Systems and methods for bioengineering hypoimmunogenic organs, tissues and cells

Abstract

Systems and methods for bioengineering organs, tissues and cells universally immunocompatible with recipients and organs, tissues and cells obtained or obtainable with the system and by methods, including a system comprising: a) a donor cell having one or more genomic mutations which suppress expression of major histocompatibility complex I (MHCI) and / or major histocompatibility complex II (MHCII); and b) a recipient fertilized egg of a non-human mammal, wherein the recipient fertilized egg is a gene-edited fertilized egg having one or more genomic mutations which suppress innate-like immunity and / or primitive hematopoiesis, said gene-edited fertilized egg capable of producing an embryo having suppressed innate-like immunity and / or primitive hematopoiesis when said gene-edited fertilized egg is implanted into a pseudopregnant non-human female mammal; and wherein the recipient fertilized egg has one or more genetic mutations which suppress development of one or more of organs, tissues or cells in the embryo.

Description

Systems And Methods For Bioengineering Hypoimmunogenic Organs, Tissues And CellsCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to the U.S. provisional patent application 63 / 659,971 filed June 14, 2024, the entire disclosure of which is herein incorporated by reference in its entirety.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format. Said .XML copy, created on June 6, 2025, is named “4227-155776PCT.xml” and is 30,910 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0003] This disclosure relates to the field of tissue and organ bioengineering and provides systems and methods for bioengineering organs, tissues and cells that may be universally compatible with recipients, and transplantation organs, tissues and cells obtainable by using the systems and methods.BACKGROUND

[0004] Organ transplantation may be the only treatment that can prolong a life of a patient with the end-stage organ failure. However, successful treatment of these patients is hindered by a shortage of immunocompatible organs. Despite advances in immunosuppressive regimens, graft rejection and side effects still remain a major challenge.

[0005] To address these issues, US patent publication US 2022 / 0338452 describes a blastocyst complementation method for generating pluripotent stem cell (PSC)- derived organs in rodents. It was reported that a PSC-derived pancreas could be generated within the empty embryonic pancreatic niche of a knockout (KO) animal incapable of generating a pancreas. The blast complementation approach also allows for generation of functional exogenic organs of one species within another (e.g., mouse organ in a rat) (Kobayashi et al. 2010). The exogenic pancreatic islet cells derived from these xeno-chimeras efficiently maintain long-term glucose homeostasis when transplanted into a syngeneic host without requiring immunosuppression (Yamaguchi et al. 2017). These findings highlight a therapeutic potential of autologous exogenic organs bioengineered by interspecies blastocyst complementation. By using this method, several functional organs, including kidney, vascular endothelial cells and hematopoietic cells, and lungs have been bio-engineered in rodents (Usui et al. 2021; Hamanaka et al. 2018; and Mori et al. 2019) and in pigs (Matsunari et al. 2013; Matsunari 2020), further highlighting the broad applicability of the blast complementation method.

[0006] In application to bio-engineering tissues and organs for human patients, one of the main challenges is still considered to be the need for producing a personalized and immuno-compatible organ for each particular human patient, which may be timeconsuming, technologically challenging and / or economically unsustainable.

[0007] Thus, there remains the need in the field for systems and methods that may be used to bioengineer a hypoimmune organ or tissue which may be universally immunocompatible with a number of human transplant recipients such that atransplant rejection by human transplant recipients can be avoided, or at least minimized.SUMMARY

[0001] At least some of these needs may be addressed by the present disclosure which provides systems and methods for bioengineering organs, tissues and cells that are hypoimmunogenic and universally compatible with recipients, including inter-species recipients such that, these bioengineered organs, tissues and / or cells can be used off- the-shelf for transplantation to multiple recipients, including allogeneic transplantations, and preferably without the need for an immunosuppressive drug or HLA -matching, and also eliminating the need for producing an organ or tissue separately for each recipient. In embodiments, these systems and methods may be used for bioengineering an organ, tissue or cells for intra and inter-species transplantation, including embodiments in which an organ, tissue or cells for transplantation into a human recipient is bioengineered in a non-human mammal.

[0002] In one aspect, this disclosure provides a system for bioengineering a hypoimmunogenic organ, tissue or cell, the system comprising: a) a donor cell wherein the donor cell is a progenitor cell, stem cell, pluripotent stem cell or induced pluripotent stem cell, said donor cell having one or more genomic mutations which suppress expression of major histocompatibility complex I (MHCI) and / or major histocompatibility complex II (MHCII); and b) a recipient fertilized egg of a non- human mammal, wherein the recipient fertilized egg is a gene-edited fertilized egg having one or more genomic mutations which suppress innate-like immunity and / or primitive hematopoiesis, said gene-edited fertilized egg capable of producing anembryo having suppressed innate-like immunity and / or primitive hematopoiesis when said gene-edited fertilized egg is implanted into a pseudopregnant non-human female mammal; and wherein the recipient fertilized egg has one or more genetic mutations which suppress development of one or more of organs, tissues or cells in the embryo.

[0003] Embodiments of the system include those, wherein the donor stem cell has a knock-out mutation in CIITA gene and a knock-out mutation in at least one of the following genes: CITA (NLRC5) gene or B2m gene.

[0004] Preferably, the fertilized egg carries a knock-out mutation in one or more of the following genes: Tall, GATA2, Runxl, and / or PU.l.

[0005] Particularly preferred embodiments include those wherein the donor cell is a human progenitor cell, stem cell, pluripotent stem cell or induced pluripotent stem cell.

[0006] In some embodiments, the system may further comprise a recipient non-human female mammal of the same species as the recipient fertilized egg, the recipient non- human female mammal suitable for being a surrogate parent and for implantation of a blastocyst grown in vitro from the fertilized egg into which the donor stem cell was injected.

[0007] In particularly preferred embodiments, the system may further include one or more single guide RNAs (sgRNAs) suitable for genome editing the donor cell or the recipient fertilized egg.

[0008] In particularly preferred embodiments, the fertilized egg may carry a mutation which blocks embryonic development of one or more organs or tissues, e.g. Sall-1 orPdx-1 gene, and / or wherein the organ may be preferably a kidney, pancreas or thymus.

[0009] In some preferred embodiments, the donor stem cell may be an induced pluripotent stem cell.

[0010] In other aspect, this disclosure relates to an organ, tissue or cells obtainable or obtained by a method comprising at least the following steps performed with the system according to this disclosure, the method comprising:1) injecting the fertilized egg at the blastocyst stage with the donor cell and obtaining a chimera;2) implanting the chimera obtained in step 1) into a pseudo-pregnant nonhuman female mammal of the same species as that for the fertilized egg;3) growing a chimeric embryo; and4) harvesting an organ, tissue or cells, wherein the harvested organ, tissue or cells is / are composed of cells having the same genotype as the donor cell.

[0011] In yet another aspect, this disclosure relates to a method for treating a subject in need of a transplant, the method comprising: transplanting into the subject the organ, tissue and / or cells obtained or obtainable by the methods according to this disclosure, wherein the subject is of the same species as the donor cell.

[0012] Preferably, the method can be performed without using an immunosuppressive drug and / or without HLA-matching the subject with the organ, tissue and / or cells to be transplanted.

[0013] The methods and systems according to this disclosure may be useful for treating various diseases, including for treating a subject having an organ failure. The methods and systems may be also used for treating at least some types of infertility.

[0014] In yet another aspect, this disclosure relates to a method for engineering a hypoimmunogenic organ, tissue or a cell, the method comprising: a) genome editing a fertilized egg of a non-human mammal at the two- pronucleus zygote stage to suppresses innate-like immunity and primitive hematopoiesis when an embryo is developing from the fertilized egg; b) growing the fertilized egg to a blastocyst stage in vitro,- c) contacting a blastocyst of step b) with a gene-edited donor cell having suppressed expression of MHCI and / or MHCII genes to produce a chimeric blastocyst; d) implanting the chimeric blastocyst into a pseudo-pregnant non- human female mammal; e) feeding the non-human mammal of step d) through prenatal gestation of implanted chimeric blastocysts; and f) harvesting the organ, tissue and / or cells comprising the off-spring of the donor cell after prenatal gestation of step e) is completed partially or completely.

[0015] In embodiments, the method may further comprise prior to step c), genome-editing the donor cell to carry a knock-out mutation in CIITA gene and aknock-out mutation in at least one of the following genes: CITA (NLRC5) gene or B2m gene.

[0016] In some embodiments of the method, genome editing in the fertilized egg may include a knock-out mutation in one or more of the following genes: Tall, GATA2, Runxl, and / or PU.1. Preferably, the donor cell and the fertilized egg are not from the same species and more preferably, the donor cells may originate from a human species, while the fertilized egg may originate from a mammalian non-human species.

[0017] In embodiments of the method, the genome editing may be carried out by transfecting the donor cell with Cas9 enzyme and one or more of the following single guide RNAs (sgRNAs): SEQ ID NOs: 1-14, or any combination thereof; and / or by transfecting the recipient fertilized egg, preferably at the one cell zygote stage, with Cas 9 enzyme and one or more of the following sgRNAs: SEQ ID NOs: 15-17, or any combination thereof.

[0018] In particularly preferred embodiments of the method, the fertilized egg may carry a mutation which blocks embryonic development of one or more organs or tissues, preferably, organs such as a kidney, pancreas or thymus. Preferably, the donor cell may be an induced pluripotent stem cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figs. 1A-1D show a Single Cell Gene Expression Analysis of MHCRegulators in Mouse Embryos. A preliminary analysis of published mouse embryo single-cell RNA sequencing data from E6.5 to E8.5 stages (PMID: 30787436) illustrates differential expression patterns of Major Histocompatibility Complex(MHC) I and II regulators across various cell types. The panel is a UMAP showing single-cell gene expression patterns of genes in the mouse embryo (E6.5-E8.5). Fig. 1A reveals that the MHC I regulator, B2m, is ubiquitously expressed across all analyzed cell types, suggesting a widespread role during these early developmental stages. In contrast, Fig. IB shows minimal to no expression of the MHC II regulator, Ciita, across the cell types examined. Similarly, Fig. 1C displays the limited expression of Nlrc5 (Cita), the MHC I master regulator, indicating its more restricted regulation or function at these stages of embryogenesis. Fig. ID showcases a UMAP plot highlighting various embryonic lineages, each marked as distinct clusters, serving as the foundation for representing the expression patterns observed in the above figures, elucidating the diverse cellular landscape within mouse embryos during early development stages (E6.5-E8.5).

[0020] Fig. 2 is a schematic overview of knockout (KO) generation of MHC regulators in mouse pluripotent stem cells.

[0021] Fig. 3 reports a population doubling time (PDT) of MHC engineered mouse PSCs (mPSCs) in which regulators of MHCI and MHC II contain genomic knock-out mutations.

[0022] Fig. 4 reports a significant reduction in chimerism from MHC knock-out (KO) mPSCs.

[0023] Fig. 5 reports an unexpected role of MHCs at the matemo-fetal interface during early gestation.

[0024] Fig. 6 reports the impact of MHC modifications on chimerism and embryonic development.

[0025] Fig. 7 reports evaluation of inducible MHC expression on chimerism and cell fate.

[0026] Fig. 8 reports flow cytometry analysis of cellular composition of El 1.5 mouse embryos.

[0027] Figs. 9A and 9B report a rescue of MHC dKO cell elimination in Pu.1 (Spil) KO embryos; with a representative image being shown in Fig. 9A and a dot plot quantifying this analysis being shown in 9B.

[0028] Fig. 10 reports a rescue of organ chimerism in Pu.1 (Spil) knock-out (KO) embryos.DETAILED DESCRIPTION

[0008] In this disclosure, molecular biology, genetics, embryology and biochemistry terms are used in their common meanings, unless specified otherwise.

[0009] A knock-out (KO) mutation of a gene of interest refers to a genomic mutation introduced into the gene, e.g. into its coding or non-coding sequence, said mutation resulting in no functional protein being produced from the mutated gene. Examples of genomic mutations include, but are not limited to, a deletion, insertion, replacement of one or more nucleotides, or any combination thereof.

[0010] A human gene may be referred to by a gene name with letter “h” before the gene name and a corresponding mouse gene may be referred to with letter “m” before the gene name. For example, hCIITA stands for human CIITA gene, while mCIITA stands for mouse CIITA gene.

[0011] In this disclosure, the term “hypoimmune organ, tissue or cell” may be used interchangeably with the term “universally immuno-compatible organ, tissue or cell.” These terms refer to an organ, tissue or cell which is genetically manipulated, preferably by gene editing, in order to suppress expression of major histocompatibility complex (MHC) class I and / or class II proteins.

[0012] Genome or gene editing refers to methods in which a cellular genome is modified by introducing a deletion, insertion and / or substitution of one or more nucleotides in a gene of interest by transfecting cells with a DNA nuclease such as CAS 9 or its variants or and a guide RNA having a matching sequence to the gene of interest, guiding the nuclease to cleave the gene of interest and gene editing the gene of interest with an insertion-deletion (IND EL). (Nishimura et al. Cell Stem Cell. 2021 Jan. 7; 28(1): 141-149)

[0013] In this disclosure, the term “suppressed expression of a gene, such as for example as MHC class I and / or MHC class II” means that RNA and / or protein expression from the gene is reduced in comparison to a control cell which was not gene edited to produce a gene suppression.

[0014] The MHC of human origin may be referred to as the human leukocyte antigen (HLA). (Immunology: the Immune System in Health and Disease, 5thedition (2001)). MHC glycoproteins are used by the immune system to identify cells and tissues within the body as “self’ or “non-self ’ and to initiate an immune response aiming at destroying cells and tissues identified as “non-self.” MHC class I proteins are expressed on all cell surfaces and play a central role in antigen presentation, T cell maturation and selection (Becar and Kasi, 2022). MHC class II proteins are expressedon immune cells, including B cells, monocytes, macrophages and dendritic cells and epithelial cells following inflammatory signals (Rock et al et. Trends Immunol. 2016 Nov; 37(11): 724-737. As genes encoding the MHCs are highly polymorphic, it is rare to find two individuals with the same MHC identity, thus making organ transplantation extremely challenging because a transplanted organ is likely to be identified as “non-self,” and then may be targeted for distraction. The present disclosure provides systems and methods helpful in overcoming this challenge and thereby minimizing a rejection of transplants and also eliminating the need that a donor and recipient would be compatible in their HLA expression profile.

[0015] With the disclosed herein systems and methods, hypoimmunogenic organs, tissues and cells may be bioengineered from a donor cell having one or more genomic mutations which suppress expression of MHC type I and / or MHC II genomic locus. Preferably, the donor cell is gene-edited and has one or more loss-of-function genomic mutations in MHCI and MHCII regulators, and / or in the MHC type I and / or type II genes. In preferred embodiments, the donor cell may contain a genomic-loss- of-function mutation in one or more of the following genes: CIITA and one or more from CITA or B2m gene. However, other genes, including, but not limited to, TAPI, TAP2 may be also used for producing a loss-of-function mutation in MHCI and MHCII regulators. In at least some other embodiments, some other MHCI and MHCII regulators, such as for example viral proteins, including human cytomegalovirus (HCMV) US2, US11, US3, or US6, human immunodeficiency virus (HIV) Nef, or adenovirus E3 / 18.5K may be overexpressed, resulting in downregulation of MHCI or MHCII expression.

[0016] Referring to Table 1, it provides a list of MHCI and MHCII regulators, including incorporation by reference of gene sequences by NCBI ID and Ensemble ID numbers.Table 1.

[0017] CIITA is a human gene which encodes a protein called the class II, major histocompatibility complex, transactivator. This protein contains an acidic transcriptional activation domain, 4 LRRs (leucine rich repeats) and a GTP binding domains. The donor cells according to this disclosure may have a knock-out mutation in CIITA gene by which RNA expression and / or production of functional CIITA protein is eliminated. In order to knock-out CIITA gene, a gene editing technology can be used by which one or more nucleotide deletions, insertions and / or nucleotide replacements are introduced into CIITA gene, resulting in no functional CIITA protein being produced and therefore leading to blocking expression of HLA class II in the donor cells.

[0018] CITA / NLRC5 gene is a critical transcriptional regulator of MHC class I gene expression. The donor cells according to this disclosure may have a knock-out mutation in CITA gene by which RNA expression and / or production of functional CITA protein is eliminated. In order to knock-out CITA gene, a gene editing technology can be used by which one or more nucleotide deletions, insertions and / or nucleotide replacements are introduced into CITA gene, resulting in no functional CITA protein being produced and therefore leading to blocking expression of HLA class I in the donor cells.

[0019] B2m gene is beta two macroglobulin (B2M) is a component of MHC class I molecules. The donor cells according to this disclosure may have a knock-out mutation in B2m gene by which RNA expression and / or production of functional B2m protein is eliminated. In order to knock-out B2m gene, a gene editingtechnology can be used by which one or more nucleotide deletions, insertions and / or nucleotide replacements are introduced into B2m gene, resulting in no functional B2m protein being produced and therefore leading to blocking expression of HLA class I in the donor cells.

[0020] The donor cells according to this disclosure may include, but are not limited to, donor cells of human origin. Preferably, the donor cells may be progenitor cells, stem cells, including embryonic stem cells, pluripotent stem cells, hematopoietic stem cells, induced pluripotent stem cells or any other undifferentiated cells suitable for bioengineering tissues, organs and / or cells in chimeric embryos. Mostly preferred stem cells are pluripotent stem cells, which include induced pluripotent stem cells which can be produced according to a protocol reported in Ang et al. Cell, 2022 Jul 7;185(14):2523-2541.

[0021] In some preferred embodiments, the donor cells, preferably pluripotent stem cells and more preferably induced pluripotent stem cells are manipulated, preferably by one or more of gene-editing technologies which may include, but are not limited to CRISPRi (CRSPR interference which uses a deactivated Cas9 nuclease fused to a repressor construct to block transcription of target genes, without cutting DNA), shRNA (a short hairpin RNA which can used to silence target gene expression via RNA interference (RNAi), RNAi (RNA interference), TALEN (transcription activator-like effector nucleases), ZFN (zine-finger nucleases) or utilizing CRISPR- Cas9 or Cpf-1 DNA nucleases, to inactivate genes which encode transcriptional regulators of MHC type I and MCH type II genes or to express viral proteins, including those listed in Table 1. In particularly preferred embodiments, theinactivated regulators may be CIITA gene (a regulator of MHC class II genes) and one or more of B2m gene or CITA (two known regulators of MHC class I genes, Downs et al. Biofactors. 2016 Jul 8;42(4):349-57; and Zijlstra et al. Nature. 1989 Nov 23;342(6248):435-8; Koller et al. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8932- 5.). However, it should be understood that any other transcriptional regulators of MHC I or MHCII, e.g. TAPI or TAP2, can be also inactivated instead of CIITA and / or B2m or CITA, so long as eliminating these regulators downregulates (suppresses or blocks) expression of MHCI and / or MHCII in a donor cell.

[0022] Preferably, the regulars may be inactivated by gene editing and more preferably by transfecting cells with a ribonuclear complex comprising a CAS9 nuclease and single guide RNA (sg RNA) sequence specific for inserting an indel and thereby knocking-out expression of the CIITA gene and then also using one or more of sg RNA specific to inserting an indel and thereby knocking-out expression of the B2m gene or CITA gene. The MHCI and MHCII regulators may by gene-edited stepwise wherein one regulator is knocked out first, and then the second regulator is knocked out in cells that already carry a knock out mutation for the first regulator. In the alternative embodiments, MHCI and MHCII regulators may be gene-edited at the same time.

[0023] One embodiment of this method is shown in Fig. 2 which is a schematic overview of knockout (KO) generation of mouse pluripotent stem cells, but the same technology can be also used for generating a knockout of CIITA and at least one from B2m and CITA genes in any donor stem cell. In order to disrupt MHCII expression, CIITA gene is knocked out. Subsequent knockouts target either the B2m or Cita(Nlrc5) genes to eliminate MHC I expression in the MHC II KO cells. This sequential knockout strategy aims to delineate the roles of MHC expression in embryonic development and its influence on the integration of cells within the host.

[0024] In some embodiments of this method, pluripotent stem cells, e.g. mouse PSCs (mPSCs) or human pluripotent stem cells (hPSCs) may be cultured in tissue culture and subjected to a knock-out process in which cells may be electroporated with a ribonucleoprotein (RNP) complex composed of Cas9 enzyme and a sgRNA including a sequence for recognition and binding to CIITA gene. After the knockout is completed, single-cell clones (SCC) are isolated and analyzed using ICE analysis (a bioinformatic tool for analyzing sequencing data to infer and quantify the efficiency and types of edits (mutations) generated by CRISPR-Cas9 genome editing experiments developed by Synthego). A clonal analysis is then performed to identify those with the most effective KO phenotype. These CIITA knock-out (KO) clones are then used in second round of genome-editing in which one or CITA or B2m genes is knocked out.

[0025] Referring to Table 2, it provides nucleotide sequences for sgRNAs which may be used in combination CRISPR-Cas9 in systems and methods according to this disclosure.Table. 2

[0026] In addition to gene-edited MHCI and MHCII regulators, donor cells preferably are also modified to overexpress BCL2, CD47, CD24, or any combination thereof, or some other gene having a function similar to CD47, which can be defined as preventing elimination of donor cells by embryo innate immune cells like macrophages.

[0027] As reported in Figs. 3-8, it was unexpectedly found that suppressing expression of MHCI and MHCII results in a significant decrease of chimerism and minimized, if at all, contribution from donor cells.

[0028] Fig. 3 reports the population doubling time (PDT) of MHC engineered mouse PSCs (mPSCs). This bar graph compares the PDT of wild-type (WT) mPSCs with those genetically engineered to lack specific Major Histocompatibility Complex (MHC) components: Ciita knockout (KO) resulting in MHC II loss, Ciita+B2m indicating MHC I and II loss, and Ciita+Cita (Nlrc5) also denoting MHC I and II loss. For this analysis, cells were seeded at a density of 14K / cm2on irradiated mouse embryonic fibroblasts (MEF) and counted every 3 days using NucleoView NC3000 slides. mPSCs were cultured in GMEM supplemented with 10% FBS, 2- mercaptaethanol, NEAA, GlutaMAX, sodium pyruvate, PD0325901 (luM), CHIR99021 (3uM), and LIF (either hLIF at 20ng / ml or ESGRO mouse LIF at IX). The PDT calculation revealed no significant differences among the cell types, indicating that MHC modification does not affect the proliferation rate of mPSCs under these culture conditions. The analysis reveals no significant differences in PDT among the various cell types, suggesting that alterations in MHC expression do not impact the proliferation rate of mPSCs.

[0029] Fig. 4 reports chimerism results from pups bom from mouse blastocysts injected with wild type (WT) and MHC double knockout (dKO) mPSCs derived from C57B6 mice, leading to black coat color, injected into white coat color CD-I blastocysts. Approximately 8-10 mPSCs were injected per blastocyst, with a total of 20 blastocysts transferred into pseudopregnant sows for each experimental group (B and C). Chimerism was assessed based on coat color contribution, with panel 4A showing extensive black coat color chimerism from WT mPSCs, indicating successful integration and contribution to embryonic development. In contrast, panels 4B {Ciita + B2m KO) and 3C {Ciita + Cita / Nlrc5 KO) exhibit minimal to no black coat color chimerism, demonstrating the critical impact of MHC expression loss on the cells' ability to contribute to the development, likely due to early embryonic rejection.

[0030] Fig. 5 illustrates the increased rate of resorption of MHC dKO chimeric embryos at El 1.5, marked by arrows. Embryo development requires immune tolerance from maternal tissues, which adapt to support a genetically foreign fetus. Post-implantation, uterine stromal cells envelop the embryo, forming a decidua that sustains development until placentation (around E9.5). The decreased contribution of MHC KO chimeric cells might involve complex maternal immune interactions. Notably, NK cell dysfunction within these maternal immune populations, which make up 30-40% of all cells during early gestation, could lead to early embryonic loss. This underscores the significance of innate-like cells and those of myeloid origin in maternal-fetal immune surveillance, suggesting potential avenues for enhancing outcomes in reproductive technologies such as IVF by fostering a more conducive maternal environment.

[0031] Fig. 6 reports an impact of MHC modifications on chimerism and embryonic development. (A) Representative images of mPSC integration into wiltd- type (WT) mouse embryos, showcasing the effects of various mPSC modifications on embryonic development. Modifications include WT mESC, B2m knockout (KO), Ciita KO, Ciita "t- B2m double knockout dF O), and double F Os with constitutive overexpression of BCL2, Cd47, or both. (B) Quantitative analysis of chimerism in El 1.5 mouse embryos. This dot plot depicts chimerism levels achieved under different mPSC modification conditions, calculated as the percentage ratio of donor (B6-derived mPSCs) to host (CD-I embryo) cells assessed by ddPCR. Mean chimerism levels are as follows: WT mESC at 21.67%, B2m KO at 14.40%, Ciita KO at 6.41%, dKO at 1.59%, dKO + BCL2 at 3.59%, dKO + Cd47 at 9.78%, and dKO + BCL2 + Cd47 at 19.60%. The loss of MHC expression surprisingly leads to significant reductions in chimerism, suggesting a critical role for MHC in embryonic acceptance and integration. While individual overexpression of BCL2 or Cd47 partially mitigates this loss, their combination significantly enhances chimerism. However, this is accompanied by abnormal development patterns in embryos, as indicated by the arrowheads in the dKO + BCL2 + Cd47 condition. These observations underscore the complex implications of manipulating immune evasion mechanisms in embryonic development and highlight the challenges of engineering hypoimmunogenic organs without unintended developmental consequences.

[0032] Fig. 7 reports evaluation of inducible MHC expression on chimerism and cell fate. (A) Representative images of embryos at El 1.5 after Tet-On inducible expression of B2m, Ciita, and both (dKO + dOE) in dKO cells. (B) Effects ofdoxycycline induction for three days specifically in the dKO + dOE condition, showcasing abnormal and random differentiation patterns in mESC culture media. This phenotype was not observed with WT, dKO and dKO + single gene inductions or (data not shown). (C) A dot plot from ddPCR assays quantifying chimerism, indicating that inducible ectopic expression of MHC genes via doxycycline does not rescue the cell elimination phenotype, following a protocol described in Suchy et al. Sci Rep. 2022 Jun 17; 12(1): 10223, and by using the primer probe for quantification as described. Briefly, Tyr gene is used (the gene for coat color). Host embryo (from CD-I mouse) in these experiments are Albino (white color) and donor mPSC (from C57BL6 mouse) are Wildtype prob (black coat color). Forward Primer; mTyr-F / 1;1.8 pM; AAT AGG ACC TGC CAG TGC TC (SEQ ID NO: 18) Reverse Primer; mTyr-R / 1; 1.8 pM; TCA AGA CTC GCT TCT CTG TACA (SEQ ID NO: 19) Albino probe; mTyr-alb-P / 1; 0.25 pM; FAM-cttaGagtttccgcagttgaaaccc-Zen / IowaBlack (SEQ ID NO: 20) Wildtype probe; mTyr-wt-P / 1; 0.25 pM; HEX-cttaCagtttccgcagttgaaaccc- Zen / IowaBlack (SEQ ID NO: 21).

[0033] Throughout the experiments, both the cells and pseudopregnant recipients were maintained on doxycycline. The data reveal that dKO + dOE (double overexpression) leads to further reduction in cell survival, likely due to enhanced differentiation prompted by induced expression of the genes based on mean chimerism levels of dKO + B2m: 2.45%, dKO + Ciita: 4.07%, and dKO + dOE:1.80% respectively. This unexpected finding highlights that, contrary to previous beliefs, MHC molecules may influence embryonic stem cells (ESCs) and embryonicdevelopment, as their induced overexpression leads to abnormal phenotypes not typically associated with their known functions.

[0034] Fig. 8 provides cellular composition of El 1.5 mouse embryos by flow cytometry. Whole El 1.5 CD-I mouse embryos were dissociated into single cells and subjected to live cell flow cytometry to identify various hematopoietic lineages: Pan hematopoietic cells (CD45+), Myeloid lineage (CD45+, CDl lb+), Macrophages (CD45+, CDl lbfy F4 / 80 High), Macrophage Precursors (CD45+, CDl lb+, F4 / 80 Low), Granulocytes (CD45+, CDl lb+, F4 / 80-, Ly6G+). While no detectable staining for T cells, B cells, and NK cells (using markers CD3+, B220+, and NK1.1+ respectively) was observed, the predominant presence of macrophages and their precursors indicates a substantial innate immune activity. Each dot representing an embryo showed approximately 50% of macrophages and 40% macrophage precursors in the CD45+ population. This suggests that other innate or innate-like cells, possibly including novel non-professional immune cells, may be active at this or earlier gestational stages, contributing to the potential elimination of the MHC dKO or other outlier cells with the developing embryo.

[0035] In order to address the problem of innate-like immune response to donor cells which carry loss-of-function mutations in MHCI and MHCII regulators, the inventors gene-edited recipient fertilized eggs.

[0036] It has been unexpectedly found that introducing a loss-of-function mutation into one or more of the following genes: Tall, GATA2, Runxl, and / or PU.1 suppresses the innate-like immune response to donor cells and increases chimerism and contribution from donor cells with gene-edited MHCI and MHCII regulations intodifferent organs, thereby making it possible to form embryos developing hypoimmune organs, tissues and cells in a non-human mammal recipient. Referring to Table 3, it summarizes information about Tall, GATA2, Runxl, and / or PU. l regulators of embryonic innate-like immunity and / or primitive hematopoiesis, while DNA sequences for these mouse and human genes are listed in Table 1 by reference to NCBI Gene ID and Ensemble Gene ID.Table 3. Regulators Of Embryonic Innate-Like Immunity And / Or Primitive Hematopoiesis.

[0037] Referring to Figs. 9A and 9B, they report results of rescuing MHC I andII double knockout (dKO) cell elimination in Pu.l KO embryos. Fig. 9A depicts arepresentative image showing a significant reversal of MHC dKO cell elimination when injected into Pu.1 KO embryos at El 1.5. Fig. 9B is a dot plot quantifying this effect using ddPCR assay, demonstrating that the absence of Pu.1 -a key regulator of myeloid lineage development during early gestation - markedly reduces the rejection of MHC dKO cells. The data shown includes mean chimerism levels for WT (21.67%), dKO (1.59%), and dKO cells injected into PU.l KO embryos (14.53%). This significant rescue effect suggests that combining PU.1 KO embryos with hypoimmunogenic cells (dKO+CD47), which already show reduced elimination, could facilitate the generation of hypoimmune organs. Such an approach may revolutionize organ transplantation by enabling the development of universally compatible organs without the need for lifelong immunosuppression.

[0038] Referring to Fig. 10, it reports a rescue of organ chimerism, as assessed in the ddPCR analysis at stage El 8.5. This dot plot illustrates the enhanced chimerism levels across various organs from El 8.5 mouse embryos when comparing MHC dKO cells injected into wild-type CD-I embryos versus PU.l KO embryos. Each dot represents the chimerism level in a specific organ of an individual embryo. Assessed organs included blood, brain, heart, intestine, kidney, liver, lung, pancreas, spleen, thymus and the 'shell,' which refers to the remaining embryonic carcass excluding the organs listed and primarily comprises developmental cell lineages like fibroblasts and residual mesenchymal cells. Notably, all organs in PU.l KO embryos exhibit significantly higher chimerism compared to those in CD-I embryos. Mean chimerism levels are: Blood (7.4% in CD-I, 59.75% in PU.l KO), Brain (1.12%, 15.13%), Heart (3.14%, 20.38%), Intestine (3.22%, 19.97%), Kidney (2.38%,29.13%), Liver (7.12%, 48.72%), Lung (5.94%, 41.40%), Pancreas (6.14%, 41.37%), Spleen (9.1%, 63.37%), Thymus (0.7%, 67.60%), and Shell (2.96%, 28.85%). These results significantly highlight the successful rescue of MHC dKO cell chimerism through PU.1 targeting, indicating PU.1 critical role in enhancing the compatibility and survival of hypoimmunogenic precursor cells.

[0039] As illustrated in Fig. 10, a combination of hypoimmune cells (MHC dKO + CD47 overexpression) with PU.1 knockout could further optimize the generation of hypoimmunogenic organs, potentially eliminating the need for lifelong immunosuppression. Notably, the enhanced chimerism across all embryonic lineages in PU.l KO host embryos injected with MHC dKO cells underscores the widespread improvement in donor cell integration during embryonic development.

[0040] Importantly, in the methods according to the present disclosure when a hypoimmune donor cell is injected into a blastocyst composed of blastomeres in which PU.1 gene has been knocked out, mesoderm-derived tissues or organs such as the heart, spleen, thymus, and kidney, which develop from the mesoderm, show enhanced chimerism from the donor cell progeny. This could be due to the role of PU.1 in regulating hematopoiesis and immune cell maturation, which are crucial in mesodermal tissues. By knocking out PU.1, the local immune environment within these mesodermal organs is altered, potentially reducing resistance against the engraftment of donor cells.

[0041] As is also shown in Fig. 10, endoderm-derived tissues and organs such as the liver, intestine, lung, and pancreas also exhibit increased chimerism. WhilePU.1 does not directly influence endodermal cell differentiation, its impact on theimmune landscape — especially on organ-specific macrophages — can modify how these tissues respond to foreign cells. For instance, in the liver and lungs, where resident macrophages play a critical role in tissue homeostasis and immune surveillance, reduced PU.l expression may lessen the immune barriers, facilitating better integration and survival of hypoimmunogenic cells.

[0042] As is further reported in Fig. 10, the brain, primarily derived from the ectoderm, also shows enhanced chimerism under PU.l knockout conditions. Without wishing to be bound by this theory, the inventors believe that this improvement may be attributed at least in part to a modulation of microglial activity — brain-specific immune cells derived from mesodermal lineage — whose function and maturity are known to be influenced by PU.1. By mitigating the microglial-mediated immune response, the neuronal environment may become more conducive to the integration of these injected foreign or outlier donor cells.

[0043] As is further reported in Fig. 10, the shell, representing residual non- organ-specific tissues comprising multiple embryonic origins, indicates a generalized improvement in the acceptance of the injected MHC dKO donor cells across tissues. This suggests that the PU.1 knockout enhances overall tissue receptiveness to foreign cells by dampening potential immune-like responses across embryonic lineages.

[0044] This categorization by embryonic lineage underscores the pervasive influence of PU.1 in modulating embryonic immune environments, thus facilitating a wider acceptance and integration of hypoimmunogenic cells across diverse organ systems derived from all primary germ layers.

[0045] Figs. 1-10 support a conclusion that injected MHC null (double MHC KO) or foreign (from a different species) cells into a developing embryo undergo an immune -like surveillance, which results in elimination of the injected cells. Fig. 5 also reports a higher-than-expected resorption rate in MHC dKO chimeras. The inventors report that the myeloid lineage and its derivatives may be affecters of this phenotype. However, the Pu.1 KO in a recipient fertilized egg results in perturbing this linage within a developing embryo. As demonstrated in Figs. 9 and 10, MHC null cells injected into a Pu.1 KO embryo produce a higher pan-embryonic lineage contribution. This should enable not only intra, but also interspecies organogenesis by blastocyst complementation for generation of hypoimmunogenic organs. Given the impact of immune cells in embryonic developments, these results also have implications for improving in vitro fertilization (IVF) outcomes.

[0046] In order to further increase a contribution from donor cell toward a particular lineage, the fertilized recipient eggs may carry one or more mutations in one or more genes, blocking expression of which prevents contribution from recipient cells in that organ or tissues, making it possible to bioengineer an organ, tissue or cells having predominantly a genotype of donor cells, while the rest of the chimeric embryo may have a significant contribution from recipient cells.

[0047] Referring to Table 4, it provides a list of genes which may be knocked out in a recipient fertilized egg in order to produce an organ or cell type having a genotype of the donor cell.Table 4.Organ / Cell Type Gene(s)Pancreas Pdxl, Nkx6.1, Pax4, MafA, Sox9Nephrogenesis Sall 1, Pax2, Wtl, Six2, Gdnf, EyalHepatogenesis Sekl (Map2k4), Hhex, Hnf4a, Foxa2, HnflbVasculogenesis Flkl (Kdr), Tie2 (Tek), Vegfa, PecamlLungs FgflO, Nkx2.1, Sox2, Wnt7b, Tbx4, FoxalGermline Nanos3, Prdml4, Dazl, Stra8, Oct4 (Pou5fl)Spil (Pu. l), Cebpa, Gatal, Runxl, Irf8, Tall (Scl),(Pan) Myeloid Gata2No T or B Cells Rag2, 112rg (Common gamma chain), Jak3Myeloid Csflr, Itgam (Cdl lb), Ly6g (Gr-1), MpoVasculogenesis andHematogenesis Etv2, Tall (Scl), Gata2, Runxl, Lmo2Keratinocyte Trp63 (p63), Krtl4, Krt5, Tp73, KrtlThymus Foxnl, Tbxl, Paxl, 117, Ccl25Vein Ephb4, Coup-TFII (Nr2f2), ProxlArtery Notch 1, EphrinB2, Hey2, D114Spleen Nkx2-3, Tlxl (Hoxl l), Bapxl (Nkx3.2), SpilHematopoietic Stem Cells (HSC) Runxl, Gata2, Scl (Tall), Cd34, C-Kit, FlklEye Pax6, Rax, ChxlO (Vsx2), Mitf, CrxBrain (General) Sox2, Pax6, Emx2, Otx2, NeurodiBrain (Forebrain) Foxgl, Lhx2, Dlxl, EmxlBrain (Midbrain) Enl, Otx2, Wntl, Pax3Brain (Hindbrain) Hoxbl, Krox20 (Egr2), Gbx2, Fgf8Heart Nkx2.5, Tbx5, Gata4, Mef2c, Hand2, MyocdPlacenta Gcml, Esxl, Handl, Tpbpa, Mash2 (Ascl2)Epiblast Oct4 (Pou5fl), Nanog, Sox2, MGA, Klf4T Cells Cd3, Tcra, LekB Cells Cdl9, Pax5, EbflSkeletal Muscle MyoD, Myf5, MyogeninCardiac Muscle Nkx2.5, Tbx5, Mef2c, MyocdIntestine Cdx2, Lgr5, Sox9Skin Krtl4, Krt5, Lor, FigAdipose Tissue Pparg, Cebpa, Fabp4Cartilage Sox9, Col2al, AcanBone Runx2, Osx (Sp7), Bglap (Osteocalcin)

[0048] The invention will now be further described with the following non- limiting examples.Example 1. Bioengineering Donor Cells.

[0049] With reference to Fig. 2, mouse pluripotent stem cells (mPSCs) were cultured at the density of 14K / cm2on irradiated mouse embryonic fibroblasts (MEF) and counted every 3 days using NucleoView NC3000 slides. Cells were cultured in GMEM supplemented with 10% FBS, 2-mercaptoethanol, NEAA, GlutaMAX, sodium pyruvate, PD0325901 (luM), CHIR99021 (3uM), and LIF (either hLIF at 20ng / ml or ESGRO mouse LIF at IX). For the knock out (KO) process, cells underwent electroporation using NEON (Thermofisher) using lOul tips, with the electroporation conditions set at 1600V, 10ms, and 3 pulses. Each reaction consisted of -100K cells, with a ribonucleoprotein (RNP) complex composed of 6ug HiFI Cas9 V3 (IDT) and 3.2ug sgRNA (Synthego).

[0050] Following electroporation, cells were seeded into 12-well plates at a density of ~1. IxlO5cells / well of MEFs. The knockout procedure involved initially targeting the CIITA gene using designated single-guide RNAs (sgRNAs, listed in Table 2), followed by the isolation of single-cell clones (SCC) which were analyzed using the ICE analysis (a bioinformatic tool for analyzing sequencing data to infer and quantify the efficiency and types of edits (mutations) generated by CRISPR-Cas9 genome editing experiments developed by Synthego). A clonal analysis was then performed to identify those clones with the most effective KO CIITA phenotype, signified in notations such as sgl#2 for gRNA 1, clone 2.

[0051] KO clones in which no functional CIITA protein was produced, were then subjected to a second round of genome editing with a ribonucleoprotein (RNP) complex composed of 6 pg HiFI Cas9 V3 (IDT) and 3.2 pg sgRNA (synthego). Asshown in Fig. 2, the KO CIITA cells were genome edited in order to suppress production of functional protein for either B2m or CITA with either sgRNA specific to B2m (listed in Table 2) or sgRNA specific to CITA (listed in Table 2), respectively. A clone selection for double-mutants was then performed to identify those clones with no functional CITA or B2m protein in addition to no functional CIITA protein.

[0052] Referring to Fig. 3 which reports population Doubling Time (PDT) of MHC engineered mouse PSCs (mPSCs). This bar graph compares the PDT of wildtype (WT) mPSCs with those genetically engineered to lack specific Major Histocompatibility Complex (MHC) components: Ciita knockout (KO) resulting in MHC II loss, Ciita+B2m indicating MHC I and II loss, and Ciita+Cita (Nlrc5) also denoting MHC I and II loss. Cells were seeded at a density of 14K / cm2on irradiated mouse embryonic fibroblasts (MEF) and counted every 3 days using NucleoView NC3000 slides. mPSCs were cultured in GMEM supplemented with 10% FBS, 2- mercaptaethanol, NEAA, GlutaMAX, sodium pyruvate, PD0325901 (luM), CHIR99021 (3uM), and LIF (either hLIF at 20ng / ml or ESGRO mouse LIF at IX). The PDT calculation revealed no significant differences among the cell types, indicating that MHC modification does not affect the proliferation rate of mPSCs under these culture conditions. The analysis reveals no significant differences in PDT among the various cell types, suggesting that alterations in MHC expression do not impact the proliferation rate of mPSCs.Example 2. Bioengineering Recipient Fertilized Eggs and Hypoimmunogenic Organs, Tissues and Cells.

[0053] Donor cells produced in Example 1 were injected into recipient fertilized eggs produced by the blastocyst complementation method, essentially as was described in US 2022 / 0338452.Embryo Culture and Cell Injection

[0054] Wild-type mouse embryos from CD-I mice were prepared as described (PMID: 33373620). In brief, zygotes were obtained by flushing the oviduct with M2 medium (EMD Millipore or CytoSpring) from superovulated CD-I mice the morning after confirmation of the vaginal plug (E0.5). Collected zygotes were rinsed three times in M2 medium before being transferred and cultured in KSOM-AA medium (EMD Millipore or CytoSpring) for 1-4 hours. Two-pronucleus zygotes were then used for embryo manipulation as described below. Following electroporation, zygotes were transferred back to KSOM-AA medium and incubated for 3-5 days. For micromanipulation, PSCs were trypsinized and suspended in their respective culture media. Using a piezo-driven micromanipulator (Prime Tech, Tsuchiura, Japan), the zona pellucida and trophectoderm were drilled under microscopy, and 8-10 PSCs were introduced into the blastocyst cavities near the inner cell mass. After the injection, embryos were cultured for an additional 1-2 hours before being transferred.Embryo Manipulation

[0055] Cas9 ribonucleoproteins were introduced into zygotes according to published protocols (PMID: 33373620). In brief, two-pronuclear zygotes were washed three times with Opti-MEM I medium (GIBCO, Waltham, MA). Groups of 20-30 zygotes were transferred into 5 pL of Opti-MEM I medium containing 100ng / pL Cas9 protein (IDT, Coralville, IA) and 100 ng / pL sgRNA (Synthego, Redwood City, CA) on an LF501PT1-10 electrode (BEX, Tokyo, Japan). Electroporation was performed using the Genome Editor (BEX) under the following conditions: 25 V, 3 ms ON, 97 ms OFF, Pd Alt 3 times.

[0056] Embryos were collected, cultured, and engineered as described above.After the WT and Pu.1 KO embryos were injected with MHC dKO mPSCs and transferred into pseudopregnant sows, the embryos were harvested at E17.5 and analyzed by ddPCR as described (PMID: 35715477) for chimerism quantification in Fig. 9b and Fig. 10. Briefly, the primer probe for quantification was described as follows: Tyr gene is used (the gene for coat color). The host embryo (from CD-I mouse) in our experiments is Albino (white color) and the donor mPSC (from C57BL6 mouse) is Wildtype (black coat color).

[0057] Forward Primer: mTyr-F / 1 ; 1.8 pM; AAT AGG ACC TGC CAG TGC TC (SEQ ID NO: 18)

[0058] Reverse Primer: mTyr-R / 1; 1.8 pM; TCA AGA CTC GCT TCT CTG TACA (SEQ ID NO: 19)

[0059] Albino probe: mTyr-alb-P / 1; 0.25 pM; FAM-cttaGagtttccgcagttgaaaccc- Zen / IowaBlack (SEQ ID NO: 20)

[0060] Wildtype probe: mTyr-wt-P / 1; 0.25 pM; HEX- cttaCagtttccgcagttgaaaccc-Zen / IowaBlack (SEQ ID NO: 21)References:1 Kobayashi, T. el al. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 142, 787-799, doi:10.1016 / j.cell.2010.07.039 (2010).2 Yamaguchi, T. et al. Interspecies organogenesis generates autologous functional islets. Nature 542, 191-196, doi: 10.1038 / nature21070 (2017).3 Usui, J. et al. Generation of kidney from pluripotent stem cells via blastocyst complementation. The American journal of pathology 180, 2417-2426, doi:10.1016 / j.ajpath.2012.03.007 (2012).4 Hamanaka, S. et al. Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation. Stem Cell Reports 11, 988-997, doi:10.1016 / j.stemcr.2018.08.015 (2018).5 Mori, M. et al. Generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells. Nat Med 25, 1691-1698, doi:10.1038 / s41591-019-0635-8 (2019).6 Matsunari, H. et al. Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proceedings of the National Academy of Sciences of the United States of America 110, 4557-4562, doi: 10.1073 / pnas.1222902110 (2013).7 Matsunari, H. et al. Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses by Blastocyst Complementation. Stem Cell Reports 14, 21- 33, doi: 10.1016 / j.stemcr.2019.11.008 (2020).8 Deuse, T. et al. Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nature biotechnology 37 , 252-258, doi: 10.1038 / s41587-019-0016-3 (2019).9 Han, X. et al. Generation of hypoimmunogenic human pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America 116, 10441-10446, doi: 10.1073 / pnas.1902566116 (2019).10 Xu, H. et al. Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility. Cell Stem Cell 24, 566-578 e567, doi: 10.1016 / j. stem.2019.02.005 (2019).11 Grusby, M. J. et al. Mice lacking major histocompatibility complex class I and class II molecules. Proceedings of the National Academy of Sciences of the United States of America 90, 3913-3917, doi: 10.1073 / pnas.90.9.3913 (1993).12 Masaki, H. et al. Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos. Cell Stem Cell 19, 587-592, doi: 10.1016 / j.stem.2016.10.013 (2016).13 Koller, B. H. & Smithies, O. Inactivating the beta 2-microglobulin locus in mouse embryonic stem cells by homologous recombination. Proceedings of the National Academy of Sciences of the United States of America 86, 8932-8935, doi: 10.1073 / pnas.86.22.8932 (1989).14 Zijlstra, M., Li, E., Sajjadi, F., Subramani, S. & Jaenisch, R. Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologousrecombination in embryonic stem cells. Nature 342, 435-438, doi:10.1038 / 342435a0 (1989). Pijuan-Sala, B. et al. A single-cell molecular map of mouse gastrulation and early organogenesis. Nature 566, 490-495, doi: 10.1038 / s41586-019-0933-9 (2019). Wu, Y. & Hirschi, K. K. Tissue-Resident Macrophage Development and Function. Front Cell Dev Biol 8, 617879, doi: 10.3389 / fcell.2020.617879 (2020). Palis, J. Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo. FEBS letters 590, 3965-3974, doi: 10.1002 / 1873-3468.12459 (2016). Suchy, F. P. et al. Streamlined and quantitative detection of chimerism using digital PCR. Sci Rep 12, 10223, doi: 10.1038 / s41598-022-14467-5 (2022). Nishimura, T. et al. Generation of Functional Organs Using a Cell-Competitive Niche in Intra- and Inter-species Rodent Chimeras. Cell Stem Cell 28, 141-149 el43, doi: 10.1016 / j.stem.2020.11.019 (2021). Larsen, E. C., Christiansen, O. B., Kolte, A. M. & Mackion, N. New insights into mechanisms behind miscarriage. BMC Med 11, 154, doi: 10.1186 / 1741- 7015-11-154 (2013). Mori, M., Bogdan, A., Balassa, T., Csabai, T. & Szekeres-Bartho, J. The decidua-the maternal bed embracing the embryo-maintains the pregnancy. Semin Immunopathol 38, 635-649, doi: 10.1007 / s00281-016-0574-0 (2016).

Claims

ClaimsWe claim:

1. A system for bioengineering a hypoimmunogenic organ, tissue or cell, the system comprising: a) a donor cell wherein the donor cell is a progenitor cell, stem cell, pluripotent stem cell or induced pluripotent stem cell, said donor cell having one or more genomic mutations which suppress expression of major histocompatibility complex I (MHCI) and / or major histocompatibility complex II (MHCII); and b) a recipient fertilized egg of a non-human mammal, wherein the recipient fertilized egg is a gene-edited fertilized egg having one or more genomic mutations which suppress innate-tike immunity and / or primitive hematopoiesis, said gene-edited fertilized egg capable of producing an embryo having suppressed innate-like immunity and / or primitive hematopoiesis when said gene-edited fertilized egg is implanted into a pseudopregnant non-human female mammal; and wherein the recipient fertilized egg has one or more genetic mutations which suppress development of one or more of organs, tissues or cells in the embryo.

2. The system of claim 1, wherein the donor cell contains a knock-out mutation in CIITA gene and a knock-out mutation in at least one of the following genes: CITA (NLRC5) gene or B2m gene.

3. The system of claim 1 or 2, wherein the recipient fertilized egg contains a knock-out mutation in one or more of the following genes: Tall, GATA2, Runx 1 , and / or PU.1.

4. The system of claim 1, wherein the donor cell is a human induced pluripotent stem cell.

5. The system of claim 1, wherein the system further comprises a recipient nonhuman female mammal of the same species as the fertilized egg, the recipient non-human female mammal suitable for being a surrogate parent and for implantation of a blastocyst grown in vitro from the fertilized egg into which the donor stem cell was injected.

6. The system of claim 1, wherein the system further includes one or more single guide RNAs (sgRNAs) suitable for guiding a CAS9 nuclease to gene edit the donor cell or the recipient fertilized egg.

7. The system of claim 1, wherein the fertilized egg has a genomic loss-of- function mutation in Sall 1 gene or Pdx-1 gene.

8. The system of claim 1, wherein the organ is a kidney, pancreas or thymus.

9. The system of claim 1, wherein the recipient fertilized egg is a mouse or pig fertilized egg.

10. The system of claim 1, wherein the donor cell is an induced pluripotent stem cell.

11. An organ, tissue or cells obtainable or obtained by a method comprising at least the following steps performed with the system of claim 1 :1) injecting the fertilized egg at the blastocyst stage with the donor cell and obtaining a chimera;2) implanting the chimera obtained in step 1) into a pseudo-pregnant nonhuman female mammal of the same species as that for the recipient fertilized egg;3) growing a chimeric embryo; and4) harvesting an organ, tissue or cells, wherein the harvested organ, tissue or cells are composed of cells having the same gene as the donor cell.

12. A method for treating a subject in need of a transplant, the method comprising: transplanting into the subject the organ, tissue and / or cells of claim 11, wherein the subject is of the same species as the donor cell.

13. The method of claim 12, wherein the method is performed without using an immunosuppressive drug and / or without HLA-matching the subject with the organ, tissue and / or cells to be transplanted.

14. A use of the system of claim 1 for treating organ failure or infertility wherein the use comprises transplanting an organ obtained or obtainable in the system to a subject in need of treatment for organ failure or infertility.

15. A method for engineering a hypoimmunogenic organ, tissue or a cell, the method comprising: a) genome editing a fertilized egg of a non-human mammal at the two- pronucleus zygote stage to suppresses innate-like immunity and primitive hematopoiesis when an embryo is developing from the fertilized egg;b) growing the fertilized egg to a blastocyst stage in vitro,- c) contacting a blastocyst of step b) with a gene-edited donor cell having suppressed expression of MHCI and / or MHCII genes to produce a chimeric blastocyst; d) implanting the chimeric blastocyst into a pseudo-pregnant non-human female mammal; e) feeding the non-human mammal of step d) through prenatal gestation of implanted chimeric blastocysts; and f) harvesting the organ, tissue and / or cells comprising cells having the same genotype as the donor cell after prenatal gestation of step e) is completed partially or completely.

16. The method of claim 15, wherein the method further comprises prior to step c), gene-editing the cell to have a knock-out mutation in CIITA gene and a knockout mutation in at least one of the following genes: CITA (NLRC5) gene or B2m gene.

17. The method of claim 15, wherein gene editing in the fertilized egg includes introducing a knock-out mutation in one or more of the following genes: Tall, GATA2, Runxl, and / or PU.l.

18. The method of claim 15, wherein the donor cell is a human pluripotent stem cell and the fertilized egg is from a non-human mammal species.

19. The method of claim 15, wherein gene editing is carried out by transfecting the donor cell with Cas 9 enzyme and one or more of the following single guide RNAs (sgRNAs): SEQ ID NOs: 1-14, or any combination thereof; and / or bytransfecting the recipient fertilized egg with Cas 9 enzyme and one or more of the following sgRNAs: SEQ ID NOs: 15-17, or any combination thereof.

20. The method of claim 15, wherein the fertilized egg has a genomic mutation which blocks embryonic development of one or more organs or tissues.

21. The method of claim 15, wherein the organ is a kidney, pancreas or thymus and / or the donor cell is an induced pluripotent stem cell.

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