or any other inactivation, disruption, or alteration.
  • the cells, tissues, organs, and animals may comprise a knockout, inactivation, or disruption of asialoglycoprotein receptor 1 (ASGR1) .
  • ASGR1 asialoglycoprotein receptor 1
  • the cells, tissues, organs, and animals may be genetically modified to exhibit a reduced carbohydrate antigen response.
  • the cells, tissues, organs, or animals may comprise a knockout, inactivation, or disruption of one or more carbohydrate antigen genes (e.g., glycoprotein ⁇ -galactosyltransferase 1 (GGTA) , ⁇ 1, 4 N-acetylgalactosaminyltransferase 2 (B4GalNT2) , cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) ) .
  • carbohydrate antigen genes e.g., glycoprotein ⁇ -galactosyltransferase 1 (GGTA) , ⁇ 1, 4 N-acetylgalactosaminyltransferase 2 (B4GalNT2) , cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)
  • the isolated cells, tissues, organs, and animals provided herein comprise the transgenes CD46, CD55, CD59, HLA-E, B2M, CD47, CD39, THBD, TFPI, A20, PD-L1, and HO-1, and further comprise a knockout, inactivation, or disruption of GGTA, B4GalNT2, and CMAH.
  • these cells, tissues, organs, and animals exhibit enhanced immunological compatibility comprising reduced carbohydrate antigen response and enhanced coagulation, complement, inflammatory, and/or immune response.
  • the isolated cells, tissues, organs, and animals provided herein are porcine, i.e., a porcine cell, porcine tissue, porcine organ, or a pig or progeny thereof.
  • the cells, tissues, organs, or animals are free of porcine endogenous retroviruses ( “PERV-free” ) .
  • the PERV-free cells, tissues, organs, and animals comprise the transgenes CD46, CD55, CD59, HLA-E, B2M, CD47, CD39, THBD, TFPI, A20, PD-L1, and HO-1, and in certain of these embodiments the cells, tissues, organs, and animals further comprise a knockout, inactivation, or disruption of GGTA, B4GalNT2, and CMAH.
  • the cells or tissues are kidney or liver cells or tissues.
  • the organ is a kidney or a liver.
  • the present disclosure provides vectors comprising a plurality of transgenes of at least two types selected from the group consisting of inflammatory response transgenes, immune response transgenes, immunomodulator transgenes, or combinations thereof.
  • the plurality of transgenes comprises three types selected from the group consisting of inflammatory response transgenes, immune response transgenes, immunomodulator transgenes, or combinations thereof.
  • the present disclosure provides for a vector comprising a plurality of transgenes, wherein the plurality of transgenes comprises at least one inflammatory response transgene, at least one immune response transgene, and at least one immunomodulator transgene.
  • the inflammatory response transgene is selected from the group consisting of A20, HO-1, CD47, and combinations thereof.
  • the immune response transgene is selected from the group consisting of HLA-E, B2M, and combinations thereof.
  • the immunomodulator transgene is selected from the group consisting of PD-L1, FasL, and combinations thereof.
  • the plurality of transgenes further comprises at least one coagulation response transgene.
  • the coagulation response transgene is selected from the group consisting of CD39, THBD, TFPI, and combinations thereof.
  • the plurality of transgenes further comprises at least one complement response transgene.
  • the complement response transgene is selected from the group consisting of CD46, CD55, CD59, and combinations thereof.
  • the present disclosure provides vectors for use in genetically modifying cells, tissues, organs, or animals to produce the cells, tissues, organs, or animals provided herein, including, for example, vectors for inserting (i.e., knocking in) one or more complement response, coagulation response, inflammatory response, immune response, and/or immunomodulator transgenes.
  • the vectors comprise at least 6, 7, 8, 9, 10, 11, or 12 of the transgenes. In some of these embodiments, at least six of the transgenes are expressed from a single locus.
  • CRISPR-based editing components such as guide RNAs (gRNAs) or endonucleases.
  • the vectors provided herein comprise the transgenes CD46, CD55, CD59, HLA-E, B2M, CD47, CD39, THBD, TFPI, A20, PD-L1, and HO-1, and in certain of these embodiments the vectors comprise the sequence set forth in FIGs. 17-20, 31, or 48-50.
  • kits for generating the isolated cells, tissues, organs, and animals provided herein comprise introducing one or more of the vectors provided herein. Accordingly, in certain embodiments, the cells, tissues, organs, and animals provided herein comprise one or more of the vectors disclosed herein.
  • the methods disclosed and described herein comprise single copy polycistronic transgene integration through transposition, mono/bi-allelic site-specific integration through recombinase-mediated cassette exchange (RMCE) , genomic replacement, endogenous gene humanization, or any combination thereof.
  • RMCE recombinase-mediated cassette exchange
  • the methods further comprise knocking out or otherwise disrupting or inactivating one or more PERV genes, for example PERV pol, and in certain of these embodiments the resultant porcine cells, tissues, organs, or animals are PERV-free.
  • the present disclosure provides a transgenic pig liver having reduced liver damage and/or stable coagulation when exposed to non-pig blood, wherein reduced liver damage is assessed by determining the levels of bile production, one or more metabolic enzymes, and/or one or more serum electrolytes, and wherein stable coagulation is assessed by determining the levels of Prothrombin Time (PT) and International Normalized Ratio (PT-NIR) , fibrinogen levels (FIB) , and/or lower activated partial thromboplastin time (APTT) .
  • the metabolic enzymes are selected from the group consisting of alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , and albumin (ALB) .
  • the serum electrolytes are potassium (K) and/or sodium (Na) .
  • the transgenic pig livers disclosed and described herein comprise native metabolic enzymes selected from the group consisting of alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , and albumin (ALB) .
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ALB albumin
  • FIGs. 1A-1C are charts displaying genotyping results of a complement factor 3 knockout ( “C3-KO” ) pig.
  • FIG. 1A shows the sizes of the deletions introduced.
  • FIG. 1B illustrates the position of the indels.
  • FIG. 1C lists sequences of the indels generated.
  • FIG. 2 is a block diagram of a scheme depicting a Major Histocompatibility Complex class I ( “MHC class I” ) replacement strategy where the locus containing the SLA-1, SLA-2, and SLA-3 genes was flanked with loxP sites.
  • MHC class I Major Histocompatibility Complex class I
  • FIGs. 3A and 3B are charts displaying genotyping results of a Major Histocompatibility Complex (MHC) class II knockout ( “MHCII-KO” ) pig genotype, specifically the MHCII gene DQA.
  • FIG. 3A shows the positions and sized and indels having two insertions of 1bp in positions 126 and 127 of the amplicon.
  • FIG. 3B illustrates the position of one of the insertions.
  • MHC Major Histocompatibility Complex
  • FIGs. 4A and 4B are charts displaying genotyping results of another MHC class II-KO pig genotype, specifically the MHCII gene DRA.
  • FIG. 4A shows the positions and sized and indels having two insertions of 1bp in positions 106 and 107 of the amplicon.
  • FIG. 4B illustrates the position of one of the insertions.
  • FIG. 5 includes six charts showing the results of a fluorescence assisted cell sorting (FACS) analysis of an MHCII-KO pig ( “H3-9P01” ) and a wild-type ( “WT” ) pig.
  • FACS fluorescence assisted cell sorting
  • FIG. 6 is a series of images depicting one or more phenotypes associated with the MHCII-KO phenotype.
  • FIG. 7 is a series of block diagrams illustrating a scheme for altering the PD-L1 gene.
  • FIG. 8 is a chart illustrating expression of PD-L1 as measured by qPCR using two amplicons.
  • FIG. 9 is a sequence listing showing alignment of porcine and human vWF protein.
  • the A1 domain is highlighted in the box, whereas the potential glycosylation sites in the flanking region are labeled by dashes.
  • the human specific residues that are deleted in pvWF is labeled with a horizontal line.
  • the A1 and flanking region that were humanized is labeled with the half parenthesis.
  • FIG. 10 depicts a design of a homology-directed repair ( “HDR” ) vector targeting pvWF and two sgRNAs.
  • FIG. 11 shows the screening results for HDR via SphI and BspEI digestion.
  • FIGs. 12A and 12B show sequencing results of a biallelic HDR clone obtained from FIG. 11 where vWF was targeted. The chromatography of both sequencing results is illustrated with one line of overlapping sequences. The humanized A1 and flanking region is labeled with half parenthesis.
  • FIG. 13 is a graph depicting a species-specific platelet aggregation response induced by shear stress and monitored by light transmission for platelets isolated from WT (porcine A1-domain) or HDR targeted (human A1-domain) pigs.
  • FIG. 14 is a schematic of the porcine MHC class I locus. All classical MHCI genes are color coded. Unique flanking regions immediately next to the UTRs of the MHCI genes are labeled as green parenthesis. Four highly active sgRNAs (SEQ ID NOs: 1-4) selected from these regions are also shown.
  • FIG. 15 depicts fragmental deletion of the MHCI classical cluster induced using the sgRNAs in FIG. 14.
  • FIG. 15A shows PCR amplicon across the unique regions of MHCI 5’, 3’ and 5’-3’ deletion junctions in the population of sgRNA transfected cells.
  • FIG. 15B shows that the 5’-3’ junction PCR was TOPO cloned and the sequencing results were aligned to the expected MHCI 5’-3’ junctions generated by MHC5’_sg1 and MHC3’_sg2.
  • FIG. 16 shows enrichment of MHCI negative cells using a porcine specific SLA-1 antibody.
  • FIG. 17 shows a transgene expression vector for expressing multiple transgenes (e.g. humanized transgenes) according to an embodiment disclosed and described herein.
  • Payload 5 (Pig2.1) : 12 transgenes, ubiquitous expression.
  • FIG. 18 shows a transgene expression vector for expressing multiple transgenes (e.g. humanized transgenes) according to an embodiment disclosed and described herein.
  • Payload 9 (Pig2.2) : 12 transgenes, endothelial-specificity.
  • FIG. 19 shows a transgene expression vector for expressing multiple transgenes (e.g. humanized transgenes) according to an embodiment disclosed and described herein.
  • Payload 10 (Pig2.3) : 12 transgenes, endothelial/islet-specificity.
  • FIG. 20 shows a transgene expression vector for expressing multiple transgenes (e.g. humanized transgenes) according to an embodiment disclosed and described herein.
  • Payload 10-Exo (Pig2.4) : 12 transgenes, endothelial-/islet-specificity, with pancreatic exocrine ablation.
  • FIG. 21 is a schematic showing pedigrees of genetically engineered source donor pigs described herein.
  • FIG. 22 demonstrates that genetically engineered pig fibroblasts having enhanced compatibility with human tissues show a significantly reduced binding affinity to human antibodies.
  • FIG. 23 demonstrates tissue-specific mRNA expression from genetically engineered pig primary fibroblasts or endothelial cells described herein.
  • FIG. 23A is a schematic of a transgenic construct assembled using molecular cloning techniques.
  • the CD46, CD55, and CD59 cassette was placed under control of the ubiquitous EF1 ⁇ promoter
  • the HLA-E, B2M, and CD47 cassette was placed under control of the ubiquitous CAG promoter
  • the A20, PD-L1, HO-1 cassette was placed under control of the islet specific NeuroD promoter
  • the THBD, TFPI, and CD39 cassette was placed under control of endothelial specific ICAM2 promoter.
  • the transgenic construct was electroporated into porcine primary fibroblasts (FIG. 23B) or an immortalized porcine aortic endothelial cell line (PEC-A) (FIG. 23C) and mRNA expression determined by qRT-PCR.
  • FIG. 24 depicts transgene protein expression in Pig 2.0 spleen and fibroblast cells.
  • FIG. 25 demonstrates that the genetically engineered pig fibroblasts having enhanced compatibility with human cells exhibited a significantly lower level of complement-mediated cell death.
  • FIG. 26 demonstrates that pig fibroblasts genetically engineered to express human HLA-E exhibit a reduced susceptibility to NK-mediated lysis.
  • FIG. 27 demonstrates that endothelial cells derived from GGTAKO +CD55KI pigs exhibit decreased formation of thrombin-antithrombin III (TAT) complexes.
  • TAT thrombin-antithrombin III
  • FIG. 28 demonstrates that livers isolated from 4-7 pigs and perfused with human blood have increased bile production as compared to wild type (WT) livers.
  • FIG. 29 demonstrates that livers isolated from 4-7 pigs and perfused with human blood have improved liver function as assessed by makers of liver damage and serum electrolyte levels as compared to WT livers.
  • FIG. 30 demonstrates that livers isolated from 4-7 pigs and perfused with human blood have improved coagulation as compared to WT livers.
  • FIG. 31 shows a transgene expression vector according to an embodiment disclosed and described herein.
  • Payload 13 (Pig2.5) : 10 transgenes, bicistronic.
  • FIGs. 32A-B demonstrate that host monkeys transplanted with kidneys isolated from Payload 9 (A) and Payload 10 (B) donor pigs exhibit stable serum creatinine levels.
  • FIGs. 33A-B show hematocrit levels in host monkeys transplanted with kidneys isolated from Payload 9 (A) and Payload 10 (B) donor pigs.
  • FIGs. 34A-B show platelet counts in host monkeys transplanted with kidneys isolated from Payload 9 (A) and Payload 10 (B) donor pigs.
  • FIGs. 35A-B show fluctuations in white blood cell (WBC) counts in host monkeys transplanted with kidneys isolated from Payload 9 (A) and Payload 10 (B) donor pigs
  • FIG. 36 shows RNAseq expression data showing complement and cellular toxicity genes are expressed in samples collected from Payload 9 and Payload 10 pigs.
  • FIG. 37 shows FACS data showing complement and cellular toxicity proteins expressed in samples collected from Payload 5, Payload 9, and Payload 10 pigs.
  • FIGs. 38A-I show clinical labs following pig-to-baboon orthotopic liver xenotransplants (OLTx) .
  • FIGs. 39A-F are representative images of H+E staining liver samples from OLTx.
  • FIGs. 40A-E demonstrate clinical labs following ex vivo xenoperfusion of genetically modified pig livers with human whole blood.
  • FIGs. 41A-H are representative images of H+E staining of xenoperfused pig livers.
  • FIG. 42 demonstrates that pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in ‘untreated’ Pig 2.0 lungs perfused with human blood, relative to GalTKO. hCD55 lungs.
  • PVR pulmonary vascular resistance
  • FIGs. 43A-D demonstrate binding of a panel of human serum to human T cells (A) and B cells (C) , showing that high PRA sera are more likely than low PRA to stain human cells and binding of a panel of human sera to porcine T cells (B) and B cells (D) . Sera from both low PRA patients and high PRA patients show high levels of binding to porcine targets.
  • FIG. 44 shows a panel of high PRA human sera show significantly lower levels of binding to genetically modified porcine aortic endothelial cells (Pig 2.0 pAEC) compared to wild-type cells (WT pAEC) .
  • the Pig 2.0 cells lack ⁇ -1, 3-galactosyl-galactose ( ⁇ Gal) , N-glycolylneuraminic acid (Neu5Gc) , and Sda.
  • FIGs. 45A-C demonstrate staining of Pig 2.0 pAEC with serum taken from kidney (A) , heart (B) , and liver (C) xenotransplant recipient animals at various time points. Serum samples taken post-transplantation show a reduced level of binding, particularly the post liver xenotransplants.
  • FIGs. 46A-C demonstrate binding of human serum to wild-type (WT) and Pig 2.0 pAEC (A) , binding of human serum (B) or cynomolgus serum (C) to pAEC before and after IdeS treatment.
  • IdeS effectively reduces human and cynomolgus IgG binding, while having no impact on the binding of intact IgM.
  • FIG. 47 shows a transgene expression vector according to an embodiment disclosed and described herein.
  • Payload 12F 12 transgenes.
  • FIG. 48 shows a transgene expression vector according to an embodiment disclosed and described herein.
  • Payload 12G 12 transgenes.
  • FIG. 49 shows a transgene expression vector according to an embodiment disclosed and described herein.
  • Payload 13A 10 transgenes.
  • FIG. 50 shows RNAseq results demonstrating expression of complement & cellular toxicity genes.
  • pig , “swine” and “porcine” are used herein interchangeably to refer to anything related to the various breeds of domestic pig, species Sus scrofa.
  • biologically active when used to refer to a fragment or derivative of a protein or polypeptide means that the fragment or derivative retains at least one measurable and/or detectable biological activity of the reference full-length protein or polypeptide.
  • a biologically active fragment or derivative of a CRISPR/Cas9 protein may be capable of binding a gRNA, sometimes also referred to herein as a single guide RNA (sgRNA) , binding a target DNA sequence when complexed with a guide RNA, and/or cleaving one or more DNA strands.
  • gRNA single guide RNA
  • treatment when used in the context of a disease, injury or disorder, are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect, and may also be used to refer to improving, alleviating, and/or decreasing the severity of one or more symptoms of a condition being treated.
  • the effect may be prophylactic in terms of completely or partially delaying the onset or recurrence of a disease, condition, or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for a disease or condition and/or adverse effect attributable to the disease or condition.
  • Treatment covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., arresting its development) ; or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms) .
  • spontaneous is used herein to refer to an event that occurs at the same time as another event, such as within seconds, milliseconds, microseconds, or less when compared to the occurrence of another event.
  • KO knockout
  • KO knocking out
  • KO can also refer to a method of performing, or having performed, a deletion, deactivation or ablation of a gene or portion thereof.
  • KI knockin
  • knocking in is used herein to refer to an addition, replacement, or mutation of nucleotide (s) of a gene in a pig or other animal or any cells in the pig or other animal.
  • KI as used herein, can also refer to a method of performing, or having performed, an addition, replacement, or mutation of nucleotide (s) of a gene or portion thereof.
  • Porcine xenografts are broadly compatible with human organ size and physiology and are ethically acceptable to the US general population.
  • xenotransplanted porcine tissue elicits a complex series of events leading to graft rejection including: hyperacute rejection due to the presence of preformed antibodies to pig antigens, complement activation and hypercoagulability, and heightened innate and adaptive immune responses due to molecular incompatibilities.
  • the present disclosure uses genetic engineering approaches to address current shortcomings of xenotransplantation.
  • Complement-and coagulation-mediated dysfunction arises due to molecular incompatibility between the donor porcine tissue and human physiology and leads to acute xenograft failure.
  • Pre-formed antibodies to ⁇ Gal epitopes initiate hyperacute graft rejection through activation of complement.
  • Genetic inactivation of the glycoprotein ⁇ -galactosyltransferase 1 gene (GGTA1) can reduce this rapid graft destruction. Protection is further improved through over-expression of genes for human complement regulatory proteins (hCRPs) CD46 (membrane cofactor protein) , CD55 (complement decay accelerating factor) , and CD59 (MAC-inhibitory protein) .
  • hCRPs human complement regulatory proteins
  • CMAH cytidine monophosphate-N-acetylneuraminic acid hydrolase
  • Coagulation dysfunction including thrombotic microangiopathy and systemic consumptive coagulopathy, has persisted even with GTKO and overexpression of hCRP due primarily to molecular incompatibilities in the coagulation system between pig and non-human primates (NHP) .
  • transgenic pigs for safe xenotransplantation, these transgenic pigs carried only a limited number of transgenes due to construct capacity constraints and transcription interference between transgenes. These methods proved insufficient to overcome xenograft incompatibility.
  • US Patent Publ. No. 2018/0249688 utilized multi-cistronic expression vectors with different combinations of transgenes. Importantly, these multi-cistronic vectors comprised only 4 transgenes and were used to produce pigs having 6 genetic modifications, including KO of alpha Gal (GTKO) . In the present disclosure, a combination of KO, KI, and genomic replacement strategies are utilized. For the first time, PERV-free pigs have been produced expressing more than 6 transgenes from a single locus.
  • porcine complement factors can be KO'd and that viable pigs can be produced having one or more modified MHC Class I genes, inactivation of MHC Class II genes, KI of PD-L1 to reduce adaptive immunity-based rejections, modified porcine vWF to modulate platelet aggregation, and deletions of porcine MHC Class I genes.
  • porcine cells were genetically modified with more than six transgenes to generate immunologically compatible cells, tissues, organs, pigs, and progeny.
  • CRISPR-Cas9 multiple genes were knocked out, including GGTA1, CMAH, and B4GALNT2, to eliminate the glycans that are recognized by human preformed anti-pig antibodies.
  • twelve human transgenes including CD46, CD55, CD59, CD39, CD47, A20, PD-L1, HLA-E, B2M, THBD, TFPI, HO-1, were integrated into a single multi-transgene cassette in the pig genome.
  • a pig has been produced utilizing CRISPR-mediated non-homologous end joining (NHEJ) to delete the 3 major carbohydrate antigen genes ( “3KO” ; GGTA1, B4GALNT2 and CMAH) coupled with PiggyBAC-mediated random integration of the 12 selected transgenes (12TG: CD46, CD55, CD59, HLA-E, B2M, CD47, CD39, THBD, TFPI, A20, PD-L1, and HO-1) into the porcine genome.
  • NHEJ CRISPR-mediated non-homologous end joining
  • the present disclosure provides cells, tissues, organs, and animals having multiple modified genes, and methods of generating the same.
  • the cells, tissue, organs are obtained from an animal, or is an animal.
  • the animal is a mammal.
  • the mammal is a non-human mammal, for example, equine, primate, porcine, bovine, ovine, caprine, canine, or feline.
  • the mammal is a porcine.
  • Modification of genes in accordance with the present disclosure serves to improve molecular compatibility between the donor and the recipient and to reduce adverse events, including hyperacute rejection, acute humoral rejection, thrombotic microangiopathy, and chronic vasculopathy.
  • hyperacute rejection occurs in a very short time span, typically within minutes to hours after transplantation and results from pre-formed antibodies that activate complement and graft endothelial cells, in turn causing pro-coagulation changes that lead to hemostasis and eventually destruction of the grafted organ.
  • the cells, tissues, organs, and animals generate a reduced hyperacute rejection.
  • the present disclosure provides for one or more cells, tissues, organs, or animals having multiple modified genes.
  • the cell, tissue, organ, or animal has been genetically modified such that multiple genes have been added, deleted, inactivated, disrupted, a portion thereof has been excised, or the gene sequence has been altered.
  • the cell, tissue, organ, or animal has 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes that have been modified.
  • the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes that have been modified are expressed from a single locus.
  • the 5, 10, or 12 genes that have been modified are expressed from a single locus.
  • the 12 genes that have been modified are expressed from a single locus.
  • the cell, tissue, organ, or animal has more than 20, more than 15, more than 10, more than 5, more than 3, or 2 genes that have been modified.
  • the cell, tissue, organ, or animal has more than 10, more than 5, more than 3, more than 2, or more than 1 gene that has been modified.
  • the cell, tissue, organ, or animal has one copy of the modified gene and in other embodiments, the cell, tissue, organ, or animal has more than one copy of the one or more modified genes, such as more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20, more than 25, more than 30, more than 35, more than 40, more than 50, more than 60, more than 70, more than 80, more than 90, or more than 100 copies of the modified gene.
  • the cell has between 100 copies and about 1 copy, 90 copies and about 1 copy, 80 copies and about 1 copy, about 70 copies and about 1 copy, 60 copies and about 1 copy, between about 50 copies and about 1 copy, between about 40 copies and about 1 copy, between about 30 copies and about 1 copy, between about 20 copies and about 5 copies, between about 15 copies and about 10 copies, or between about 5 copies and about 1 copy of one or more modified genes.
  • the present disclosure provides for one or more cells, tissues, organs, or animals having multiple copies of one or more of the modified genes.
  • the cells, tissues, organs, or animals may have 2, 3, 4, 5, 6, 7, 8, 9, about 10, about 15, about 20, about 25, about 30, or more of one or more of the modified genes.
  • the one or more cells is a primary cell. In some embodiments, the one or more cells is a somatic cell. In some embodiments, the one or more cells is a post-natal cell. In some embodiments, the one or more cells is an adult cell (e.g., an adult ear fibroblast) . In some embodiments, the one or more cells is a fetal/embryonic cell (e.g., an embryonic blastomere) . In some embodiments, the one or more cells is a germ line cell. In some embodiments, the one or more cells is an oocyte. In some embodiments, the one or more cells is a stem cell. In some embodiments, the one or more cells is a cell from a primary cell line.
  • the one or more cells is selected from the group consisting of: an epithelial cell, a liver cell, a granulosa cell, a fat cell.
  • the one or more cells is a fibroblast.
  • the fibroblast is a female fetal fibroblast.
  • the one or more cells is in vitro.
  • the one or more cells is in vivo.
  • the one or more cells is a single cell.
  • the one or more cells is a member of a cell colony.
  • the one or more cells is a porcine cell.
  • Non-limiting examples of the breeds a porcine cell originates from or is derived from includes any of the following pig breeds: American Landrace, American Yorkshire, Aksai Black Pied, Angeln saddleback, Appalachian English, Arapawa Island, Auckland Island, Australian Yorkshire, Babi Kampung, Ba Xuyen, Bantu, Basque, Bazna, Beijing Black, else Black Pied, Belgian Landrace, Bengali Brown Shannaj, Bentheim Black Pied, Berkshire, Bisaro, Bangur, Black Slavonian, Black Canarian, Breitovo, British Landrace, British Lop, British Saddleback, Bulgarian White, Cambrough, Cantonese, Celtic, Chato Murciano, Chester White, Chiangmai Blackpig, Choctaw Hog, Creole, Czech Improved White, Danish Landrace, Danish Protest, Dermantsi Pied, Li Yan, Duroc, Dutch Landrace, East Landrace, East Balkan,
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes has been modified by addition, deletion, inactivation, disruption, excision of a portion thereof, or a portion of the gene sequence has been altered.
  • the cells, tissues, organs or animals of the disclosure comprise one or more mutations that inactivate one or more genes.
  • the cells, tissues, organs or animals comprise one or more mutations or epigenetic changes that result in decreased or eliminated expression of one or more genes having the one or more mutations.
  • the one or more genes is inactivated by genetically modifying the nucleic acid (s) present in the cells, tissues, organs or animals.
  • the inactivation of one or more genes is confirmed by means of an assay.
  • the assay is an infectivity assay, reverse transcriptase PCR assay, RNA-seq, real-time PCR, or junction PCR mapping assay.
  • the cells, tissues, organs, and animals are genetically engineered to have enhanced complement (i.e., complement toxicity) , coagulation, inflammatory (i.e., apoptosis/inflammation) , immune (i.e., cellular toxicity) , and/or immunomodulation systems that render them compatible in humans.
  • enhanced complement i.e., complement toxicity
  • coagulation i.e., apoptosis/inflammation
  • immune i.e., cellular toxicity
  • immunomodulation systems that render them compatible in humans.
  • Novel combinations of knockout (KO) , knockin (KI) , and/or genomic replacement strategies provide the enhanced complement, coagulation, inflammatory, immune, and/or immunomodulation systems.
  • Insertion of key complement, coagulation, inflammatory, immune, and/or immunomodulation factors into one or more genomic loci will aid in regulating the human complement system, and natural killer (NK) , macrophage, and T cell function.
  • safe harbor genomic loci such as AAVS1
  • NK natural killer
  • Nonlimiting examples include, overexpression by KI of hCD46, hCD55, and hCD59 to inhibit the human complement cascade; humanization of vWF to prevent unregulated platelet sequestration and thrombotic microangiopathy, for example, by humanizing the A1 domain and/or flanking regions of the porcine vWF sequence; KI of B2M-HLA-E SCT to provide protection against human NK cell cytotoxicity and humanization of porcine cells; and KI of CD47, CD39, THBD, TFPI, A20 to function as immunosuppressants, immunomodulators, and/or anticoagulants.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes has been modified by addition, deletion, inactivation, disruption, excision of a portion thereof, or a portion of the gene sequence has been altered.
  • the present disclosure provides an isolated cell, tissue, organ, or animal having multiple modified genes.
  • the modified genes include one or more of alpha 1, 3, galactosyltransferase (GGTA) , Beta-1, 4-N-Acetyl-Galactosaminyltransferase 2 (B4GalNT2) , cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) , THBD, TFPI, CD39, HO-1, CD46, CD55, CD59, major histocompatibility complex, class I, E single chain trimer (HLA-E SCT) , A20, PD-L1, CD47, swine leukocyte antigen 1 (SLA-1) , SLA-2, SLA-3, vWF, B2M, DQA, DRA, and CD47.
  • GGTA galactosyltransferase
  • Beta-1 4-N-Acetyl-Galactosaminyltransferase 2
  • CMAH cytidine monophosphate
  • the modified genes are GGTA, B4GalNT2, CMAH, or any combination thereof. In some embodiments, the GGTA, B4GalNT2, and/or CMAH are genetically KO. In some embodiments, the modified genes are THBD, TFPI, CD39, HO-1, or any combination thereof. In some embodiments, the THBD, TFPI, CD39, and/or HO-1 are genetically KI. In some embodiments, the modified genes are CD46, CD55, CD59, B2M-HLA-E SCT, A20, PD-L1, CD47, or any combination thereof.
  • the CD46, CD55, CD59, B2M-HLA-E SCT, A20, PD-L1, and/or CD47 are genetically KI.
  • the modified genes are SLA-1, SLA-2, SLA-3, B2M, or any combination thereof.
  • the modified genes are DQA and/or DRA.
  • the modified genes are PD-L1, exogenous vWF, HLA-E, HLA-G, B2M, CIITA-DN, and or any combination thereof.
  • the modified genes are TBM, PD-L1, HLA-E, CD47, or any combination thereof.
  • the TBM, PD-L1, HLA-E, and/or CD47 are genetically KI.
  • the modified genes are MHC-I genes SLA-1, SLA-2, and SLA-3, MHC-II genes DQA and DRA, endogenous vWF, CD9, asialoglycoprotein receptor, at least one complement inhibitor gene (e.g., C3, CD46, CD55, and CD59) , and any combination thereof.
  • the CD46, CD55 and/or CD59 are genetically KI.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified with a transgene expression vector comprising B2M, HLA-E, CD47, SCT, THBD, TFPI, CD39, A20, PD-L1, FasL, CD46, CD55, CD59, or any combination thereof.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified with a transgene expression vector comprising each of B2M, HLA-E, CD47, SCT, THBD, TFPI, CD39, A20, PD-L1, FasL, CD46, CD55, and CD59.
  • a transgene expression vector is depicted in FIG. 17.
  • the cells, tissues, organs or animals of the present disclosure have been further genetically modified to have reduced or no expression of GGTA, B4GalNT2, CMAH, or any combination thereof, for example by genetic KO.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified with a transgene expression vector comprising B2M, HLA-E, SCT, CD47, THBD, TFPI, CD39, A20, PD-L1, HO-1, CD46, CD55, CD59, or any combination thereof.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified with a transgene expression vector comprising each of B2M, HLA-E, SCT, CD47, THBD, TFPI, CD39, A20, PD-L1, HO-1, CD46, CD55, and CD59.
  • a transgene expression vector is depicted in FIG. 18.
  • the cells, tissues, organs or animals of the present disclosure have been further genetically modified to have reduced or no expression of GGTA, B4GalNT2, CMAH, or any combination thereof, for example by genetic KO.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified with a transgene expression vector comprising B2M, HLA-E, SCT, CD47, PD-L1, HO-1, THBD, TFPI, CD39, A20, CD46, CD55, CD59, or any combination thereof.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified with a transgene expression vector comprising each of B2M, HLA-E, SCT, CD47, PD-L1, HO-1, THBD, TFPI, CD39, A20, CD46, CD55, and CD59.
  • a transgene expression vector is depicted in FIG. 19.
  • the cells, tissues, organs or animals of the present disclosure have been further genetically modified to have reduced or no expression of GGTA, B4GalNT2, CMAH, or any combination thereof, for example by genetic KO.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified with a transgene expression vector comprising CD46, CD55, CD59, A20, THBD, TFPI, CD39, HO-1, 2xFKBP (fusion of s FK506 binding protein) , hCaspase8, PD-L1, B2M, HLA-E, SCT, CD47, or any combination thereof.
  • a transgene expression vector comprising CD46, CD55, CD59, A20, THBD, TFPI, CD39, HO-1, 2xFKBP (fusion of s FK506 binding protein) , hCaspase8, PD-L1, B2M, HLA-E, SCT, CD47, or any combination thereof.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified with a transgene expression vector comprising each of CD46, CD55, CD59, A20, THBD, TFPI, CD39, HO-1, 2xFKBP, hCaspase8, PD-L1, B2M, HLA-E, SCT, and CD47.
  • a transgene expression vector is depicted in FIG. 20.
  • the cells, tissues, organs or animals of the present disclosure have been further genetically modified to have reduced or no expression of GGTA, B4GalNT2, CMAH, or any combination thereof, for example by genetic KO.
  • the cells, tissues, organs or animals of the present disclosure can be genetically modified by any method.
  • suitable methods for the knockout (KO) , knockin (KI) , and/or genomic replacement strategies disclosed and described herein include CRISPR-mediated genetic modification using Cas9, Cas12a (Cpf1) , or other CRISPR endonucleases, Argonaute endonucleases, transcription activator-like (TAL) effector and nucleases (TALEN) , zinc finger nucleases (ZFN) , expression vectors, transposon systems (e.g., PiggyBac transposase) , or any combination thereof.
  • the cells, tissues, organs or animals of the present disclosure can be further modified to have PERV copies functionally deleted from their genome.
  • PERVs represent a risk factor if porcine cells, tissues, or organs were to be transplanted into human recipients.
  • PERVs are released from normal pig cells and are infectious.
  • PERV-A and PERV-B are polytropic viruses infecting cells of several species, among them humans; whereas PERV-C is an ecotropic virus infecting only pig cells.
  • Non-limiting methods for functionally deleted PERV copies are disclosed and described in Niu 2017 and WIPO Publ. No. WO2018/195402, both of which are incorporated by reference herein in their entireties.
  • the pigs are genetically engineered to be PERV-A, PERV-B, or PERV-C (or any combination thereof) free.
  • additional genes of cells, tissues, organs, or animals of the present disclosure are modified by addition, deletion, inactivation, disruption, or excision or alteration of a portion thereof.
  • the modified genes include deletions of MHC-I genes SLA-1, SLA-2, or SLA-3, MHC-II genes DQA or DRA, endogenous vWF, CD9, asialoglycoprotein receptor, or C3, and/or addition of one or more of the following transgenes: PD-L1, exogenous vWF, HLA-E, HLA-G, B2M, and CIITA-DN.
  • the modified genes include deleting one or more of GGTA, B4GalNT2, and CMAH, and expressing one or more of the following transgenes: CD46, CD55, CD59, CD47, HO-1, A20, TNFR1-Ig, CD39, THBD, TFPI, endothelial protein C receptor (EPCR) , PD-1, CTLA-Ig, CD73, SOD3, CXCL12, FasL, CXCR3, CD39L1, GLP-1R, M3R, IL35, IL12A and EB13.
  • EPCR endothelial protein C receptor
  • the modified genes are CD46, CD55, CD59, CD47, HO-1, A20, TNFR1-Ig, CD39, THBD, TFPI, EPCR, PD-1, CTLA-Ig, CD73, SOD3, CXCL12, FasL, CXCR3, CD39L1, GLP-1R, M3R, IL35, IL12A and EB13.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes has been modified by addition, deletion, inactivation, disruption, excision of a portion thereof, a portion of the gene sequence has been altered, or introducing a transgene or a portion thereof.
  • the present disclosure provides an isolated cell, tissue, organ, or animal has one or more modified genes.
  • the modified genes are MHC Class I genes.
  • the modified MHC Class I genes include one or more of the following SLA-1, SLA-2, SLA-3, and B2M.
  • the modified genes are SLA-1, SLA-2, and/or SLA-3.
  • the modified gene is B2M.
  • the modified MHC Class I genes include one or more of the following SLA-1, SLA-2, SLA-3, and B2M.
  • the modified B2M, SLA-1, SLA-2, and/or SLA-3 genes, and/or a portion thereof are replaced with a human HLA-E gene, a human HLA-G gene, a human B2M gene, and/or a human (dominant-negative mutant class II transactivator) CIITA-DN gene, and/or a portion thereof.
  • the modified genes are conditionally and/or inducibly modified.
  • a conditional promoter and/or an inducible promoter is used to conditionally and/or inducibly modify the one or more modified genes.
  • the isolated cell, tissue, organ, or animal comprises conditionally altering B2M, SLA-1, SLA-2, or SLA-3 genes, or a combination thereof, and replacing the conditionally altered genes with at least a portion of a human HLA-E gene, a human HLA-G gene, a human B2M gene, and/or a human CIITA-DN gene.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes has been modified by addition, deletion, inactivation, disruption, excision of a portion thereof, a portion of the gene sequence has been altered, or introducing a transgene or a portion thereof.
  • the present disclosure provides an isolated cell, tissue, organ, or animals has one or more modified genes.
  • the modified genes are MHC Class II genes.
  • the modified MHC Class II genes are DRQ, DRA, or a combination thereof.
  • DRQ and/or DRA is modified by addition, deletion, inactivation, disruption, excision of a portion thereof, a portion of the gene sequence has been altered.
  • the modified genes are conditionally and/or inducibly modified.
  • a conditional promoter and/or an inducible promoter is used to conditionally and/or inducibly modify the one or more modified genes.
  • the isolated cell, tissue, organ, or animal comprises conditionally altering DRQ and/or DRA genes, or a combination thereof.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes has been modified by addition, deletion, inactivation, disruption, excision of a portion thereof, a portion of the gene sequence has been altered, or introducing a transgene or a portion thereof.
  • the present disclosure provides an isolated cell, tissue, organ, or animal having a modified vWF gene.
  • the modified genes are vWF genes and vWF-related genes.
  • the modified vWF gene, and/or a portion thereof is replaced with a human vWF gene and/or a portion thereof.
  • the modified vWF gene, modified vWF-related genes, and/or a portion (s) thereof is replaced with a human vWF gene, one or more human vWF-related genes, and/or a portion thereof.
  • the modified vWF gene and/or vWF-related genes are conditionally and/or inducibly modified.
  • a conditional promoter and/or an inducible promoter is used to conditionally and/or inducibly modify the one or more modified genes.
  • the isolated cell, tissue, organ, or animal comprises conditionally altering vWF, vWF-related genes, a portion (s) thereof, or a combination thereof, and replacing the conditionally altered genes with the human vWF gene, at least a portion of the human vWF gene, one or more other human vWF-related genes, at least a portion of one or more human vWF-related genes, or a combination thereof.
  • the vWF gene is modified using gRNAs designed to initiate the HDR replacement in the endogenous porcine genome and cut near the region to be replaced by the human sequences.
  • suitable gRNAs are any one or more of SEQ ID NOs: 5-157.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes has been modified by addition, deletion, inactivation, disruption, excision of a portion thereof, a portion of the gene sequence has been altered, or introducing a transgene or a portion thereof.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified by introduction of one or more exogenous genes, or portions thereof, into the cells, tissues, organs, or animals, such as a transgene.
  • the present disclosure provides an isolated cell, tissue, organ, or animal having one or more modified genes.
  • the modified genes are programmed death genes.
  • the modified gene is PD-L1.
  • the cells, tissues, organs, or animals are modified to express an exogenous PD-L1 gene, or portion thereof, such as a transgene.
  • the modified genes are conditionally and/or inducibly modified.
  • a conditional promoter and/or an inducible promoter is used to conditionally and/or inducibly modify the one or more modified genes.
  • the isolated cell, tissue, organ, or animal comprises conditionally altering PD-L1.
  • the PD-L1 comprises the sequence described in SEQ ID NO: 211 or any variant or portion thereof.
  • the cells, tissues, organs or animals of the present disclosure have been genetically modified such that one or more genes has been modified by addition, deletion, inactivation, disruption, excision of a portion thereof, a portion of the gene sequence has been altered, or introducing a transgene or a portion thereof.
  • the present disclosure provides an isolated cell, tissue, organ, or animal has one or more modified genes.
  • the modified genes are complement genes.
  • the modified gene is C3.
  • C3 is modified by addition, deletion, inactivation, disruption, excision of a portion thereof, a portion of the gene sequence has been altered.
  • the modified C3 gene and/or complement-related genes are conditionally and/or inducibly modified.
  • a conditional promoter and/or an inducible promoter is used to conditionally and/or inducibly modify the one or more modified genes.
  • the isolated cell, tissue, organ, or animal comprises conditionally altering C3, complement-related genes, a portion (s) thereof, or a combination thereof.
  • the C3 gene is modified using gRNAs.
  • suitable gRNAs include any one or more of SEQ ID NOs: 158-210.
  • the modified gene is a knockout of C3. In some embodiments, the modified gene is a knock-in of PD-L1. In some embodiments, the modified gene is a humanized vWF of the porcine vWF. In some embodiments, the modified gene is a conditional knock-in of MHC-I genes SLA-1, SLA-2, and SLA-3.
  • no or substantially no immune response is elicited by the host against the genetically modified cell, tissue or organ.
  • the disclosure provides for nucleic acids obtained from any of the cells disclosed herein.
  • the nucleic acid (s) in the cell are genetically modified such that one or more genes in the cell are altered or the genome of the cell is otherwise modified.
  • the genetic modification system is a TALEN, a zinc finger nuclease, and/or a CRISPR-based system.
  • the genetic modification system is a CRISPR-Cas9 system.
  • the cell is genetically modified such that one or more genes or portions thereof in the cell are inactivated, and the cell is further genetically modified such that the cell has reduced expression of one or more genes, or portions thereof, that would induce an immune response if the cell (or a tissue or organ cloned/derived from the cell) were transplanted to a human.
  • the cell is genetically modified to have increased expression of one or more human genes, or portions thereof.
  • the cell is genetically modified to have increased expression of one or more humanized genes, or portions thereof.
  • the cell is genetically modified such that one or more genes, or portions thereof, in the cell are inactivated, and the cell is further genetically modified such that the cell has increased expression of one or more genes that would suppress an immune response if the cell (or a tissue or organ cloned/derived from the cell) were transplanted to a human.
  • the cell is genetically modified such that one or more genes, or portions thereof, in the cell are inactivated, and the cell is further genetically modified such that the cell has reduced expression of one or more genes that would induce an immune response if the cell (or a tissue or organ cloned/derived from the cell) were transplanted to a human, and the cell is further genetically modified such that the cell has increased expression of one or more genes that would suppress an immune response if the cell (or a tissue or organ cloned/derived from the cell) were transplanted to a human.
  • the disclosure provides for an embryo that was cloned from the genetically modified cell.
  • the genetically modified nucleic acid (s) are extracted from the genetically modified cell and cloned into a different cell.
  • the genetically modified nucleic acid from the genetically modified cell is introduced into an enucleated oocyte.
  • oocytes can be enucleated by partial zona dissection near the polar body and then pressing out cytoplasm at the dissection area.
  • an injection pipette with a sharp beveled tip is used to inject the genetically modified cell into an enucleated oocyte arrested at meiosis 2.
  • Oocytes arrested at meiosis-2 are frequently termed “eggs. ”
  • an embryo is generated by fusing and activating the oocyte. Such an embryo may be referred to herein as a “genetically modified embryo. ”
  • the genetically modified embryo is transferred to the oviducts of a recipient female pig.
  • the genetically modified embryo is transferred to the oviducts of a recipient female pig 20 to 24 hours after activation. See, e.g., Cibelli 1998 and U.S. Patent No. 6,548,741.
  • recipient females are checked for pregnancy approximately 20-21 days after transfer of the genetically modified embryo.
  • the genetically modified embryo is grown into a post-natal genetically modified animal.
  • the post-natal genetically modified animal is a neo-natal genetically modified animal.
  • the genetically modified pig is a juvenile genetically modified animal.
  • the genetically modified animal is an adult genetically modified animal (e.g., older than 5-6 months) .
  • the genetically modified animal is a female genetically modified animal.
  • the animal is a male genetically modified animal.
  • the genetically modified animal is bred with a non-genetically modified animal.
  • the genetically modified animal is bred with another genetically modified animal.
  • the genetically modified pig is bred with another genetically modified animal that has reduced or no active virus. In some embodiments, the genetically modified animal is bred with a second genetically modified animal that has been genetically modified such that the cells, tissues or organs from the second genetically modified animal are less likely to induce an immune response if transplanted to a human.
  • the genetically modified animal is an animal having one or more modified genes and maintains a same or similar level of expression or inactivation of the modified gene (s) for at least a month, at least 6 months, at least 1 year, at least 5 years, at least 10 years post-gestation. In some embodiments, the genetically modified animal remains genetically modified having one or more modified genes as a genetically modified pig even after delivery from a non-viral-inactivated surrogate or after being in a facility/space with other non-viral-inactivated animals.
  • the disclosure provides for cells, tissues, or organs obtained from any of the post-natal genetically modified pigs described herein.
  • the cell, tissue, or organ is selected from the group consisting of liver, kidney, lung, heart, pancreas, muscle, blood, and bone.
  • the organ is liver, kidney, lung or heart.
  • the cell from the post-natal genetically modified pig is selected from the group consisting of: pancreatic islets, lung epithelial cells, cardiac muscle cells, skeletal muscle cells, smooth muscle cells, hepatocytes, non-parenchymal liver cells, gall bladder epithelial cells, gall bladder endothelial cells, bile duct epithelial cells, bile duct endothelial cells, hepatic vessel epithelial cells, hepatic vessel endothelial cells, sinusoid cells, choroid plexus cells, fibroblasts, Sertoli cells, neuronal cells, stem cells, and adrenal chromaffin cells.
  • pancreatic islets lung epithelial cells, cardiac muscle cells, skeletal muscle cells, smooth muscle cells, hepatocytes, non-parenchymal liver cells, gall bladder epithelial cells, gall bladder endothelial cells, bile duct epithelial cells, bile duct endothelial cells, hepatic vessel epit
  • the genetically modified organs, tissues or cells have been separated from their natural environment (i.e., separated from the pig in which they are being grown) .
  • separation from the natural environment means a gross physical separation from the natural environment, e.g., removal from the genetically modified donor animal, and alteration of the genetically modified organs', tissues' or cells' relationship with the neighboring cells with which they are in direct contact (e.g., by dissociation) .
  • the disclosure provides for methods of generating any of the cells, tissues, organs, or animals having one or more modified genes disclosed herein.
  • the disclosure provides a method of inactivating, deleting, or otherwise disrupting one or more genes, or portions thereof, in any of the cells disclosed herein, comprising administering to the cell a gene editing agent specific to a gene, wherein the agent disrupts transcription and/or translation of the gene.
  • the agent targets the start codon of the gene and inhibits transcription of the gene.
  • the agent targets an exon in the gene and the agent induces a frameshift mutation in the gene.
  • the agent introduces an inactivating mutation into the gene.
  • the agent represses transcription of the gene.
  • the disclosure provides a method of altering one or more genes, or a portion thereof, in vivo, comprising administering to the cell a gene editing agent specific to a gene, wherein the agent alters a sequence of the gene, such as by humanizing the gene or otherwise changing a native (e.g., wild-type) sequence of the gene.
  • the disclosure provides a method of expressing one or more genes, or a portion thereof, such as a transgene (e.g., non-native gene) comprising administering to the cell a gene editing agent specific to the transgene gene, wherein the agent introduces a sequence of the transgene.
  • the agent is a nucleic acid sequence, such as a plasmid, a vector, or the like.
  • the nucleic acid sequence includes one or more nucleic acid sequences, such as a promoter, a transgene, and/or additional genes.
  • the nucleic acid sequence, or a portion thereof is derived from one or more species and/or one or more sources.
  • the species is a species that will receive the genetically modified cell, tissue, or organ. In some embodiments, the species is a human. In other embodiments, the species is non-human, such as a mammal, an animal, a bacteria, and/or a virus.
  • any of the agents disclosed herein is a polynucleotide.
  • the polynucleotide encodes one or more of the nucleases and/or nickases and/or RNA or DNA molecules described herein.
  • the polynucleotide agent is introduced to one or more cells.
  • the polynucleotide is introduced to the one or more cells in a manner such that the polynucleotide is transiently expressed by the one or more cells.
  • the polynucleotide is introduced to the one or more cells in a manner such that the polynucleotide is stably expressed by the one or more cells.
  • the polynucleotide is introduced in a manner such that it is stably incorporated in the cell genome. In some embodiments, the polynucleotide is introduced along with one or more transposonal elements. In some embodiments, the transposonal element is a polynucleotide sequence encoding a transposase. In some embodiments, the transposonal element is a polynucleotide sequence encoding a PiggyBac transposase. In some embodiments, the transposable element is inducible. In some embodiments, the transposable element is doxycycline-inducible. In some embodiments, the polynucleotide further comprises a selectable marker. In some embodiments, the selectable marker is a puromycin-resistant marker. In some embodiments, the selectable marker is a fluorescent protein (e.g., GFP) .
  • GFP fluorescent protein
  • the agent is a nuclease or a nickase that is used to target DNA in the cell. In some embodiments, the agent specifically targets and suppresses expression of a gene. In some embodiments, the agent comprises a transcription repressor domain. In some embodiments, the transcription repressor domain is a Krüppel associated box (KRAB) .
  • KRAB Krüppel associated box
  • the agent is any programmable nuclease. In some embodiments, the agent is a natural homing meganuclease. In some embodiments, the agent is a TALEN-based agent, a ZFN-based agent, or a CRISPR-based agent, or any biologically active fragment, fusion, derivative or combination thereof. In some embodiments, the agent is a deaminase or a nucleic acid encoding a deaminase. In some embodiments, a cell is genetically engineered to stably and/or transiently express a TALEN-based agent, a ZFN-based agent, and/or a CRISPR-based agent.
  • any of the genetically modified cells, tissues or organs disclosed herein may be used to treat a subject of a different species as the genetically modified cells.
  • the disclosure provides for methods of transplanting any of the genetically modified cells, tissues or organs described herein into a subject in need thereof.
  • the subject is a human.
  • the subject is a non-human primate.
  • a genetically modified organ for use in any of the methods disclosed herein may be selected from the heart, lung, , liver, , eye, pituitary, thyroid, parathyroid, esophagus, thymus, adrenal glands, appendix, bladder, gallbladder, small intestine, large intestine, small intestine, kidney, pancreas, spleen, stomach, skin, and/or prostate, of the genetically modified pig.
  • a genetically modified tissue for use in any of the methods disclosed herein may be selected from cartilage (e.g., esophageal cartilage, cartilage of the knee, cartilage of the ear, cartilage of the nose) , muscle such as, but not limited to, smooth and cardiac (e.g., heart valves) , tendons, ligaments, bone (e.g., bone marrow) , cornea, middle ear and veins of the genetically modified pig.
  • a genetically modified cell for use in any of the methods disclosed herein includes blood cells, skin follicles, hair follicles, and/or stem cells. Any portion of an organ or tissue (e.g., a portion of the eye such as the cornea) may also be administered the compositions of the present disclosure.
  • a heart, lung, liver, kidney, pancreas, or spleen is isolated from a pig that has been genetically modified to comprise (a) deletions of GGTA1, CMAH, and B4GALNT2; (b) CD46, CD55, CD59, CD39, CD47, A20, PD-L1, HLA-E, B2M, THBD, TFPI, and HO transgenes expressed from a single multi-transgene cassette in the pig genome; and (c) functional deletion of all PERV copies.
  • the pig has been further genetically modified to have humanized vWF, deletion of ASGR1, and/or deletion of B2M genes.
  • the xenotransplanted organ e.g., heart, lung, liver, kidney, pancreas, spleen
  • the disclosure provides for treating a subject having a disease, disorder or injury that results in a damaged, deficient or absent organ, tissue or cell function.
  • the subject has suffered from an injury or trauma (e.g., an automobile accident) resulting in the damage of one or more cells, tissues or organs of the subject.
  • the subject has suffered a fire or acid burn.
  • the subject has a disease or disorder that results in a damaged, deficient or absent organ, tissue or cell function.
  • the subject is suffering from an autoimmune disease.
  • the disease is selected from the group consisting of: heart disease (e.g., atherosclerosis) , dilated cardiomyopathy, severe coronary artery disease, scarred heart tissue, birth defects of the heart, diabetes Type I or Type II, hepatitis, cystic fibrosis, cirrhosis, kidney failure, lupus, scleroderma, IgA nephropathy, polycystic kidney disease, myocardial infarction, emphysema, chronic bronchitis, bronchiolitis obliterans, pulmonary hypertension, congenital diaphragmatic hernia, congenital surfactant protein B deficiency, and congenital cystic emphysematous lung disease, primary biliary cholangitis, sclerosing cholangitis, biliary atresia, alcoholism, Wilson’s disease, hemochromatosis, and/or alpha-1 antitryps
  • heart disease
  • any of the genetically modified cells, tissues and/or organs of the disclosure are separated from the genetically modified donor and administered into a non-donor subject host.
  • “Administering” or “administration” includes, but is not limited to, introducing, applying, injecting, implanting, grafting, suturing, and transplanting.
  • the genetically modified cells, tissues and/or organs may be administered by a method or route which results in localization of the organs, tissues, cells or compositions of the disclosure at a desired site.
  • the organs, tissues, cells or compositions of the disclosure can be administered to a subject by any appropriate route which results in delivery of the cells to a desired location in the subject where at least a portion of the cells remain viable.
  • the cells remain viable after administration to the subject.
  • Methods of administering organs, tissues, cells or compositions of the disclosure are well-known in the art.
  • the cells, tissues and/or organs are transplanted into the host.
  • the cells, tissues and/or organs are injected into the host.
  • the cells, tissues and/or organs are grafted onto a surface of the host (e.g., bone or skin) .
  • the transplanted heart, lung, liver, kidney, pancreas, spleen, or a portion thereof survive and are functional for a period of time of about 1 day, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 9 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more.
  • the genetically modified cell (s) , tissue (s) or organ (s) is administered with a matrix or coating (e.g., gelatin) to protect the genetically modified cell (s) , tissue (s) or organ (s) from an immune response from the host.
  • a matrix or coating e.g., gelatin
  • the matrix or coating is a biodegradable matrix or coating.
  • the matrix or coating is natural. In other embodiments, the matrix or coating is synthetic.
  • the genetically modified cell (s) , tissue (s) or organ (s) is administered with an immunosuppressive compound.
  • the immunosuppressive compound is a small molecule, a peptide, an antibody, and/or a nucleic acid (e.g., an antisense or siRNA molecule) .
  • the immunosuppressive compound is a small molecule.
  • the small molecule is a steroid, an mTOR inhibitor, a calcineurin inhibitor, an antiproliferative agent or an inosine monophosphate dehydrogenase (IMDH) inhibitor.
  • IMDH inosine monophosphate dehydrogenase
  • the small molecule is selected from the group consisting of corticosteroids (e.g., prednisone, budesonide, prednisolone) , calcineurin inhibitors (e.g., cyclosporine, tacrolimus) , mTOR inhibitors (e.g., sirolimus, everolimus) , IMDH inhibitors (azathioprine, leflunomide, mycophenolate) , antibiotics (e.g., dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin) and methotrexate, or salts or derivatives thereof.
  • corticosteroids e.g., prednisone, budesonide, prednisolone
  • calcineurin inhibitors e.g., cyclosporine, tacrolimus
  • mTOR inhibitors e.g., sirolimus, everolimus
  • IMDH inhibitors
  • the immunosuppressive compound is a polypeptide selected from the group consisting of: CTLA4, anti-b7 antibody, abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab, daclizumab, and muromonab.
  • CTLA4 anti-b7 antibody
  • abatacept abatacept
  • adalimumab anakinra
  • certolizumab certolizumab
  • etanercept golimumab
  • golimumab infliximab
  • ixekizumab natalizumab
  • rituximab secukinumab
  • the genetically modified cell (s) , tissue (s) or organ (s) to be administered to the subject have been further genetically modified such that they are less likely to induce an immune response in the subject. In some embodiments, the genetically modified cell (s) , tissue (s) or organ (s) have been further genetically modified such that they do not express functional immunostimulatory molecules.
  • C3 A highly conserved region of C3 was selected and two sgRNAs that target a C3 domain were designed.
  • the sequences of the two gRNAs sequences are TCTCCAGACGCAGGACGTTG (SEQ ID NO: 158) and GGAGGCCCACGAAGGGCAAG (SEQ ID NO: 159) .
  • the C3 sgRNA was transiently transfected together with GGTA sgRNA (GAGAAAATAATGAATGTCAA (SEQ ID NO: 210) ) plasmid and cas9 plasmid into porcine fetal fibroblast cells using the neon transfection machine and reagents.
  • C3-KO Cells lacking C3 ( “C3-KO” ) were selected using a GGTA antibody counter selection method to co-enrich the C3-KO cells which were then single cell sorted and genotyped to determine the efficiency of knocking down the C3 target using deep sequencing.
  • FIG. 1A shows the sizes of deletions introduced into C3
  • FIG. 1B shows the position of the indels
  • FIG. 1C shows the sequence of the indels generated in the C3-KO pig.
  • the C3-KO pig described above would not have produced any functional C3 protein. Due to the lack of functional C3 protein, the C3-KO pig’s complement system would not be activated thereby decreasing the C3-KO pig’s innate immune system. In addition, it is expected that the C3-KO pig might be more prone to bacterial and/or viral infections compared to a wild-type pig. Moreover, xenotransplantation of a C3-KO pig’s cell, tissue, and/or organ into a human is not expected to activate the human complement system. This should therefore minimize the human innate immune response to the C3-KO pig xenograft.
  • Example 2 Pigs Having One or More Modified MHC Class I Genes
  • a pig’s MHC major class I alleles were conditionally replaced with human MHC minor class I alleles ( “MHC-I pigs” ) . To do so, a region of the pig’s genome containing the SLA-1, SLA-2, and SLA-3 genes was replaced with a modified version of the human minor allele HLA-E.
  • FIG. 2 depicts a scheme of the MHC class I replacment strategy: the locus containing SLA-1, SLA-2, and SLA-3 genes was flanked with loxP sites. After treatment with Cre, SLA-1, SLA-2, and SLA-3 were excised and replaced by human HLA-E, such as various combinations of HLA-E, HLA-G, B2M, and CIITA-DN genes. The MHC-I pigs were viable and severely immunocompromised.
  • the MHC I region of the pig was sequenced using long reads technology. Probes to capture the SLA-1, SLA-2, and SLA-3 genes were designed and used to capture the MHC-I genetic region. Pacbio sequencing and 10X sequencing were used to accurately determine the MHC-I genetic region.
  • the configuration of SLA-1, SLA-2, and SLA-3 is illustrated in FIG. 9. Two cassettes having loxP sites to flank the MHC-I region were designed. Cassette 1 contains a promoter, a loxP site, and a selection agent (i.e., puromycin) .
  • Cassette 2 contains a second marker (GFP) , a loxP site, and a promoter-less cassette of genes including HLA-E, B2M and CIITA-DN.
  • Cassettes 1 and 2 were synthesized from individual components using a Golden Gate Assembly strategy (New England BioLabs) and were flanked with a 800bp homology sequence corresponding to the insertion sites. Two consecutive rounds of CRISPR-cas9 were used to insert both sites 17 . Puromycin selection and GFP FACS sorting were used to isolate clones and junction PCR was used to validate insertions.
  • Cells were transfected with Cre recombinase and expression of Cre recombinase was induced. Single cell sorting was performed and sorted cells were screened using junction PCR to isolate cells having biallelic replacement of SLA-1, SLA-2, and SLA-3 with human MHC-1.
  • an alternative cassette 1 has been designed and includes a Cre recombinase under control of a tissue specific promoter or an inducible promoter.
  • a tissue specific promoter or an inducible promoter By using a tissue specific promoter or an inducible promoter, the SLA-1, SLA-2, and SLA-3 genes will be excised in cell, tissue and/or organ of interest or excision can be induced in the animal prior to harvesting the cell, tissue, and/or organ.
  • Pigs having SLA-1, SLA-2, and SLA-3 replaced with the human MHC-I can be generated by somatic cell nuclear transfer (SCNT) and piglets encoding conditional and/or tissue specific conditionally replaced genes can be generated.
  • SCNT somatic cell nuclear transfer
  • Pigs lacking expression of the MHC-II alpha chain were generated by excising DQA genes and inactivating DRA genes using established gRNA technology in porcine cells which were then transferred into host pigs via SCNT. Briefly, following gRNA transfer into the porcine cells, the genome was sequenced and variation at the MHC-II loci were identified. Cas9 was delivered to these cells, which were then sorted to isolate single cells. These single cells were sequenced to genotype the targeted DQA and DRA genes. In single cells having DQA and DRA inactivation, embryos were generated following SCNT and were subsequently implanted into a pig to generate the MHC-II KO pig. Four weeks after birth, the MHC-II KO pig remained healthy.
  • FIGs. 3A and 3B illustrate the genotype of the MHC-II KO based on the DQA gene.
  • the MHC-II KO pig was genotyped by exonic targeting-based amplification and sequencing of the DQA gene as well as sequencing of the DRA gene. As shown in the left panel, the sizes and positions of the indels are located in the DRA gene. Inactivation of the DRA gene was caused by the two single nucleotide insertions at each of positions 126 and 127 in the DRA amplicon as illustrated in the right panel.
  • FIGs. 4A and 4B illustrate another genotype of an MHC-II KO pig.
  • the DRA genotype was determined using exonic targeting-based amplification and sequencing of the DRA gene.
  • the exonic targeting area from DRA has been amplified and sequenced.
  • the sizes and positions of the indels are located in the DRA gene.
  • Inactivation of the DRA gene was caused by the two single nucleotide insertions at each of positions 106 and 107 in the DRA amplicon as illustrated in the right panel.
  • the MHC-II KO pig Similar to a human lacking MHC-II expression, the MHC-II KO pig has a decreased population of CD4 + T cells however, the CD8 + T cell population remains intact (FIG. 5) . In addition, the MHC-II KO pig is immunosuppressed, has increased autoimmunity, and lymphoid defects, amongst other issues. These phenotypes are known to be associated with the MHC-II KO phenotype and have been observed in mice lacking MHC-II expression. These similarities confirm that the MHC-II KO pig is a valid MHC-II KO rather than an active gene modification (FIG. 6) .
  • a human PD-L1 gene (e.g., PD-L1 transgene) was delivered to a pig genome. See scheme with structure in FIG. 7. Expression of the human PD-L1 transgene was confirmed by qPCR using two different PD-L1 amplicons (FIG. 8) .
  • Porcine tissues expressing PD-L1 may have reduced rejection by a host, such as a human, following xenotransplantation.
  • An HDR vector that contains the homology arms from pvWF, the A1 domain, and the certain residues in the flanking regions from hvWF was designed and constructed. (FIG. 10) .
  • Two sgRNAs were also designed to initiate the HDR replacement in the endogenous porcine genome and cut near the region to be replaced by the human sequences: TCTCACCTGTGAAGCCTGCG (SEQ ID NO: 5) and CACAGTGACTTGGGCCACTA (SEQ ID NO: 6) .
  • the HDR vector is composed of ⁇ 1 kb homology arms from porcine vWF and the human A1 and flanking domains as well as inactivating mutations in the sgRNA cutting sites to prevent sgRNA from cutting the donor and modified porcine genome.
  • the HDR vector also contains SphI and BspEI sites that can distinguish the HDR vector from the endogenous porcine genome near the sgRNA cutting sites.
  • Porcine primary fibroblast cells were transfected using the Neon Transfection System (Invitrogen) with 8 ⁇ g of Cas9, 1 ⁇ g of sgRNA1, and 1 ⁇ g of sgRNA2, as well as 10 ⁇ g of the HDR vector. Two days after transfection, cells were single cell sub-cloned using FACS. The single cells were cultured for additional 12 days until the episomal form of the HDR vectors are lost during cell division. The A1 and flanking regions of the hvWF were amplified using flanking primers.
  • the PCR product was subjected to SphI and BspEI sequential digestions to screen the clones having HDR replacement which would add novel SphI and BspEI sites to the PCR products having fragments sized at 700bp, 323bp and 258bp following sequential digestion (FIG. 11) .
  • the complete bi-allelic HDR eliminates a wild-type product of 1281bp as well as any partial digestion products larger than 700bp.
  • a cell having a bi-allelic HDR was isolated from about 150 single-cell colonies (FIG. 11) . As confirmed by sequencing, both alleles of the porcine A1 domain and flanking regions were replaced with the human counterpart (FIGs. 12A and 12B) . The A1 domain is highlighted, whereas the potential glycosylation sites in the flanking region are labeled with dashes. The human specific residues that are deleted in pvWF are labeled with a bar and the humanized A1 domain and flanking regions are labeled with half parenthesis.
  • This isolated cell has been expanded into a cell line and may be used to generate a genetically modified pig by SCNT.
  • Cells expressing the A1-humanized pvWF had a significantly reduced aggregation response against human platelets during a platelet activation assay (FIG. 13) . Briefly, the cells were incubated with human platelets and aggregation was induced by shear stress. The cells expressing the A1-humanized pvWF showed a milder and inducible aggregation curve whereas the cells expressing wildtype pvWF had a stronger aggregation response towards human platelets.
  • porcine organs having A1 hvWF will likely induce a milder coagulation response in human blood compared to porcine organs expressing pvWF and might ameliorate the vascular incompatibility observed in pig-to-human xenotransplantation.
  • MHC class I molecules play a vital role in the rejection of allotransplantation through their peptide presentation to CD8+ T cells.
  • deletion of the entire ⁇ 200kb classical MHC class I locus in porcine primary fibroblast cells prevented CD8+ T cell mediated toxicity in xenotransplantation.
  • Classical MHC class I genes encode highly polymorphic proteins that are widely expressed in cell surface. They present foreign peptides to CD8+ T lymphocytes leading to the lysis of target cells. Also, mismatched MHCI molecules also serve as antigens in transplantation. Different strategies of removing the classical MHCI molecular in donor porcine organs for xenotransplantation have been explored. In one attempt, the Tector group knocked out the conserved Exon4 of the SLA-1, SLA-2 and SLA-3 molecular using Cas9 and 3 sgRNAs (Reyes 2014) . However, this exon is also share by other classical and non-classical MHCI molecular and it may generate unpredicted off-target effects.
  • the remaining Exon1-3 may still be presented as cell surface antigens.
  • the heterodimerization partner B2M was knocked out using TALENs (Wang 2016) . This method may also affect the non-classical MHCI molecules and the remaining MHCI may still be presented de-structured proteins on the cell surface.
  • human HLA-E/B2M molecules are usually complemented in the MHCI deficient cells to prevent NK cell mediated toxicity.
  • the human B2M might dimerize with porcine SLAs and restore their antigenicity in the B2M knockout pigs.
  • the MHC classical class I cluster with unique flanking sequences in the porcine genome were first identified (FIG. 14) .
  • This ⁇ 200kb cluster contains all the 8 classical MHCI genes without any other protein coding genes.
  • sgRNAs SEQ ID NOs 1-4
  • enrichment strategies were also designed to isolate bi-allelic deletion clones.
  • Porcine primary fibroblast cells were transfected with 1.25 ⁇ g of TrueCut Cas9 protein and 7.5nmole of crRNA/tracrRNA duplex (Invitrogen) using the Neon transfection system (Invitrogen) .
  • genomic DNA was harvested from the transfected cells and subject to PCR using designated primer pairs shown in FIG. 15A. Fragmental deletion was detected using primers flanking the expected deletion junction.
  • This PCR product was subcloned using Toposiomerase based cloning ( “TOPO cloning” ) and the individual TOPO clones were Sanger sequenced to confirm the sequence of the deletion junctions. The sequences were aligned to the expected junction shown in FIG. 15B.
  • TOPO cloning Toposiomerase based cloning
  • the cells containing bi-allelic deletion can be used to produce classical MHCI knockout pigs via somatic cells nuclear transfer. It is contemplated that the pigs are completely deficient in all classical MHCI molecules and proficient for the non-classical MHCI molecules, which might be involved in fertility and other physiological functions. The remaining B2M molecules are unlikely to be antigenic because they are non-polymorphic and highly conserved to the human counterpart. Also, the exogenous expression of human HLA-E/B2M cannot rescue the deficiency of classical MHCI molecules. The resultant pig should have the cleanest classical MHCI knockout background compared to previous reports.
  • Example 7 Generation of Immunologically Compatible Porcine Cells, Tissue, Organs, Pigs, And Progeny
  • transgenic pigs for safe xenotransplantation carried a limited number of transgenes due to the compacity of the constructs and transcription interference between transgenes.
  • a combination of KO, KI, and genomic replacement is utilized.
  • CRISPR-Cas9 multiple genes have been knocked out (GGTA1, CMAH, and B4GALNT2) to eliminate the glycans that will be recognized by human preformed anti-pig antibodies. It is anticipated that the functional loss of these genes will largely eliminate the binding of preformed anti-pig antibodies to the endothelium of the porcine graft.
  • Human transgenes (CD46, CD55, CD59, CD39, CD47, A20, PD-L1, HLA-E, B2M, THBD, TFPI, HO-1) have been integrated into a single multi-transgene cassette in the pig genome. (See, FIGs. 17-20, 31, 47-49) .
  • the transgenes were arranged into 4 different cistrons with desired ubiquitous or tissue-specific promoters.
  • the transgenes within each cistron were separated with ribosomal skipping 2A peptides to ensure expression in a similar molar ratio.
  • cis-elements such as ubiquitous chromatin opening elements (UCOEs) were introduced to prevent transgene silencing and insulators with strong polyadenylation sites and terminators to minimize the interaction among transgenes and between transgenes and the flanking chromosome.
  • UCOEs ubiquitous chromatin opening elements
  • Donor pigs were created through several iterations of genetic modifications.
  • FIG. 21 outlines the progression of donor pig generations through sequential gene editing.
  • Pig 2.0 have been generated through CRISPR-mediated NHEJ to delete the 3 major carbohydrate antigen genes (3KO; GGTA1, B4GALNT2 and CMAH) coupled with PiggyBAC-mediated random integration of the 12 selected transgenes (12TG: CD46, CD55, CD59, HLA-E, B2M, CD47, CD39, THBD, TFPI, A20, PD-L1, and HO-1) into the porcine genome.
  • the 12 human genes were inserted into a safe harbor site, such as the AAVS1 genomic locus.
  • transgenes in adjacent cistrons demonstrated desired tissue-specificity in fibroblast and endothelial cell lines without detectable transcription interference.
  • all transgenes showed highly consistent expression levels across clones with various locations of genomic integration, which indicates that the transgene expression is independent of chromosomal context.
  • the transgenes will be randomly integrated into the genome using PiggyBac transposase. The integration site and copy number will be determined using junction capture based on inverted PCR. Clones with single copy integration into intergenic regions with no predictable consequences will be used for pig production. For clinical development, homozygous female/male pigs will be generated with biallelic site-specific transgene integration into a safe harbor prior to scaled up breeding and production of source donor pigs.
  • assessments of innate and adaptive immune cell function and complement and coagulation cascades will include antibody reactivity profiling, mixed lymphocyte reaction, complement-dependent cytotoxicity, NK cell cytotoxicity, macrophage phagocytosis, and effects on coagulation factors and platelet aggregation.
  • Preformed antibodies that bind wild-type pig tissues are the major initial immunologic barrier to xenotransplantation.
  • Three genes have been identified as being largely responsible for producing antigens targeted by preformed antibodies: GGTA1, CMAH, and B4GALNT2 (Byrne 2014, Lai 2002, Lutz 2013, Martens 2017, Tseng 2006) . These three genes were eliminated by CRISPR-Cas9 in Pig 2.0 fibroblasts and demonstrated to have decreased binding of host antibodies to target fibroblasts by flow cytometry (FIG. 22) .
  • pigs were genetically engineered to express 12 human genes to address immunologic, coagulation, and species incompatibilities.
  • Transgene expression levels and tissue-specific promoter-driven expression was determined using qPCR (FIG. 23) .
  • the genes inserted under control of a ubiquitous promoter including the complement regulatory genes (CD46, CD55, and CD59; EF1 ⁇ promoter) as well as beta-2-microglobulin, HLA-E, and CD47 (CAG promoter) were expressed in both fibroblast and endothelial cells.
  • human complement regulatory proteins were over-expressed. Briefly, genetically engineered pig fibroblasts and pig splenocytes were incubated with 25%human complement for one hour. Cells were stained with propidium iodide and analyzed by flow cytometry to quantify cell death. Wild-type fibroblasts and splenocytes demonstrated the highest percentage of cell death after culture with human complement. 4-7P and 4-7H cells (3KO +12 KI) are derived from Pig 2.0 piglets; 4-7F cells (3KO +12 KI) are derived from Pig 2.0 fetus.
  • 3-9 is triple carbohydrate antigen KO, HLA-DQA KO, HLA-DRA KO, and human complement regulatory factor C3 KO.
  • pig fibroblasts and splenocytes genetically engineered to express human CD46, CD55, and CD59 exhibited significantly lower levels of complement-mediated cell death compared to control human fibroblasts.
  • NK cells are susceptible to targeted cell killing by NK cells.
  • human HLA-E which ligates human NK KIR receptors, was overexpressed in pig cells. Seventy percent of WT pig fibroblast and K562 cells (human MHC-deficient cell line) were targeted for killing by NK cells. As shown in FIG. 26, human HLA-E+ engineered pig fibroblast cells demonstrated significantly lower NK-mediated cell killing. In contrast, HLA-E+ pig fibroblasts demonstrated significantly lower killing by NK cells, suggesting that expression of HLA-E protected these cells from lysis.
  • the over-expression of human CD55 in pig cells reduces complement-mediated toxicity which may diminish coagulation and improve xenograft survival.
  • the activation of coagulation ultimately leads to the formation of thrombin which is inactivated by binding antithrombin in a stable thrombin-antithrombin (TAT) complex.
  • TAT thrombin-antithrombin
  • wild-type, CD55 KI + GGTA1-deficient cells, and human endothelial cells were cultured with human blood. As shown in FIG. 27, human blood alone or human blood incubated with human endothelial cells for 60 min generated approximately 10ng/mL TAT protein.
  • RNAseq was performed on samples isolated from pigs genetically modified with Payload 9 or Payload 10. Results demonstrated increased expression of several of the payload immune modifications transgenes, namely the complement transgenes, along with cellular toxicity genes (B2M, HLA-E, CD47) (FIG. 36) .
  • Example 8 Antibodies in xenotransplantation and the potential of enzymatic cleavage to prevent functional binding
  • Antibody-mediated rejection has historically been the primary hinderance to the development of xenotransplantation as a viable treatment for end stage organ failure.
  • recent genetic advancements have allowed for development of multiple-gene knockout pigs, which lack established xenoantigen targets.
  • Knockout of ⁇ Gal, Neu5Gc, and SDa have been linked to improved graft survival.
  • further work is needed to fully understand the impact of residual antibody binding to other xenoantigen targets and if the removal of these antigens protects tissues from highly sensitized human serum.
  • it was investigated whether xenoantigen knockout decreases high PRA serum binding and whether functional antibody binding is decreased by enzymatic degradation.
  • Human and porcine PBMCs were collected from peripheral blood using Ficoll separation. Porcine aortic endothelial cells (pAECs) were processed from WT pigs and genetically modified pigs lacking ⁇ Gal/Neu5Gc/SDa, denoted as “Pig 2.0” . Anonymous high and low PRA serum samples were generously provided by the Massachusetts General Hospital HLA laboratory. Serum was collected from heart, liver, and kidney xenotransplant recipients. Serum antibody was enzymatically cleaved by IdeS (Genovis Inc. ) .
  • FIGs. 46A-46C show that the IgG-specific protease, IdeS, effectively reduces the binding of functional IgG from human and cynomolgus serum to background levels.
  • Example 9 Generation of PERV-Free and Immunologically Compatible Porcine Cells, Tissue, Organs, Pigs, And Progeny
  • Porcine organs are considered a favorable resource for xenotransplantation since they are similar to human organs in size and function, and pigs can be bred in large numbers.
  • porcine organs has been hindered by the potential risk of porcine endogenous retrovirus (PERV) transmission, and by immunological incompatibilities.
  • PERVs are gamma retroviruses found in the genome of all pig strains. Pig genomes contain from a few to several dozen copies of PERV elements (Lee 2011) .
  • PERVs are an integral part of the pig genome. As such, they cannot be eliminated by bio-secure breeding (Schuurman 2009) .
  • PERVs can infect and propagate in human cells through “copy-and-paste” mechanisms.
  • viral particles can be released and can infect human cells and randomly integrate into the human genome, preferentially in intragenic regions and in areas of active chromatin remodeling (Armstrong 1971, Moalic 2006, Niu 2017, Patience 1997) .
  • both PERV-A and PERV-B can infect human cells.
  • PERV-C is ecotropic, the recombinant viral type (A/C) demonstrates the greatest infectivity.
  • PERVs adapt to the new host genome environment through elongation of the LTR sequence, infectivity potential may increase.
  • PERVs can also pass horizontally from infected human cells to other human cells that have had no contact with porcine cells.
  • PERV can pass from pig cells to mouse cells (Clémenceau 2002) .
  • PERV integration could potentially lead to immunodeficiency and tumorigenesis, as reported with other retroviruses.
  • Recent breakthroughs in genetic engineering have demonstrated genome-wide inactivation of PERV in an immortalized pig cell line (Yang 2015, WIPO Publication No. WO2017/062723) and production of PERV-free pigs (Niu 2017, WIPO Publication No. WO2018/195402) .
  • PERV copy number was monitored both in a population and in clones of PERV-infected HEK293T-GFP cells (iHEK293T-GFP) for greater than 4 months. PERV copy number was observed to increase over time, as determined by ddPCR (Pinheiro 2012) .
  • PERV-Free and Immunologically Compatible Pigs A study will be undertaken to engineer donor pigs that do not harbor any active PERVs in the genome as well as have enhanced immunological, inflammatory, and coagulation systems compatible with human tissues. Specifically, the function of all the PERVs in the pig genome were eradicated using CRISPR-Cas9 to remove the catalytic domain of the reverse transcriptase gene in the PERV elements (using the methods as described in Niu 2017 and WIPO Publication No. WO2018/195402) and using a combination of knockout (KO) , knockin (KI) , and genomic replacement to provide human tissue compatible organs.
  • KO knockout
  • KI knockin
  • the three major carbohydrate antigens that trigger humoral rejection, GGTA1, CMAH, and B4GALNT2, will be genetically KO. It is contemplated that the functional loss of these genes will largely eliminate the binding of preformed anti-pig antibodies to the endothelium of the porcine graft.
  • key immunological modulatory factors will optionally be inserted into a safe harbor genomic locus such as AAVS1 to regulate the human complement system, and NK, macrophage, and T cell functions.
  • a safe harbor genomic locus such as AAVS1 to regulate the human complement system
  • NK macrophage, and T cell functions.
  • an overexpression KI for hCD46, hCD55, and hCD59 to will be genetically engineered to inhibit the human complement cascade.
  • the methodology can be either single copy polycistronic transgene integration through transposition or mono/bi-allelic site-specific integration through recombinase-mediated cassette exchange (RMCE) .
  • RMCE recombinase-mediated cassette exchange
  • the A1 domain and flanking regions of the porcine vWF sequence may be humanized using CRISPR-Cas9 induced homologous recombination to prevent unregulated platelet sequestration and thrombotic microangiopathy.
  • Donor pigs were created through several iterations of genetic modifications.
  • FIG. 21 outlines the progression of donor pig generations through sequential gene editing.
  • Pig 1.0 porcine fibroblasts have been genetically engineered, using CRISPR-Cas9 mediated non-homologous end joining (NHEJ) , to have all PERV copies functionally deleted from the genome.
  • NHEJ CRISPR-Cas9 mediated non-homologous end joining
  • Pig 2.0 have been generated through CRISPR-mediated NHEJ to delete the 3 major carbohydrate antigen genes (3KO; GGTA1, B4GALNT2 and CMAH) coupled with PiggyBAC-mediated random integration of the 12 selected transgenes (12TG: CD46, CD55, CD59, HLA-E, B2M, CD47, CD39, THBD, TFPI, A20, PD-L1, and HO-1) into the porcine genome.
  • the 12 human genes will be inserted into a safe harbor site, such as the AAVS1 genomic locus.
  • source donor pigs have been generated to carry the 3KO and 12 specified transgenes, on the PERV-free background.
  • the next generation of source donor pigs (Pig 3.1, 3.2, etc. ) will be genetically engineered to carry additional modifications, such as humanization of the vWF gene and deletion of the asialoglycoprotein receptor 1 (ASGR1) and endogenous B2M genes.
  • ASGR1 asialoglycoprotein receptor 1
  • 3KO+12TG pigs (Pig 3.0, FIG. 21) have been genetically engineered, these pigs will be crossbred to generate progeny and/or a drift, drove, litter, and/or sounder of swine.
  • Liver perfusion experiments were performed with immunologically compatible pig livers isolated from Pig 2.0 (4-7; 3KO +12 KI ) as a proxy experiment to xenotransplantation for analyzing organ function. Wild type livers and 4-7 livers (approximately 80 kg) were isolated from 12-month-old pigs. Livers were perfused with human whole blood and human fresh frozen plasma (FFP) . A brief liver perfusion protocol is outlined in Table 1.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ARB albumin
  • Preclinical renal transplant studies For preclinical renal transplant studies, safety and efficacy studies will be in NHP. For safety and efficacy examination, kidneys from 8-to 10-week-old Pig 2.0 donors will be transplanted to NHP (cynomolgus monkey) recipients that will undergo bilateral nephrectomy at the time of transplant. Xenograft function will be monitored by serum creatinine values, complete blood counts, and urine analysis for protein as well as serial biopsies and examinations for weight and general well-being. Immunosuppression will consist of clinically relevant reagents in a combination and intensity that would be acceptable in allotransplantation.
  • In vitro antibody reactivity and mixed lymphocyte reaction (MLR) assays will be used to demonstrate lack of reactivity following xenotransplantation in preclinical models.
  • MLR mixed lymphocyte reaction
  • flow cytometry cross-matching will be performed using serum from male NHP receiving kidney transplants from normal pig and Pig 2.0 donors as described above.
  • the reactivity of serum to lymphocytes from a panel of NHP donors as well as to lymphocytes from the porcine donors will be tested.
  • Reponses to the porcine cells will confirm that a xeno-sensitizing event has occurred by elevations in anti-porcine antibody levels.
  • Samples from the NHP pretransplant will be compared with post-rejection samples to assess for changes in antibody binding to the NHP lymphocyte panel.
  • direct and indirect T cell responses by pre-and posttransplant (post-rejection) NHP recipient T cells to a panel of allogeneic stimulators will be evaluated to determine if the cell-mediated allogeneic response is augmented post-rejection of a xenograft (Baertschiger 2004, Cooper 2004, Ye 1995) .
  • Biodistribution The migration of donor cells to distal tissues/organs in the recipient remains a possible consequence of xenotransplantation. Chimera studies demonstrate that this may actually increase the success of engraftment reducing the probability of rejection (Starzl 1993, Vagefi 2015) . However, there may be unknown consequences of pig donor cell migration and therefore strategies have been developed to determine if migration of cells has occurred. Biodistribution will be studied as part of the pig-NHP xenotransplant research studies according to principles outlined in FDA guidance documents including Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans, Dec 2016 (Section IX.C.
  • Tumorigenicity All animals included in the SCNT and assisted reproduction facilities will be routinely monitored for evidence of tumorigenesis. All animals found moribund or dead will have a full necropsy and gross and microscopic pathology examinations by a veterinary pathologist. Records of all genetically engineered animal health and pathology will be maintained and compiled to determine the risk for tumorigenicity potential due to specific or unintended genetic modification.
  • Renal xenotransplantation has been studied for several decades and porcine xenografts have been evaluated in early clinical trials (Starzl 1964) .
  • the challenge is to enable xenograft procedures that provide clinical benefit equivalent to allograft survival.
  • the proposed clinical study population will include transplant patients age 18-65 with end-stage renal disease who are unlikely to find a suitable kidney donor in a timely manner due to the presence of high levels of panel reactive anti-HLA antibodies (PRA) .
  • PRA panel reactive anti-HLA antibodies
  • High PRA creates substantial challenges in matching a suitable deceased or live donor, causing extended waiting times for a transplant and excess morbidity from additional years on hemodialysis.
  • >90%PRA patients still experience markedly prolonged wait times compared to lesser sensitized patients.
  • Subjects that have >90%PRA sensitization to HLA antigens and who manifest a negative flow cross-match to porcine donor lymphocytes (or endothelial cells) will be targeted.
  • porcine donor kidneys of 120 ⁇ 10 gm providing an expected glomerular filtration rate (GFR) of 40-50 mL/min/1.73m 2 .
  • GFR glomerular filtration rate
  • Single porcine kidneys from 9-to 12-month-old donors will be transplanted to the right or left iliac fossa, in a manner identical to that used for allogeneic renal transplantation.
  • the primary endpoint will be freedom from hemodialysis for one year post transplant.
  • Protocol-designated graft biopsies will be performed every three months and for-cause based on >20%rise in creatinine from baseline, defined as the mean of the best three consecutive creatinine measures in the first month post-transplant, or proteinuria greater than 300 mg/day.
  • Safety measures will include monitoring of coagulation parameters, clinical chemistry, hematology, and adventitious infections.
  • a single adult kidney may be transplanted successfully into a 10kg infant equating to a 12-17 gm of kidney/kg, which is approximately 3-4 times the renal mass ratio for an average adult (3-4 gm of kidney/kg; Donati-Bourne 2014) .
  • This upper graft weight to recipient weight range is relevant to the proposed preclinical studies detailed below.
  • 50-75 gm kidneys will be transplanted from 8-to 10-week-old porcine donors into 5-12 kg NHP recipients ( ⁇ 10 gm of kidney/kg) .
  • Glomerular filtration rate (GFR; mL/min/1.73m2) is a standard measure of renal function or kidney potency that is used to stage the progress of chronic kidney disease (CKD) and renal failure qualifying for dialysis and/or transplantation.
  • CKD chronic kidney disease
  • the goal is to achieve a GFR of 45-60 mL/min/1.73m 2 (CKD stage 3A; Levey 2011) .
  • This target range for GFR is based on data suggesting that renal function in CKD 3A is comparable to that achieved by single kidney allotransplantation in humans and is stable, whereas lower GFR in the CKD stage 3B (GFR 30-45 mL/min/1.73m 2 ) is associated with an increase in end-stage renal disease and all-cause and cardiovascular mortality (Sharma 2010) .
  • the targeted GFR range of 45-60 mL/min/1.73m 2 is comparable to that achieved by single kidney allotransplantation in humans (50-65 mL/min/1.73m 2 ; Gourishankar 2003, Marcén 2010) .
  • kidney mass comparable to that routinely used in allotransplantation (115-170 gm) given the comparability of human and porcine kidneys in GFR per renal mass. It should be considered that some renal function may be lost in the donation process and post-transplant due to treatment of the recipient with nephrotoxic immunosuppression in the form of calcineurin inhibitors.
  • Pharmacology and Toxicology Information Efficacy and safety will be evaluated using pharmacology studies with both rodent and NHP models. A variety of integrated safety endpoints will be used, as well as an assessment of clinical pathology and pathophysiology in genetically engineered donor porcine tissues. A tiered approach will be taken involving in vitro cellular and tissue function, and assessments of clinical pathology and histopathology in donor pigs and NHP xenografts. Endpoints will include graft function and rejection, and recipient safety related to functions of innate and adaptive immunity, inflammation, as well as complement and coagulation cascades.
  • Somatic Cell Nuclear Transfer and Assisted Reproduction of Genetically Engineered Donor Pigs Genetically engineered donor pigs will be monitored routinely for safety considerations with full clinical pathology including clinical chemistry and hematology as well as gross and microscopic histopathology. Reproductive capability, embryo-fetal development, organ and tissue development, and potential tumorigenesis will be monitored and recorded for all donor pigs in the breeding colony.
  • Animals are identified by unique ear tags printed with permanent ink (placed at Place of Origin) .
  • the flow of pigs includes a quarantine area, which is an open-air, group-housed barn with a bedding of wood shavings.
  • the feed trough is wooden and kept clean from debris and waste. Fresh, free-choice water is available at all times via nipple drinkers.
  • the barn relies on outdoor wind movement to circulate the air and temperature is maintained above 10°C. Biosecurity requires at least 24 hours of no other swine contact, specific barn attire, and boot dipping in disinfectant before and after barn contact.
  • the quarantine period includes 35-40 days of quarantine, vaccination with Parvo Shield L5E, FluSure XP/ER Bac Plus, Ingelvac FLEX combo (Circovirus and Mycovirus) , and Dectomax, and includes 2 blood draws demonstrating no increase in disease antibodies (PRRSV, PRRSX3) .
  • pigs are moved into a buffer area at the facility. This area is a closed-barn, group-housed, sawdust-bedded pen in groups of up to 12. Bedding is replaced weekly. Temperature is controlled by thermostat-controlled fans and propane heater to a range of 15–24°C. Pigs are fed in a stainless-steel trough and fresh, free-choice water is available at all times via nipple drinkers. Pigs are observed at least once a day and as health status dictates.
  • Pigs with observed health issues are housed in single pens for individualized care and attention and treated as directed by the Attending Veterinarian and Director of Embryology. Biosecurity requires at least 24 hours of no other swine-herd contact. Coveralls limited to use in the barn area and boots are disinfected either with Virkon-Sor Synergize before and after barn contact. Generation of source donor pigs for use in clinical studies will follow all relevant guidance and regulations.
  • the endogenous gene KOs and human transgene expression will be validated at genomic, mRNA, and protein levels.
  • gene KOs either Sanger sequencing or deep sequencing will be performed to confirm the genetic mutations at the intended target site.
  • RNA-seq and/or RT-PCR will be performed to ensure that the mRNAs contain the intended mutations and are subject to non-sense mediated decay.
  • RT assays will be performed to demonstrate the elimination of RT activity in PERV KO cells.
  • immunohistochemistry (IHC) staining and/or flow cytometry will be performed to ensure that gene products are absent in the cell or at the cell surface.
  • Off-target mutations may still exist despite advances in the field of precision gene editing and must be understood in order to generate safe and efficacious donor organs for clinical xenotransplantation.
  • the following multi-tiered assessment approach has been employed:
  • CIRCLE-Seq A sensitive, in-vitro screening strategy that comprehensively detects genome-wide CRISPR-Cas9 off-target mutations of any given gRNA. The potential off-target sites will be censored in any derived cell line from the specific gRNA using subsequent targeted amplicon sequencing;
  • transgene expression intactness and expression of human transgenes in genomic, mRNA, and protein levels will be validated using sequencing, RT-PCR/RNA-seq, and IHC/flow cytometry technologies. Moreover, the location of random transgene integration will be determined by inverted PCR-based junction capture and the results will be validated by junction PCR.
  • Clones will be chosen with a single-copy transgene integrated into intergenic regions at least 10,000bp from any known genes and ncRNAs, and at least 50,000bp from any oncogenes and tumor suppressors.
  • biallelic site-specific integration/replacement will be validated by junction PCR and droplet digital PCR (ddPCR) .
  • Preclinical transplant studies For preclinical transplant studies, safety and efficacy studies were performed in NHP. Hearts, kidneys, and livers from 8-10 week-old Pig 2.0 donors were used for transplanted solid organ studies and liver and lungs were used for perfused organ studies. In a span of 5 months, 15 organ transplants and 11 organ perfusions were performed. Specifically, 7 kidney transplants, 4 heart transplants, 4 liver transplants were performed while 4 livers and 7 lung perfusions were performed, as summarized in Table 3.
  • Immunosuppression regimen for kidney transplantations consisted of clinically relevant reagents in a combination and intensity that was acceptable in allotransplantation.
  • hCD55 pig kidneys survived until days 76 and 93 when they were euthanized due to renal failure and weight loss, respectively. Of these two, one was found to have thrombotic microangiopathy (TMA) , chronic antibody-mediated rejection (AMR) and borderline T-cell Mediated Rejection (TCMR) ; while the other had C4d deposition, but otherwise no histologic evidence of frank rejection.
  • TMA thrombotic microangiopathy
  • AMR chronic antibody-mediated rejection
  • TCMR borderline T-cell Mediated Rejection
  • the remaining seven recipients received kidneys from Pig 2.0. In these pigs, transduced human proteins that regulate immune responses or complement activation were expressed at high levels.
  • the NHP recipients of these genetically modified pig kidneys survived >190, 72, 20, 15 and 6 days with immunosuppression regimen for kidney transplantations.
  • Compromised health of monkeys contributed to early termination of several of the xenograft monkeys. Complications included blood transfusions, injection site abscess and infection, wound healing. Several cases presented bleeding in bladder and/or ureter, possibly due to over-anti-coagulation. A summary of the Pig2.0 grafts is provided in Table 5.
  • Livers were from two genetic constructs of transgenic pigs deficient in targets of xenoantibody and containing human transgenes to address complement activation and innate immune cell function (group 1: B1, B2; group 2: B3, B4) .
  • Immunosuppression consisted of ATG, Rituximab, corticosteroids, MMF and aCD154. All recipients received an infusion of KCentra. Unlike previous studies, splenectomy was not performed, and cobra venom factor and tacrolimus were omitted.
  • B2 and B4 received a continuous infusion of a glycoprotein IIb/IIIa (GPIIbIIIa) inhibitor. Graft function was assessed with daily chemistries, lactate, CBC, INR and weekly coagulation profile.
  • GPIIbIIIa glycoprotein IIb/IIIa
  • B2 recovered uneventfully and biopsy on POD8 was nsormal. Development of hemoptysis and increased transfusion requirement, necessitated euthanasia on POD14. Pulmonary hemorrhage was identified on necropsy. H+E staining of the liver exhibited diffuse sinusoidal neutrophil infiltrate, suggesting infectious complications versus rejection, B2 was C4dnegative and the LFTs remained normal throughout (FIGs. 38C-D) . B3 was hypotensive and hypoxemic intra-op after reperfusion, requiring euthanasia.
  • Necropsy showed diffuse pulmonary hemorrhage with normal liver and patent vasculature. B4 recovered uneventfully. Only one post-op blood transfusion was required. On POD7, a rise in Tbili and LFT’s prompted exploration, where a bile leak and hepatic artery thrombosis (HAT) were identified, requiring euthanasia. Biopsy showed focal subcapsular necrosis with negative C4d and no evidence of rejection, consistent with HAT (FIGs. 38E-F) .
  • Liver Xenoperfusion Barriers to successful xenogeneic pig liver transplantation include hyperacute rejection by preformed xeno-antibody, molecular incompatibilities resulting in dysregulated complement, coagulation, and innate and adaptive immunity. Genetically modified swine may circumvent these obstacles and will require a rapid and efficient model to evaluate the effectiveness of different genetic constructs.
  • EDLXP ex-vivo liver xenoperfusion
  • WT wild type
  • hWB+P human blood and plasma
  • EVXLP was performed at 37 °C with fresh, heparinized hWB+P. Failure during EVXLP was defined by decreased blood flow due to elevated vascular resistance, severe metabolic derangements or gross necrosis.
  • CBC serum clinical chemistry, and blood gas analysis were performed. Tissue biopsies were stained with H+E and for depositions of IgG, IgM, and complement (C4d) .
  • EG liver tissue biopsies exhibited preserved hepatic architecture on H+E with mild diffuse portal and sinusoidal inflammation (FIG. 41A) .
  • WT livers manifested focal ischemic necrosis and vascular congestion on H+E (FIG. 41E) , with strong staining for IgM and IgG (FIGs. 41F-41G) and C4d-positivity (FIG. 41H) .
  • EG livers showed diffuse mild sinusoidal IgG and IgM deposition (FIGs. 41B-41C) , with negative C4d (FIG. 41D) , perhaps suggesting the reduction in pre-formed antigens and improvements in complement regulation by addition of human complement regulatory protein expression resulted in less injury.
  • Xenolivers from transgenic pigs deficient in xeno-specific antigens and containing humanized transgenes related to complement activation and immune cell function achieved significantly prolonged survival with less severe platelet sequestration, preserved RBC mass and diminished antibody and complement deposition compared to WT or GTKO.
  • CD55 xenografts This model is an efficient and informative tool to simulate pig-to-human xenotransplantation and evaluate the efficacy of specific genetic modifications.
  • Lung Xenoperfusion Ex vivo lung perfusion with human blood is a standardized method to evaluate the impact of transgene combinations. Here results associated with novel transgenic pig lines, evaluated in the context of a reference cohort, are reported.
  • Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in ‘untreated’ Pig 2.0 lungs, relative to GalTKO. hCD55 lungs (FIG. 42) .
  • RNAseq expression data showed complement and cellular toxicity genes are expressed in samples collected from Payload 9 and Payload 10 Pig 2.0 pigs (FIG. 36) .
  • FACS data showed complement and cellular toxicity proteins are expressed in samples collected from Payload 5, Payload 9 and Payload 10 pigs (FIG. 37) .
  • Payload 5 expressed CD39
  • Payload 10 expressed PDL1.
  • AVR acute vascular rejection
  • APTT activated partial thromboplastin time
  • AAVS1 adeno-associated virus integration site 1
  • ALT alanine aminotransferase
  • ARB albumin
  • AMR antibody-mediated rejection
  • ASGR1 anti-thymocyte globulin
  • ASGR1 asialoglycoprotein receptor 1
  • ASGR1 aspartate aminotransferase
  • AST aspartate aminotransferase
  • B4GalNT2 beta-2 microglobulin
  • B2M Cluster of Differentiation 39
  • CD47 Cluster of Differentiation 47
  • CRISPR class II transactivator dominant-negative
  • CMV CMV early

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Abstract

L'invention concerne des cellules, des tissus, des organes et/ou des animaux ayant un ou plusieurs gènes modifiés pour une meilleure survie et/ou tolérance aux xénogreffes. L'invention concerne également des procédés de production et d'utilisation des cellules, tissus, organes et/ou animaux.
PCT/CN2019/087314 2019-05-16 2019-05-16 Cellules, tissus, organes et/ou animaux ayant un ou plusieurs gènes modifiés pour une meilleure survie et/ou tolérance aux xénogreffes WO2020228043A1 (fr)

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PCT/CN2019/087314 WO2020228043A1 (fr) 2019-05-16 2019-05-16 Cellules, tissus, organes et/ou animaux ayant un ou plusieurs gènes modifiés pour une meilleure survie et/ou tolérance aux xénogreffes
US17/611,838 US20220267805A1 (en) 2019-05-16 2020-05-15 Cells, tissues, organs, and/or animals having one or more modified genes for enhanced xenograft survival and/or tolerance
KR1020217041209A KR20220033468A (ko) 2019-05-16 2020-05-15 강화된 이종이식편 생존 및/또는 내성을 위한 하나 이상의 변형된 유전자를 갖는 세포, 조직, 장기 및/또는 동물
AU2020274150A AU2020274150A1 (en) 2019-05-16 2020-05-15 Cells, tissues, organs, and/or animals having one or more modified genes for enhanced xenograft survival and/or tolerance
JP2021568652A JP2022532783A (ja) 2019-05-16 2020-05-15 向上した異種移植生存及び／又は耐性のための１つ以上の改変遺伝子を有する細胞、組織、器官、及び／又は動物
MX2021013914A MX2021013914A (es) 2019-05-16 2020-05-15 Células, tejidos, órganos y/o animales que tienen uno o más genes modificados para supervivencia y/o tolerancia de xenoinjerto mejorada.
CN202080050306.1A CN115176020A (zh) 2019-05-16 2020-05-15 具有一个或多个用于增强异种移植物存活和/或耐受性的经修饰基因的细胞、组织、器官和/或动物
BR112021023029A BR112021023029A2 (pt) 2019-05-16 2020-05-15 Células, tecidos, órgãos e/ou animais tendo um ou mais de genes modificados para sobrevivência e/ou tolerância de xenoenxerto potencializada(s)
SG11202112675SA SG11202112675SA (en) 2019-05-16 2020-05-15 Cells, tissues, organs, and/or animals having one or more modified genes for enhanced xenograft survival and/or tolerance
PCT/CN2020/090440 WO2020228810A1 (fr) 2019-05-16 2020-05-15 Cellules, tissus, organes et/ou animaux ayant un ou plusieurs gènes modifiés pour une survie et/ou une tolérance améliorée à la xénogreffe
CA3139928A CA3139928A1 (fr) 2019-05-16 2020-05-15 Cellules, tissus, organes et/ou animaux ayant un ou plusieurs genes modifies pour une survie et/ou une tolerance amelioree a la xenogreffe
EP20806445.1A EP3969596A4 (fr) 2019-05-16 2020-05-15 Cellules, tissus, organes et/ou animaux ayant un ou plusieurs gènes modifiés pour une survie et/ou une tolérance améliorée à la xénogreffe
IL288049A IL288049A (en) 2019-05-16 2021-11-11 Cells, tissues, organs and/or animals with one or more modified genes to improve xenograft survival and/or tolerance

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CN102548394A (zh) * 2009-08-14 2012-07-04 雷维维科公司 用于糖尿病治疗的多转基因猪
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CN102548394A (zh) * 2009-08-14 2012-07-04 雷维维科公司 用于糖尿病治疗的多转基因猪
CN107249318A (zh) * 2014-12-10 2017-10-13 明尼苏达大学董事会 用于治疗疾病的遗传修饰的细胞、组织和器官
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