Acceleration of human hepatocyte engraftment in humanized liver animals by supplementing paracrine ligands or agonists that activate human liver regeneration signals

Abstract

Provided are genetically modified non-human animals that are immunodeficient and optionally comprise xenotransplanted hepatocytes such as human hepatocytes, wherein the genetically modified non-human animal and / or the transplanted hepatocytes are modified to restore, activate, or increase interleukin-6 (IL-6) / interleukin-6 receptor (IL-6R) signaling pathway activity or interleukin-6 receptor subunit beta (GP130) signaling pathway activity and to restore, activate, or increase hepatocyte growth factor (HGF) / hepatocyte growth factor receptor (MET) signaling pathway activity in the transplanted hepatocytes. Also provided are methods of measuring or assessing the activity of human-liver-targeting reagents in such non-human animals and methods of making animals with a humanized liver (e.g., with reduced steatosis). Also provided are genetically modified, immunodeficient, non-human animals comprising a humanized HGF gene and optionally a humanized IL6 gene and methods of using and making such animals. Also provided are methods of increasing or accelerating engraftment of xenotransplanted hepatocytes in non-human animals.

Description

ACCELERATION OF HUMAN HEPATOCYTE ENGRAFTMENT IN HUMANIZED LIVERANIMALS BY SUPPLEMENTING PARACRINE LIGANDS OR AGONISTS THAT ACTIVATE HUMAN LIVER REGENERATION SIGNALSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application No. 63 / 652,546, filed May 28, 2024, which is hereby incorporated by reference in its entirety for all purposes.REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN XML FILE

[0002] The Sequence Listing written in file 629647SEQLIST.xml is 88,773 bytes, was created on May 22, 2025, and is hereby incorporated by reference in its entirety.BACKGROUND

[0003] Humanized liver mouse and rat models, in which donor human hepatocytes repopulate recipient rodent livers, have been used in studying human liver biology, diseases, and therapeutics. However, unlike rapid expansion of hepatocytes during liver regeneration, human hepatocytes engrafted in host mice or rats repopulate much slower, which greatly compromises the quality and usefulness of the model. In addition, engrafted human hepatocytes in both humanized liver mice and rats show defects, including increased lipid droplet accumulation. Ameliorating such imperfections would improve the accuracy of the models to recapitulate normal human liver biology, and accelerated repopulation by donor hepatocytes would further increase the utility of these models.SUMMARY

[0004] Provided are genetically modified non-human animals that are immunodeficient and optionally comprise xenotransplanted hepatocytes such as human hepatocytes, wherein the genetically modified non-human animal and / or the transplanted hepatocytes are modified to restore, activate, or increase interleukin-6 (IL-6) / interleukin-6 receptor (IL-6R) signaling pathway activity or interleukin-6 receptor subunit beta (GP130) signaling pathway activity and to restore, activate, or increase hepatocyte growth factor (HGF) / hepatocyte growth factor receptor (MET) signaling pathway activity in the transplanted hepatocytes. Also provided are methods ofmeasuring or assessing the activity of human-liver-targeting reagents in such non-human animals and methods of making animals with a humanized liver (e.g., with reduced steatosis). Also provided are genetically modified, immunodeficient, non-human animals comprising a humanized HGF gene and optionally a humanized IL6 gene and methods of using and making such animals. Also provided are methods of increasing or accelerating engraftment of xenotransplanted hepatocytes in non-human animals.

[0005] In one aspect, provided are genetically modified non-human animals. In some such animals, the genetically modified non-human animal comprises transplanted hepatocytes from a different species than the non-human animal, optionally wherein the hepatocyte are human hepatocytes, wherein the genetically modified non-human animal and / or the transplanted hepatocytes are modified to restore or increase interleukin-6 (IL-6) / interleukin-6 receptor (IL- 6R) signaling pathway activity or interleukin-6 receptor subunit beta (GP130) signaling pathway activity in the transplanted hepatocytes, and wherein the genetically modified non-human animal and / or the transplanted hepatocytes are modified to restore or increase hepatocyte growth factor (HGF) / hepatocyte growth factor receptor (MET) signaling pathway activity in the transplanted hepatocytes.

[0006] In some such animals, the genetically modified non-human animal is immunodeficient. In some such animals, the genetically modified non-human animal comprises an inactivated endogenous Il2rg gene. In some such animals, the genetically modified non- human animal comprises an inactivated endogenous Ragl gene or an inactivated endogenous Rag2 gene, optionally wherein the genetically modified non-human animal comprises the inactivated endogenous Rag2 gene. In some such animals, the genetically modified non-human animal comprises an inactivated endogenous Ragl gene and an inactivated endogenous Rag2 gene. In some such animals, the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the genetically modified non-human animal further comprises an inactivated endogenous Ragl gene. In some such animals, the genetically modified non-human animal comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (l>rkdc'c,d). In some such animals, the genetically modified non-human animal comprises a SCID mutation in a Prkdc gene (Prkdcscid) and an inactivated endogenous Il2rg gene.

[0007] In some such animals, the genetically modified non-human animal is genetically modified so that endogenous non-human animal hepatocytes in the liver can be selectively and conditionally ablated. In some such animals, the genetically modified non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liverspecific promoter. In some such animals, the genetically modified non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter. In some such animals, the genetically modified non-human animal comprises an inactivated endogenous Fah gene.

[0008] In some such animals, the genetically modified non-human animal comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcscldand the genetically modified non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter. In some such animals, the genetically modified non- human animal comprises a SCID mutation in a Prkdc gene (Prkdcsc,d) and an inactivated endogenous Il2rg gene, and the genetically modified non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liverspecific promoter. In some such animals, the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the genetically modified non-human animal further comprises an inactivated endogenous Ragl gene, and the genetically modified non-human animal comprises an inactivated endogenous Fah gene.

[0009] In some such animals, the transplanted hepatocytes are human hepatocytes.

[0010] In some such animals, the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, and an inactivated endogenous Fah gene, optionally wherein the genetically modified non-human animal comprises an inactivated endogenous Ragl gene.

[0011] In some such animals, the transplanted hepatocytes express non-human animal IL-6R, wherein the non-human animal IL-6R is from the same species as the genetically modified non- human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal IL-6R is mouse IL-6R, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal IL-6R is rat IL-6R. In some such animals, the transplanted hepatocytes express non-human animal oncostatin-M-specific receptor subunitbeta (OSMR), wherein the non-human animal OSMR is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal OSMR is mouse OSMR, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal OSMR is rat OSMR. In some such animals, the transplanted hepatocytes express non-human animal IL-6R and non-human animal OSMR, wherein the non-human animal IL-6R and non-human animal OSMR are from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse, the non-human animal IL-6R is mouse IL- 6R, and the non-human animal OSMR is mouse OSMR, or optionally wherein the genetically modified non-human animal is a rat, the non-human animal IL-6R is rat IL-6R, and the non- human animal OSMR is rat OSMR. In some such animals, the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal IL-6R comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter. In some such animals, the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal OSMR comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter. In some such animals, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector. In some such animals, the transplanted hepatocytes comprise in their genome a non-human animal IL-6R expression construct comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter. In some such animals, the transplanted hepatocytes comprise in their genome a non- human animal OSMR expression construct comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter. In some such animals, the promoter is a liverspecific promoter or a constitutive promoter.

[0012] In some such animals, the transplanted hepatocytes express a ligand-independent, constitutively active form of GP130. In some such animals, the transplanted hepatocytes comprise a vector comprising an expression construct for the constitutively active GP130 comprising a nucleic acid encoding the constitutively active GP130 operably linked to a promoter. In some such animals, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an AAV vector, optionally wherein the viral vector is the lentivirus vector. In some such animals, the transplanted hepatocytes comprise in theirgenome an expression construct for the constitutively active GP130 comprising a nucleic acid encoding the constitutively active GP130 operably linked to a promoter. In some such animals, the promoter is a liver-specific promoter or a constitutive promoter. In some such animals, the constitutively active GP130 is a constitutively active human GP130, optionally wherein the constitutively active human GP130 comprises a deletion of the region of GP130 from Tyrl86 to Tyrl90 (GP13OY186-Y19odel).

[0013] In some such animals, the genetically modified non-human animal further comprises a GP130-activating ligand. In some such animals, the GP130-activating ligand comprises a human IL-6R agonist antigen-binding protein or a human OSMR agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody. In some such animals, the GP130-activating ligand comprises human IL-6 or a human-IL-6R-compatible ligand or a human-IL-6R-compatible IL-6. In some such animals, the GP130-activating ligand comprises human oncostatin-M (OSM) or a human-OSMR-compatible ligand or a human-OSMR- compatible OSM. In some such animals, the genetically modified non-human animal further comprises one or more additional GP130-activating ligands, optionally wherein the GP130- activating ligands comprise: (1) human IL-6 or a human-IL-6R-compatible ligand or a human- IL-6R-compatible IL-6; and (2) human OSM or a human-OSMR-compatible ligand or a human- OSMR-compatible OSM. In some such animals, the genetically modified non-human animal comprises a vector comprising an expression construct for the GP130-activating ligand comprising a nucleic acid encoding the GP130-activating ligand operably linked to a promoter. In some such animals, the genetically modified non-human animal comprises the vector in muscle cells or in liver cells. In some such animals, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such animals, the promoter is a constitutive promoter. In some such animals, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno- associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector. In some such animals, the genetically modified non- human animal comprises in its genome a GP130-activating ligand expression construct comprising a nucleic acid encoding the GP130-activating ligand operably linked to a promoter.In some such animals, the GP130-activating ligand comprises human IL-6 or a human-IL-6R- compatible ligand or a human-IL-6R-compatible IL-6. In some such animals, the GP130- activating ligand comprises human OSM or a human-0 SMR-compatible ligand or a human- OSMR-compatible OSM. In some such animals, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such animals, the promoter is a constitutive promoter. In some such animals, the promoter is an exogenous promoter. In some such animals, the promoter is an endogenous promoter.

[0014] In some such animals, the genetically modified non-human animal comprises a humanized non-human animal IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein. In some such animals, the genetically modified non-human animal comprises a humanized non-human animal OSM gene comprising a human OSM nucleic acid encoding a human OSM protein. In some such animals, the genetically modified non-human animal comprises a humanized non-human animal IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein, and wherein the genetically modified non-human animal comprises a humanized non-human animal OSM gene comprising a human OSM nucleic acid encoding a human OSM protein. In some such animals, the human nucleic acid comprises a region of human IL6 genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human IL6 complementary DNA (cDNA). In some such animals, the human nucleic acid replaces a corresponding region of the non-human animal IL6 gene. In some such animals, the human nucleic acid is inserted into the non-human animal IL6 gene. In some such animals, the human nucleic acid is operably linked to an endogenous non- human animal IL6 promoter. In some such animals, the genetically modified non-human animal is heterozygous for the humanized IL6 gene. In some such animals, the genetically modified non- human animal is homozygous for the humanized IL6 gene. In some such animals, the genetically modified non-human animal comprises the humanized IL6 gene in its germline. In some such animals, the human nucleic acid comprises a region of human OSM genomic sequence from the start codon to the stop codon or the human nucleic acid comprises a human OSM complementary DNA (cDNA). In some such animals, the human nucleic acid replaces a corresponding region of the non-human animal OSM gene. In some such animals, the human nucleic acid is inserted intothe non-human animal OS ene. In some such animals, the human nucleic acid is operably linked to an endogenous non-human animal OSM promoter. In some such animals, the genetically modified non-human animal is heterozygous for the humanized OSM gene. In some such animals, the genetically modified non-human animal is homozygous for the humanized OSM ene. In some such animals, the genetically modified non-human animal comprises the humanized OSM gene in its germline.

[0015] In some such animals, the transplanted hepatocytes express non-human animal MET, wherein the non-human animal MET is from the same species as the genetically modified non- human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal MET is mouse MET, or optionally wherein the genetically modified non- human animal is a rat and the non-human animal MET is rat MET. In some such animals, the transplanted hepatocytes comprise a vector comprising an expression construct for the non- human animal MET comprising a nucleic acid encoding the non-human animal MET operably linked to a promoter. In some such animals, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector. In some such animals, the transplanted hepatocytes comprise in their genome a non-human animal MET expression construct comprising a nucleic acid encoding the non-human animal MET operably linked to a promoter. In some such animals, the promoter is a liver-specific promoter or a constitutive promoter.

[0016] In some such animals, the genetically modified non-human animal further comprises a MET-activating ligand. In some such animals, the MET-activating ligand comprises a human MET agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody. In some such animals, the MET-activating ligand comprises human HGF or a human- MET-compatible ligand or a human-MET-compatible HGF. In some such animals, the genetically modified non-human animal comprises a vector comprising an expression construct for the MET-activating ligand comprising a nucleic acid encoding the MET-activating ligand operably linked to a promoter. In some such animals, the genetically modified non-human animal comprises the vector in muscle cells or in liver cells. In some such animals, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is ahybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such animals, the promoter is a constitutive promoter. In some such animals, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector. In some such animals, the genetically modified non-human animal comprises in its genome a MET -activating ligand expression construct comprising a nucleic acid encoding the MET-activating ligand operably linked to a promoter. In some such animals, the MET-activating ligand comprises human HGF or a human-MET-compatible ligand or a human-MET-compatible HGF. In some such animals, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such animals, the promoter is a constitutive promoter. In some such animals, the promoter is an exogenous promoter. In some such animals, the promoter is an endogenous promoter.

[0017] In some such animals, the genetically modified non-human animal comprises a humanized non-human animal HG gene comprising a human HGF nucleic acid encoding a human HGF protein. In some such animals, the human nucleic acid comprises a region of human HGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human HGF complementary DNA (cDNA). In some such animals, the human nucleic acid replaces a corresponding region of the non-human animal HGF gene. In some such animals, the human nucleic acid is inserted into the non-human animal HGF gene. In some such animals, the human nucleic acid is operably linked to an endogenous non-human animal HGF promoter. In some such animals, the genetically modified non-human animal is heterozygous for the humanized HGF gene. In some such animals, the genetically modified non-human animal is homozygous for the humanized HGF gene. In some such animals, the genetically modified non- human animal comprises the humanized HGF gene in its germline.

[0018] In some such animals, the transplanted hepatocytes express non-human animal EGFR, wherein the non-human animal EGFR is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal EGFR is mouse EGFR, or optionally wherein the geneticallymodified non-human animal is a rat and the non-human animal EGFR is rat EGFR. In some such animals, the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal EGFR comprising a nucleic acid encoding the non-human animal EGFR operably linked to a promoter. In some such animals, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector. In some such animals, the transplanted hepatocytes comprise in their genome a non-human animal EGFR expression construct comprising a nucleic acid encoding the non-human animal EGFR operably linked to a promoter. In some such animals, the promoter is a liver-specific promoter or a constitutive promoter.

[0019] In some such animals, the genetically modified non-human animal further comprises an EGFR-activating ligand. In some such animals, the EGFR-activating ligand comprises a human EGFR agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody. In some such animals, the EGFR-activating ligand comprises human EGF or a human-EGFR-compatible ligand or a human-EGFR-compatible EGF. In some such animals, the genetically modified non-human animal comprises a vector comprising an expression construct for the EGFR-activating ligand comprising a nucleic acid encoding the EGFR-activating ligand operably linked to a promoter. In some such animals, the genetically modified non-human animal comprises the vector in muscle cells or in liver cells. In some such animals, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such animals, the promoter is a constitutive promoter. In some such animals, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector. In some such animals, the genetically modified non-human animal comprises in its genome an EGFR-activating ligand expression construct comprising a nucleic acid encoding the EGFR-activating ligand operably linked to a promoter. In some such animals, the EGFR-activating ligand comprises human EGF or a human-EGFR-compatible ligand or a human-EGFR-compatible EGF. In some such animals, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is amuscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such animals, the promoter is a constitutive promoter. In some such animals, the promoter is an exogenous promoter. In some such animals, the promoter is an endogenous promoter.

[0020] In some such animals, the genetically modified non-human animal comprises a humanized non-human animal EGF gene comprising a human EGF nucleic acid encoding a human EGF protein. In some such animals, the human nucleic acid comprises a region of human EGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human EGF complementary DNA (cDNA). In some such animals, the human nucleic acid replaces a corresponding region of the non-human animal EGF gene. In some such animals, the human nucleic acid is inserted into the non-human animal EGF gene. In some such animals, the human nucleic acid is operably linked to an endogenous non-human animal EGF promoter. In some such animals, the genetically modified non-human animal is heterozygous for the humanized EGF gene. In some such animals, the genetically modified non-human animal is homozygous for the humanized EGF gene. In some such animals, the genetically modified non- human animal comprises the humanized EGF gene in its germline.

[0021] In some such animals, the genetically modified non-human animal further comprises human R-spondin-3 (RSPO3). In some such animals, the genetically modified non-human animal comprises a vector comprising an expression construct for the human RSPO3 comprising a nucleic acid encoding the human RSPO3 operably linked to a promoter. In some such animals, the genetically modified non-human animal comprises the vector in muscle cells or in liver cells. In some such animals, the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such animals, the promoter is a constitutive promoter. In some such animals, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector. In some such animals, the genetically modified non-human animal comprises in its genome a human RSPO3 expression construct comprising a nucleic acid encoding the human RSPO3 operablylinked to a promoter. In some such animals, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such animals, the promoter is a constitutive promoter. In some such animals, the promoter is an exogenous promoter. In some such animals, the promoter is an endogenous promoter.

[0022] In some such animals, the genetically modified non-human animal does not comprise human Kupffer cells in the liver. In some such animals, the genetically modified non-human animal does not comprise a reconstituted human immune system. In some such animals, the genetically modified non-human animal further comprises a humanized non-human animal SIRPA gene. In some such animals, the humanized non-human animal SIRPA gene comprises a replacement of exons 2-4 of the non-human animal SIRPA gene with exons 2-4 of human SIRPA wherein the humanized non-human animal SIRPA gene encodes a chimeric SIRPA protein comprising an extracellular portion of a human SIRPA protein and an intracellular portion of a non-human animal SIRPA protein. In some such animals, the humanized non-human animal SIRPA gene is operably linked to an endogenous non-human animal SIRPA promoter. In some such animals, the genetically modified non-human animal is heterozygous for the humanized SIRPA gene. In some such animals, the genetically modified non-human animal is homozygous for the humanized SIRPA gene. In some such animals, the genetically modified non-human animal comprises the humanized SIRPA gene in its germline.

[0023] In some such animals, the genetically modified non-human animal is a male. In some such animals, the genetically modified non-human animal is a female. In some such animals, the genetically modified non-human animal is a mammal. In some such animals, the mammal is a rodent. In some such animals, the rodent is a rat or a mouse. In some such animals, the rodent is the rat. In some such animals, the rodent is the mouse.

[0024] In some such animals, the transplanted hepatocytes have reduced lipid droplet accumulation compared to transplanted hepatocytes in genetically modified non-human animals in which the genetically modified non-human animal and the transplanted hepatocytes are not modified to restore or increase IL-6 / IL-6R signaling pathway activity or GP130 signaling pathway activity in the transplanted hepatocytes.

[0025] In another aspect, provided are methods of measuring the activity of a human-liver- targeting reagent in vivo. Some such methods comprise: (a) administering the human-liver- targeting reagent to any of the above genetically modified non-human animals; and (b) measuring the activity of the human-liver-targeting reagent in the liver of the genetically modified non-human animal.

[0026] In some such methods, step (a) comprises AAV-mediated delivery, lipid nanoparticle (LNP)-mediated delivery, hydrodynamic delivery (HDD), or injection. In some such methods, the human-liver-targeting reagent targets a target gene expressed in the human liver. In some such methods, step (b) comprises measuring expression of a messenger RNA or a protein encoded by the target gene. In some such methods, step (b) comprises measuring modification of a genomic locus comprising the target gene, optionally wherein step (b) comprises measuring the frequency of insertions or deletions within the genomic locus comprising the target gene. In some such methods, the human-liver-targeting reagent comprises a nuclease agent designed to target a region of the target gene, optionally wherein the nuclease agent comprises a Cas protein and a guide RNA designed to target a guide RNA target sequence in the target gene, optionally wherein the Cas protein is a Cas9 protein. In some such methods, the human-liver-targeting reagent comprises an exogenous donor nucleic acid, wherein the exogenous donor nucleic acid is designed to target the target gene, and optionally wherein the exogenous donor nucleic acid is delivered via AAV. In some such methods, the human-liver-targeting reagent targets a target RNA expressed in the human liver, optionally wherein the human-liver-targeting reagent is an RNAi agent or an antisense oligonucleotide. In some such methods, the human-liver-targeting reagent targets a target protein expressed in the human liver, optionally wherein the human-liver- targeting reagent is an antigen-binding protein or a small molecule.

[0027] In another aspect, provided are methods of making a non-human animal with a humanized liver. Some such methods comprise: (a) transplanting human hepatocytes or human hepatocyte progenitors into a genetically modified non-human animal; and (b) allowing the human hepatocytes or human hepatocyte progenitors to expand, wherein the genetically modified non-human animal and / or the transplanted human hepatocytes or human hepatocyte progenitors are modified to restore or increase interleukin-6 (IL-6) / interleukin-6 receptor (IL-6R) signaling pathway activity or interleukin-6 receptor subunit beta (GP130) signaling pathway activity in the transplanted human hepatocytes or human hepatocyte progenitors, and wherein the geneticallymodified non-human animal and / or the transplanted hepatocytes are modified to restore or increase hepatocyte growth factor (HGF) / hepatocyte growth factor receptor (MET) signaling pathway activity in the transplanted hepatocytes.

[0028] In some such methods, the genetically modified non-human animal is immunodeficient. In some such methods, the genetically modified non-human animal comprises an inactivated endogenous Il2rg gene. In some such methods, the genetically modified non- human animal comprises an inactivated endogenous Ragl gene or an inactivated endogenous Rag2 gene, optionally wherein the genetically modified non-human animal comprises the inactivated endogenous Rag2 gene. In some such methods, the genetically modified non-human animal comprises an inactivated endogenous Ragl gene and an inactivated endogenous Rag2 gene. In some such methods, the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the genetically modified non-human animal further comprises an inactivated endogenous Ragl gene. In some such methods, the genetically modified non-human animal comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcscid). In some such methods, the genetically modified non-human animal comprises a SCID mutation in a Prkdc gene (Prkdcscid) and an inactivated endogenous 112rg gene.

[0029] In some such methods, the genetically modified non-human animal is genetically modified so that endogenous non-human animal hepatocytes in the liver can be selectively and conditionally ablated. In some such methods, the genetically modified non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liverspecific promoter. In some such methods, the genetically modified non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter. In some such methods, the genetically modified non-human animal comprises an inactivated endogenous Fah gene. In some such methods, the genetically modified non-human animal comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcsc,dand the genetically modified non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter. In some such methods, the genetically modified non-human animal comprises a SCID mutation in a Prkdc gene (Prkdc'c,d) and an inactivated endogenous Il2rg gene, and the genetically modified non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) codingsequence operably linked to a liver-specific promoter. In some such methods, the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous H2rg gene, optionally wherein the genetically modified non-human animal further comprises an inactivated endogenous Ragl gene, and the genetically modified non-human animal comprises an inactivated endogenous Fah gene.

[0030] In some such methods, the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, an inactivated endogenous Fah gene, and optionally an inactivated endogenous Ragl gene.

[0031] In some such methods, the transplanted hepatocytes express non-human animal IL- 6R, wherein the non-human animal IL-6R is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal IL-6R is mouse IL-6R, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal IL-6R is rat IL-6R. In some such methods, the transplanted hepatocytes express non-human animal oncostatin-M-specific receptor subunit beta (OSMR), wherein the non-human animal OSMR is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal OSMR is mouse OSMR, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal OSMR is rat OSMR. In some such methods, the transplanted hepatocytes express non-human animal IL-6R and non-human animal OSMR, wherein the non-human animal IL-6R and non-human animal OSMR are from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse, the non-human animal IL-6R is mouse IL- 6R, and the non-human animal OSMR is mouse OSMR, or optionally wherein the genetically modified non-human animal is a rat, the non-human animal IL-6R is rat IL-6R, and the non- human animal OSMR is rat OSMR. In some such methods, the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal IL-6R comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter. In some such methods, the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal OSMR comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector. In some such methods, the transplanted hepatocytes comprise in their genome a non-human animal IL-6R expression construct comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter. In some such methods, the transplanted hepatocytes comprise in their genome a non-human animal OSMR expression construct comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter. In some such methods, the promoter is a liver-specific promoter or a constitutive promoter.

[0032] In some such methods, the transplanted hepatocytes express a ligand-independent, constitutively active form of GP130. In some such methods, the transplanted hepatocytes comprise a vector comprising an expression construct for the constitutively active GP130 comprising a nucleic acid encoding the constitutively active GP130 operably linked to a promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an AAV vector, optionally wherein the viral vector is the lentivirus vector. In some such methods, the transplanted hepatocytes comprise in their genome an expression construct for the constitutively active GP130 comprising a nucleic acid encoding the constitutively active GP130 operably linked to a promoter. In some such methods, the promoter is a liver-specific promoter or a constitutive promoter. In some such methods, the constitutively active GP130 is a constitutively active human GP130, optionally wherein the constitutively active human GP130 comprises a deletion of the region of GP130 from Tyrl86 to Tyrl90 (GP130Y186’Y190del).

[0033] In some such methods, the genetically modified non-human animal further comprises a GP130-activating ligand. In some such methods, the GP130-activating ligand comprises a human IL-6R agonist antigen-binding protein or a human OSMR agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody. In some such methods, the GP130-activating ligand comprises human IL-6 or a human-IL-6R-compatible ligand or a human-IL-6R-compatible IL-6. In some such methods, the GP130-activating ligand comprises human oncostatin-M (OSM) or a human-OSMR-compatible ligand or a human-OSMR- compatible OSM. In some such methods, the genetically modified non-human animal further comprises one or more additional GP130-activating ligands, optionally wherein the GP130- activating ligands comprise: (1) human IL-6 or a human-IL-6R-compatible ligand or a human- IL-6R-compatible IL-6; and (2) human OSM or a human-OSMR-compatible ligand or a human-OSMR-compatible OSM. In some such methods, the genetically modified non-human animal comprises a vector comprising an expression construct for the GP130-activating ligand comprising a nucleic acid encoding the GP130-activating ligand operably linked to a promoter. In some such methods, the genetically modified non-human animal comprises the vector in muscle cells or in liver cells. In some such methods, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such methods, the promoter is a constitutive promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno- associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector. In some such methods, the genetically modified non- human animal comprises in its genome a GP130-activating ligand expression construct comprising a nucleic acid encoding the GP130-activating ligand operably linked to a promoter. In some such methods, the GP130-activating ligand comprises human IL-6 or a human-IL-6R- compatible ligand or a human-IL-6R-compatible IL-6. In some such methods, the GP130- activating ligand comprises human OSM or a human-0 SMR-compatible ligand or a human- OSMR-compatible OSM. In some such methods, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such methods, the promoter is a constitutive promoter. In some such methods, the promoter is an exogenous promoter. In some such methods, the promoter is an endogenous promoter.

[0034] In some such methods, the genetically modified non-human animal comprises a humanized non-human animal IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein. In some such methods, the genetically modified non-human animal comprises a humanized non-human animal OSM ene comprising a human OSM nucleic acid encoding a human OSM protein. In some such methods, the genetically modified non-human animal comprises a humanized non-human animal IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein, and wherein the genetically modified non-human animal comprises a humanized non-human animal OSM gene comprising a human OSM nucleic acidencoding a human OSM protein. In some such methods, the human nucleic acid comprises a region of human IL6 genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human IL6 complementary DNA (cDNA). In some such methods, the human nucleic acid replaces a corresponding region of the non-human animal IL6 gene. In some such methods, the human nucleic acid is inserted into the non-human animal IL6 gene. In some such methods, the human nucleic acid is operably linked to an endogenous non- human animal IL6 promoter. In some such methods, the genetically modified non-human animal is heterozygous for the humanized IL6 gene. In some such methods, the genetically modified non-human animal is homozygous for the humanized IL6 gene. In some such methods, the genetically modified non-human animal comprises the humanized IL6 gene in its germline. In some such methods, the human nucleic acid comprises a region of human OSM genomic sequence from the start codon to the stop codon or the human nucleic acid comprises a human OSM complementary DNA (cDNA). In some such methods, the human nucleic acid replaces a corresponding region of the non-human animal OSM gene. In some such methods, the human nucleic acid is inserted into the non-human animal OSM gene. In some such methods, the human nucleic acid is operably linked to an endogenous non-human animal OSM promoter. In some such methods, the genetically modified non-human animal is heterozygous for the humanized OSM gene. In some such methods, the genetically modified non-human animal is homozygous for the humanized OSM gene. In some such methods, the genetically modified non-human animal comprises the humanized OSM gene in its germline.

[0035] In some such methods, the transplanted hepatocytes express non-human animal MET, wherein the non-human animal MET is from the same species as the genetically modified non- human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal MET is mouse MET, or optionally wherein the genetically modified non- human animal is a rat and the non-human animal MET is rat MET. In some such methods, the transplanted hepatocytes comprise a vector comprising an expression construct for the non- human animal MET comprising a nucleic acid encoding the non-human animal MET operably linked to a promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector. In some such methods, the transplanted hepatocytes comprise in their genome a non-human animal MET expressionconstruct comprising a nucleic acid encoding the non-human animal MET operably linked to a promoter. In some such methods, the promoter is a liver-specific promoter or a constitutive promoter.

[0036] In some such methods, the genetically modified non-human animal further comprises a MET-activating ligand. In some such methods, the MET-activating ligand comprises a human MET agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody. In some such methods, the MET-activating ligand comprises human HGF or a human- MET-compatible ligand or a human-MET-compatible HGF. In some such methods, the genetically modified non-human animal comprises a vector comprising an expression construct for the MET-activating ligand comprising a nucleic acid encoding the MET-activating ligand operably linked to a promoter. In some such methods, the genetically modified non-human animal comprises the vector in muscle cells or in liver cells. In some such methods, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such methods, the promoter is a constitutive promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector. In some such methods, the genetically modified non-human animal comprises in its genome a MET-activating ligand expression construct comprising a nucleic acid encoding the MET-activating ligand operably linked to a promoter. In some such methods, the MET-activating ligand comprises human HGF or a human-MET-compatible ligand or a human-MET-compatible HGF. In some such methods, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such methods, the promoter is a constitutive promoter. In some such methods, the promoter is an exogenous promoter. In some such methods, the promoter is an endogenous promoter.

[0037] In some such methods, the genetically modified non-human animal comprises a humanized non-human animal HGF gene comprising a human HGF nucleic acid encoding ahuman HGF protein. In some such methods, the human nucleic acid comprises a region of human HGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human HGF complementary DNA (cDNA). In some such methods, the human nucleic acid replaces a corresponding region of the non-human animal HGF gene. In some such methods, the human nucleic acid is inserted into the non-human animal HGF gene. In some such methods, the human nucleic acid is operably linked to an endogenous non-human animal HGF promoter. In some such methods, the genetically modified non-human animal is heterozygous for the humanized HGF gene. In some such methods, the genetically modified non-human animal is homozygous for the humanized HGF gene. In some such methods, the genetically modified non-human animal comprises the humanized HGF gene in its germline.

[0038] In some such methods, the transplanted hepatocytes express non-human animal EGFR, wherein the non-human animal EGFR is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal EGFR is mouse EGFR, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal EGFR is rat EGFR. In some such methods, the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal EGFR comprising a nucleic acid encoding the non-human animal EGFR operably linked to a promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector. In some such methods, the transplanted hepatocytes comprise in their genome a non-human animal EGFR expression construct comprising a nucleic acid encoding the non-human animal EGFR operably linked to a promoter. In some such methods, the promoter is a liver-specific promoter or a constitutive promoter.

[0039] In some such methods, the genetically modified non-human animal further comprises an EGFR-activating ligand. In some such methods, the EGFR-activating ligand comprises a human EGFR agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody. In some such methods, the EGFR-activating ligand comprises human EGF or a human-EGFR-compatible ligand or a human-EGFR-compatible EGF. In some such methods, the genetically modified non-human animal comprises a vector comprising an expression construct for the EGFR-activating ligand comprising a nucleic acid encoding the EGFR-activating ligandoperably linked to a promoter. In some such methods, the genetically modified non-human animal comprises the vector in muscle cells or in liver cells. In some such methods, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such methods, the promoter is a constitutive promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector. In some such methods, the genetically modified non-human animal comprises in its genome an EGFR-activating ligand expression construct comprising a nucleic acid encoding the EGFR-activating ligand operably linked to a promoter. In some such methods, the EGFR-activating ligand comprises human EGF or a human-EGFR-compatible ligand or a human-EGFR-compatible EGF. In some such methods, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such methods, the promoter is a constitutive promoter. In some such methods, the promoter is an exogenous promoter. In some such methods, the promoter is an endogenous promoter.

[0040] In some such methods, the genetically modified non-human animal comprises a humanized non-human animal EGF gene comprising a human EGF nucleic acid encoding a human EGF protein. In some such methods, the human nucleic acid comprises a region of human EGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human EGF complementary DNA (cDNA). In some such methods, the human nucleic acid replaces a corresponding region of the non-human animal EGF gene. In some such methods, the human nucleic acid is inserted into the non-human animal EGF gene. In some such methods, the human nucleic acid is operably linked to an endogenous non-human animal EGF promoter. In some such methods, the genetically modified non-human animal is heterozygous for the humanized EGF gene. In some such methods, the genetically modified non-human animal is homozygous for the humanized EGF gene. In some such methods, the genetically modified non- human animal comprises the humanized EGF gene in its germline.

[0041] In some such methods, the genetically modified non-human animal further comprises human R-spondin-3 (RSPO3). In some such methods, the genetically modified non-human animal comprises a vector comprising an expression construct for the human RSPO3 comprising a nucleic acid encoding the human RSPO3 operably linked to a promoter. In some such methods, the genetically modified non-human animal comprises the vector in muscle cells or in liver cells. In some such methods, the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such methods, the promoter is a constitutive promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector. In some such methods, the genetically modified non-human animal comprises in its genome a human RSPO3 expression construct comprising a nucleic acid encoding the human RSPO3 operably linked to a promoter. In some such methods, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such methods, the promoter is a constitutive promoter. In some such methods, the promoter is an exogenous promoter. In some such methods, the promoter is an endogenous promoter.

[0042] In some such methods, the genetically modified non-human animal does not comprise human Kupffer cells in the liver. In some such methods, the genetically modified non-human animal does not comprise a reconstituted human immune system. In some such methods, the genetically modified non-human animal further comprises a humanized non-human animal SIRPA gene. In some such methods, the humanized non-human animal SIRPA gene comprises a replacement of exons 2-4 of the non-human animal SIRPA gene with exons 2-4 of human SIRPA, wherein the humanized non-human animal SIRPA gene encodes a chimeric SIRPA protein comprising an extracellular portion of a human SIRPA protein and an intracellular portion of a non-human animal SIRPA protein. In some such methods, the humanized non-human animal SIRPA gene is operably linked to an endogenous non-human animal SIRPA promoter. In some such methods, the genetically modified non-human animal is heterozygous for the humanizedSJRPA gene. In some such methods, the genetically modified non-human animal is homozygous for the humanized SIRPA gene. In some such methods, the genetically modified non-human animal comprises the humanized SIRPA gene in its germline.

[0043] In some such methods, the genetically modified non-human animal is a male. In some such methods, the genetically modified non-human animal is a female. In some such methods, the genetically modified non-human animal is a mammal. In some such methods, the mammal is a rodent. In some such methods, the rodent is a rat or a mouse. In some such methods, the rodent is the rat. In some such methods, the rodent is the mouse.

[0044] In some such methods, exogenous urokinase plasminogen activator or an exogenous nucleic acid encoding urokinase plasminogen activator is administered to the genetically modified non-human animal prior to step (a) to prime the liver for improved repopulation by human hepatocytes, optionally wherein the exogenous nucleic acid is an adenovirus or adeno- associated virus (AAV) encoding urokinase plasminogen activator. In some such methods, the human hepatocytes or human hepatocyte progenitors are injected into the genetically modified non-human animal intrasplenically in step (a). In some such methods, at least about ten million human hepatocytes or human hepatocyte progenitors are transplanted into the genetically modified non-human animal in step (a).

[0045] In some such methods, step (a) is done in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency. In some such methods, some or all of step (b) is done in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency. In some such methods, nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency is administered to the genetically modified non- human animal in step (b) in an on / off cycle to promote human hepatocyte repopulation. In some such methods, the on / off cycle comprises about 5 to about 7 days off and about 3 days on.

[0046] In some such methods, the humanized liver generated by the method has reduced lipid droplet accumulation or reduced steatosis compared to methods in which the genetically modified non-human animal and the transplanted human hepatocytes or human hepatocyte progenitors are not modified to restore or increase IL-6 / IL-6R signaling pathway activity or GP130 signaling pathway activity in the transplanted human hepatocytes or human hepatocyte progenitors.

[0047] In another aspect, provided are genetically modified non-human animals, non-human animal cells, or non-human animal genomes comprising a humanized HGF gene comprising a human HGF nucleic acid encoding a human HGF protein, wherein the genetically modified non- human animal, non-human animal cell, or non-human animal genome comprises one or more genetic modifications causing immunodeficiency.

[0048] Some such animals, cells, or genomes further comprise a humanized IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein. In some such animals, cells, or genomes, the human nucleic acid comprises a region of human IL6 genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human IL6 complementary DNA (cDNA). In some such animals, cells, or genomes, the human nucleic acid replaces a corresponding region of the non-human animal IL6 gene. In some such animals, cells, or genomes, the human nucleic acid is inserted into the non-human animal IL6 gene. In some such animals, cells, or genomes, the human nucleic acid is operably linked to an endogenous non-human animal IL6 promoter. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome is heterozygous for the humanized IL6 gene. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome is homozygous for the humanized IL6 gene. In some such animals, cells, or genomes, the genetically modified non-human animal comprises the humanized IL6 gene in its germline.

[0049] Some such animals, cells, or genomes further comprise a humanized OSM gene comprising a human OSM nucleic acid encoding a human OSM protein. In some such animals, cells, or genomes, the human nucleic acid comprises a region of human OSM genomic sequence from the start codon to the stop codon or the human nucleic acid comprises a human OSM complementary DNA (cDNA). In some such animals, cells, or genomes, the human nucleic acid replaces a corresponding region of the non-human animal OSM gene. In some such animals, cells, or genomes, the human nucleic acid is inserted into the non-human animal OSM gene. In some such animals, cells, or genomes, the human nucleic acid is operably linked to an endogenous non-human animal OSM promoter. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome is heterozygous for the humanized OSM gene. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genomeis homozygous for the humanized OSM gene. In some such animals, cells, or genomes, the genetically modified non-human animal comprises the humanized OSM gene in its germline.

[0050] In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Il2rg gene. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Ragl gene or an inactivated endogenous Rag2 gene, optionally wherein the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises the inactivated endogenous Rag2 gene. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Ragl gene and an inactivated endogenous Rag2 gene. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the genetically modified non-human animal, non-human animal cell, or non-human animal genome further comprises an inactivated endogenous Ragl gene. In some such animals, cells, or genomes, the genetically modified non- human animal, non-human animal cell, or non-human animal genome comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcscld' In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non- human animal genome comprises a SCID mutation in a Prkdc gene (Prkdc^)' and an inactivated endogenous Il2rg gene.

[0051] In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a genetic modification enabling selective and conditional ablation of endogenous non-human animal hepatocytes in the liver. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter. In some such animals, cells, or genomes,the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Fah gene.

[0052] In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcscldand the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a SCID mutation in a. Prkdc gene (Prkdc'iCid) and an inactivated endogenous Il2rg gene, and the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter. In some such animals, cells, or genomes, the genetically modified non-human animal, non- human animal cell, or non-human animal genome comprises an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, an inactivated endogenous Fah gene, and optionally an inactivated endogenous Ragl gene.

[0053] In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, an inactivated endogenous Fah gene, and optionally an inactivated endogenous Ragl gene.

[0054] In some such animals, cells, or genomes, the human nucleic acid comprises a region of human HGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human HGF complementary DNA (cDNA). In some such animals, cells, or genomes, the human nucleic acid replaces a corresponding region of the non-human animal HGF gene. In some such animals, cells, or genomes, the human nucleic acid is inserted into the non-human animal HGF gene. In some such animals, cells, or genomes, the human nucleic acid is operably linked to an endogenous non-human animal HGF promoter. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome is heterozygous for the humanized HGF gene. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome is homozygous for the humanized HGF gene. In some suchanimals, cells, or genomes, the genetically modified non-human animal comprises the humanized HGF gene in its germline.

[0055] Some such animals, cells, or genomes further comprise a humanized EGF gene comprising a human EGF nucleic acid encoding a human EGF protein. In some such animals, cells, or genomes, the human nucleic acid comprises a region of human EGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human EGF complementary DNA (cDNA). In some such animals, cells, or genomes, the human nucleic acid replaces a corresponding region of the non-human animal EGF gene. In some such animals, cells, or genomes, the human nucleic acid is inserted into the non-human animal EGF gene. In some such animals, cells, or genomes, the human nucleic acid is operably linked to an endogenous non-human animal EGF promoter. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome is heterozygous for the humanized EGF gene. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome is homozygous for the humanized EGF gene. In some such animals, cells, or genomes, the genetically modified non-human animal comprises the humanized EGF gene in its germline.

[0056] In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises in its genome a human RSPO3 expression construct comprising a nucleic acid encoding the human RSPO3 operably linked to a promoter, and / or wherein the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises in its genome a human EGF expression construct comprising a nucleic acid encoding the human EGF operably linked to a promoter. In some such animals, cells, or genomes, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such animals, cells, or genomes, the promoter is a constitutive promoter. In some such animals, cells, or genomes, the promoter is an exogenous promoter. In some such animals, cells, or genomes, the promoter is an endogenous promoter.

[0057] Some such animals, cells, or genomes further comprise a humanized non-human animal SIRPA gene. In some such animals, cells, or genomes, the humanized non-human animalSJRPA gene comprises a replacement of exons 2-4 of the non-human animal SIRPA gene with exons 2-4 of human SIRPA wherein the humanized non-human animal SIRPA gene encodes a chimeric SIRPA protein comprising an extracellular portion of a human SIRPA protein and an intracellular portion of a non-human animal SIRPA protein. In some such animals, cells, or genomes, the humanized non-human animal SIRPA gene is operably linked to an endogenous non-human animal SIRPA promoter. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome is heterozygous for the humanized SIRPA gene. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome is homozygous for the humanized SIRPA gene. In some such animals, cells, or genomes, the genetically modified non-human animal comprises the humanized SIRPA gene in its germline.

[0058] In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome is male. In some such animals, cells, or genomes, the genetically modified non-human animal, non-human animal cell, or non-human animal genome is female. In some such animals, cells, or genomes, the non-human animal is a mammal. In some such animals, cells, or genomes, the mammal is a rodent. In some such animals, cells, or genomes, the rodent is a rat or a mouse. In some such animals, cells, or genomes, the rodent is the rat. In some such animals, cells, or genomes, the rodent is the mouse.

[0059] In another aspect, provided are methods of making any of the above genetically modified non-human animals. Some such methods comprise: (a) introducing a genetically modified non-human animal embryonic stem (ES) cell comprising: (1) a humanized HGF gene comprising a human HGF nucleic acid encoding a human HGF protein; and (2) one or more genetic modifications causing immunodeficiency; and (b) implanting and gestating the non- human animal host embryo in a non-human animal surrogate mother, wherein the non-human animal surrogate mother produces an F0 progeny genetically modified non-human animal comprising: (1) the humanized HGF gene; and (2) the one or more genetic modifications causing immunodeficiency. Some such methods further comprise modifying a non-human animal ES cell to generate the genetically modified non-human animal ES cell comprising: (1) the humanized HGF gene; and (2) the one or more genetic modifications causing immunodeficiency prior to step (a).

[0060] In another aspect, provided are methods of making any of the above genetically modified non-human animals. Some such methods comprise: implanting and gestating a genetically modified non-human animal one-cell stage embryo comprising: (1) a humanized HGF gene comprising a human HGF nucleic acid encoding a human HGF protein; and (2) one or more genetic modifications causing immunodeficiency in a non-human animal surrogate mother, wherein the non-human animal surrogate mother produces an F0 progeny genetically modified non-human animal comprising: (1) the humanized HGF gene; and (2) the one or more genetic modifications causing immunodeficiency. Some such methods further comprise modifying a non-human animal one-cell stage embryo to generate the genetically modified non- human animal one-cell stage embryo comprising: (1) the humanized HGF gene; and (2) the one or more genetic modifications causing immunodeficiency gene prior to gestating the genetically modified non-human animal one-cell stage embryo in the non-human animal surrogate mother.

[0061] In another aspect, provided are methods of making a non-human animal with a humanized liver. Some such methods comprise: (a) transplanting human hepatocytes or human hepatocyte progenitors into any of the above genetically modified non-human animals; and (b) allowing the human hepatocytes or human hepatocyte progenitors to expand. In some such methods, exogenous urokinase plasminogen activator or an exogenous nucleic acid encoding urokinase plasminogen activator is administered to the genetically modified non-human animal prior to step (a) to prime the liver for improved repopulation by human hepatocytes, optionally wherein the exogenous nucleic acid is an adenovirus or adeno-associated virus (AAV) encoding urokinase plasminogen activator. In some such methods, the human hepatocytes or human hepatocyte progenitors are injected into the genetically modified non-human animal intrasplenically in step (a). In some such methods, at least about ten million human hepatocytes or human hepatocyte progenitors are transplanted into the genetically modified non-human animal in step (a). In some such methods, step (a) is done in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency. In some such methods, some or all of step (b) is done in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency. In some such methods, nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency is administered to the genetically modified non- human animal in step (b) in an on / off cycle to promote human hepatocyte repopulation. In some such methods, the on / off cycle comprises about 5 to about 7 days off and about 3 days on.

[0062] In another aspect, provided are methods of increasing or accelerating engraftment of transplanted human hepatocytes in a non-human animal. Some such methods comprise: (1) administering a GP130-activating ligand or a nucleic acid encoding the GP130-activating ligand to the non-human animal, wherein the GP130-activating ligand restores or increases interleukin- 6 (IL-6) / interleukin-6 receptor (IL-6R) signaling pathway activity or interleukin-6 receptor subunit beta (GP130) signaling pathway activity in the transplanted human hepatocytes; and / or (2) administering a hepatocyte growth factor receptor (MET)-activating ligand or a nucleic acid encoding the MET-activating ligand to the non-human animal, wherein the MET-activating ligand restores or increases hepatocyte growth factor (HGF) / MET signaling pathway activity in the transplanted hepatocytes.

[0063] In some such methods, the methods comprise: (1) administering the GP130-activating ligand or the nucleic acid encoding the GP130-activating ligand to the non-human animal; and (2) administering the MET-activating ligand or the nucleic acid encoding the MET-activating ligand to the non-human animal. In some such methods, the GP130-activating ligand comprises a human IL-6R agonist antigen-binding protein or a human OSMR agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody. In some such methods, the GP130-activating ligand comprises human IL-6 or a human-IL-6R-compatible ligand or a human-IL-6R-compatible IL-6. In some such methods, the GP130-activating ligand comprises human oncostatin-M (OSM) or a human-OSMR-compatible ligand or a human-OSMR- compatible OSM. In some such methods, the method further comprises administering one or more additional GP130-activating ligands or one or more nucleic acids encoding the one or more additional GP130-activating ligands, optionally wherein the GP130-activating ligands comprise: (1) human IL-6 or a human-IL-6R-compatible ligand or a human-IL-6R-compatible IL-6; and (2) human OSM or a human-OSMR-compatible ligand or a human-OSMR-compatible OSM. In some such methods, the method comprises administering a vector comprising an expression construct for the GP130-activating ligand comprising a nucleic acid encoding the GP130- activating ligand operably linked to a promoter. In some such methods, the vector is administered to muscle cells or liver cells. In some such methods, the promoter is a tissuespecific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In somesuch methods, the promoter is a constitutive promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector. In some such methods, the method comprises administering the GP130-activating ligand, optionally wherein the GP130-activating ligand is administered to the liver of the non-human animal.

[0064] In some such methods, the MET-activating ligand comprises a human MET agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody. In some such methods, the MET-activating ligand comprises human HGF or a human-MET-compatible ligand or a human-MET-compatible HGF. In some such methods, the method comprises administering a vector comprising an expression construct for the MET-activating ligand comprising a nucleic acid encoding the MET-activating ligand operably linked to a promoter. In some such methods, the vector is administered to muscle cells or liver cells. In some such methods, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such methods, the promoter is a constitutive promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector. In some such methods, the method comprises administering the MET-activating ligand, optionally wherein the MET-activating ligand is administered to the liver of the non-human animal.

[0065] In some such methods, the non-human animal is a male. In some such methods, the non-human animal is a female. In some such methods, the non-human animal is a mammal. In some such methods, the mammal is a rodent. In some such methods, the rodent is a rat or a mouse. In some such methods, the rodent is the rat. In some such methods, the rodent is the mouse.

[0066] In some such methods, the non-human animal is immunodeficient. In some such methods, the non-human animal comprises an inactivated endogenous Il2rg gene. In some such methods, the non-human animal comprises an inactivated endogenous Ragl gene or aninactivated endogenous Rag2 gene, optionally wherein the non-human animal comprises the inactivated endogenous Rag2 gene. In some such methods, the non-human animal comprises an inactivated endogenous Ragl gene and an inactivated endogenous Rag2 gene. In some such methods, the non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the non-human animal further comprises an inactivated endogenous Ragl gene. In some such methods, the non-human animal comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcscld). In some such methods, the non-human animal comprises a SCID mutation in a Prkdc gene (Prkdcsc,d) and an inactivated endogenous I12rg gene.

[0067] In some such methods, the non-human animal is genetically modified so that endogenous non-human animal hepatocytes in the liver can be selectively and conditionally ablated. In some such methods, the non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter. In some such methods, the non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter. In some such methods, the non- human animal comprises an inactivated endogenous Fah gene.

[0068] In some such methods, the non-human animal comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcscld) and the non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liverspecific promoter. In some such methods, the non-human animal comprises a SCID mutation in a Prkdc gene (Prkdcsc,d) and an inactivated endogenous Il2rg gene, and the non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter. In some such methods, the non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the non-human animal further comprises an inactivated endogenous Ragl gene, and the genetically modified non-human animal comprises an inactivated endogenous Fah gene.

[0069] In some such methods, the non-human animal comprises an inactivated endogenous Rag2 gene, an inactivated endogenous H2rg gene, and an inactivated endogenous Fah gene, optionally wherein the non-human animal comprises an inactivated endogenous Ragl gene.

[0070] In some such methods, the non-human animal comprises a humanized non-human animal IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein. In somesuch methods, the non-human animal comprises a humanized non-human animal OSM ene comprising a human OSM nucleic acid encoding a human OSM protein. In some such methods, the non-human animal comprises a humanized non-human animal IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein, and wherein the non-human animal comprises a humanized non-human animal OSM ene comprising a human OSM nucleic acid encoding a human OSM protein.

[0071] In some such methods, the human nucleic acid comprises a region of human IL6 genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human IL6 complementary DNA (cDNA). In some such methods, the human nucleic acid replaces a corresponding region of the non-human animal IL6 gene. In some such methods, the human nucleic acid is inserted into the non-human animal IL6 gene. In some such methods, the human nucleic acid is operably linked to an endogenous non-human animal IL6 promoter. In some such methods, the non-human animal is heterozygous for the humanized IL6 gene. In some such methods, the non-human animal is homozygous for the humanized IL6 gene. In some such methods, the non-human animal comprises the humanized IL6 gene in its germline.

[0072] In some such methods, the human nucleic acid comprises a region of human OSM genomic sequence from the start codon to the stop codon or the human nucleic acid comprises a human OSM complementary DNA (cDNA). In some such methods, the human nucleic acid replaces a corresponding region of the non-human animal OSM gene. In some such methods, the human nucleic acid is inserted into the non-human animal OSM gene. In some such methods, the human nucleic acid is operably linked to an endogenous non-human animal OSM promoter. In some such methods, the non-human animal is heterozygous for the humanized OSM gene. In some such methods, the non-human animal is homozygous for the humanized OSM gene. In some such methods, the non-human animal comprises the humanized OSM gene in its germline.

[0073] In some such methods, the non-human animal comprises a humanized non-human animal HGF gene comprising a human HGF nucleic acid encoding a human HGF protein. In some such methods, the human nucleic acid comprises a region of human HGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human HGF complementary DNA (cDNA). In some such methods, the human nucleic acid replaces a corresponding region of the non-human animal HGF gene. In some such methods, the human nucleic acid is inserted into the non-human animal HGF gene. In some such methods, thehuman nucleic acid is operably linked to an endogenous non-human animal HGF promoter. In some such methods, the non-human animal is heterozygous for the humanized HGF gene. In some such methods, the non-human animal is homozygous for the humanized HGF gene. In some such methods, the non-human animal comprises the humanized HGF gene in its germline.

[0074] In some such methods, the non-human animal comprises a humanized non-human animal SIRPA gene. In some such methods, the humanized non-human animal SIRPA gene comprises a replacement of exons 2-4 of the non-human animal SIRPA gene with exons 2-4 of human SIRPA wherein the humanized non-human animal SIRPA gene encodes a chimeric SIRPA protein comprising an extracellular portion of a human SIRPA protein and an intracellular portion of a non-human animal SIRPA protein. In some such methods, the humanized non-human animal SIRPA gene is operably linked to an endogenous non-human animal SIRPA promoter. In some such methods, the non-human animal is heterozygous for the humanized SIRPA gene. In some such methods, the non-human animal is homozygous for the humanized SIRPA gene. In some such methods, the non-human animal comprises the humanized SIRPA gene in its germline.

[0075] In some such methods, the method further comprises administering an EGFR- activating ligand or a nucleic acid encoding the EGFR-activating ligand to the non-human animal. In some such methods, the EGFR-activating ligand comprises a human EGFR agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody. In some such methods, the EGFR-activating ligand comprises human EGF or a human-EGFR-compatible ligand or a human-EGFR-compatible EGF. In some such methods, the method comprises administering a vector comprising an expression construct for the EGFR-activating ligand comprising a nucleic acid encoding the EGFR-activating ligand operably linked to a promoter. In some such methods, the vector is administered to muscle cells or liver cells. In some such methods, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such methods, the promoter is a constitutive promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8vector. In some such methods, the method comprises administering the EGFR -activating ligand, optionally wherein the EGFR-activating ligand is administered to the liver of the non-human animal.

[0076] In some such methods, the non-human animal comprises a humanized non-human animal EGF gene comprising a human EGF nucleic acid encoding a human EGF protein. In some such methods, the human nucleic acid comprises a region of human EGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human EGF complementary DNA (cDNA). In some such methods, the human nucleic acid replaces a corresponding region of the non-human animal EGF gene. In some such methods, the human nucleic acid is inserted into the non-human animal EGF gene. In some such methods, the human nucleic acid is operably linked to an endogenous non-human animal EGF promoter. In some such methods, the non-human animal is heterozygous for the humanized EGF gene. In some such methods, the non-human animal is homozygous for the humanized EGF gene. In some such methods, the non-human animal comprises the humanized EGF gene in its germline.

[0077] In some such methods, the method further comprises administering to the non-human animal human R-spondin-3 (RSPO3) or a nucleic acid encoding human RSPO3. In some such methods, the method further comprises administering a vector comprising an expression construct for the human RSPO3 comprising a nucleic acid encoding the human RSPO3 operably linked to a promoter. In some such methods, the vector is administered to muscle cells or liver cells. In some such methods, the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7). In some such methods, the promoter is a constitutive promoter. In some such methods, the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector. In some such methods, the method comprises administering the human RSPO3, optionally wherein the human RSPO3 is administered to the liver of the non-human animal.BRIEF DESCRIPTION OF THE FIGURES

[0078] Figures 1A-1B show c-MET antibody can induce signaling in human hepatocytes and promote liver organoid growth, but not human hepatocyte repopulation in FSRG mice. Figure 1A shows human-specific c-MET antibodies can induce downstream signaling in the human HCC cell line, HepG2, as well as primary mouse hepatocytes expressing human MET receptor (isolated from the livers of METHumInmice), but not wild-type mouse hepatocytes. Figure IB shows engraftment of primary human hepatocytes in FSRG mice cannot be enhanced by treatment with the c-MET agonist antibody, as shown by similar serum human albumin levels, and amount of FAH staining in liver sections from mice treated with Control antibody vs. c-MET antibody.

[0079] Figure 2A-2D show primary human hepatocytes expressing mIL6R show increased seeding and early expansion when treated with c-MET antibody. Figure 2A shows pSTAT3 western blot of primary human hepatocytes (PHH) transduced with lentivirus carrying mouse IL- 6 receptor (mIL6R) and treated with mouse IL6. Figure 2B shows human albumin levels at 3 weeks post-transplant of human hepatocytes. Figure 2C shows RNAscope staining for human albumin (hAlb) or GFP (to detect a GFP tag sequence in the transgene) in livers from FSRG mice engrafted with both non-infected and mIL6R-expressing PHH, at 3 weeks post-transplant. Figure 2D shows quantification of the percent human albumin positive area and percent GFP positive area shows that a larger percent of human hepatocytes express mIL6R (detected by GFP staining) in the c-MET antibody treated group, compared to the Control antibody treated group. This suggests that c-MET antibody treatment preferentially expands human hepatocytes that can respond to endogenous mouse IL6 signal.

[0080] Figures 3A-3C show enhanced re-population of FSRG-IL6Humlnmice with c-MET antibody treatment. Figure 3A shows FSRG-IL6HumInmice engrafted with primary human hepatocytes, and treated with either Control antibody or a human-specific c-MET agonist antibody starting at the time of transplant. Serum human albumin levels show a significant increase in human hepatocyte repopulation in the c-MET antibody treated group. FAH IHC on livers from engrafted mice show humanization of more than 90% at 6 weeks post-transplant is possible with c-MET antibody treatment, compared with less than 10% humanization in the Control antibody treated group at 6 weeks post-transplant. Figure 3B shows graphs showing the percent of humanized liver animals with over 2, 5, or lOmg / ml serum human albumin at differenttime-points after engraftment in FSRG, FSRG-IL6HumInmice, or FSRG-IL6HumInrnice treated with c-MET antibody. Figure 3C shows human hepatocytes engrafted in FSRG-IL6HumInmice show increased proliferation when treated with c-MET antibody vs. Control antibody. The staining shows human ASGR1+ hepatocytes in dark gray, and the proliferation marker, Ki67, in black (arrows highlight Ki67 positive proliferating hepatocytes).

[0081] Figures 4A-4B show c-MET antibody treatment of FSRG-IL6HumInmice results in highly humanized livers with decreased lipid accumulation. Figure 4A shows human hepatocytes engrafted into FSRG mice show abnormal lipid accumulation. H&E and FAH staining on humanized liver sections shows that human hepatocytes engrafted into FSRG- IL6HumInmice show decreased lipid accumulation, both with and without c-MET antibody treatment, compared with human hepatocytes engrafted into FSRG mice (“H” indicates an area of engrafted human hepatocytes and “M” indicates murine hepatocytes). Figure 4B shows quantification of the percent fatty area and percent FAH+ area confirms a decrease in liver fat in humanized FSRG-IL6HumInmice, compared to humanized FSRG mice.

[0082] Figure 5 shows c-MET antibody treatment of FSRG-IL6HumInmice results in highly humanized livers with decreased liver damage and improved metabolic profile. Serum chemistry panel shows a decrease in typical readouts of liver damage, including Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Total Bilirubin (TBIL), Alkaline Phosphatase (ALP), Bile Acids (TBA) and Ammonia (AMM), in FSRG-IL6HumInvs. FSRG humanized liver mice. Lipid panel, including Cholesterol (CHOL), Triglycerides (TRIG) and Non-esterified Fatty Acids (NEFA), also shows overall decreased levels in FSRG-IL6HumInvs. FSRG humanized liver mice.

[0083] Figures 6A-6B show c-MET antibody treatment of FSRG-IL6HumInmice results in efficient engraftment of difficult donor hepatocytes. Figure 6A shows human hepatocytes from different donors show different engraftment efficiency. At 3 months post-engraftment, donor SYF (from an adult donor, age 25 years) shows very low repopulation in FSRG mice, compared to donor HLY (from a young donor, age 4 years). Figure 6B shows serum human albumin levels show that donor SYF has improved engraftment efficiency in FSRG-IL6HuinInmice treated with c-MET agonist antibody. FAH IHC staining on liver sections from FSRG-IL6HumInrnice engrafted with either donor HLY or SYF, at 6 weeks post-transplant show very similar engraftment levels and morphology of human hepatocytes.

[0084] Figures 7A-7C show activation of additional signaling pathways can further enhance the effect of IL6 and HGF / MET signaling on hepatocyte repopulation and proliferation. Figures 7A-7B show serum human albumin levels in FSRG-IL6HumInmice engrafted with human hepatocytes and treated with c-MET antibody alone or c-MET antibody in combination with AAV8-hRSPO3 (Figure 7A) or ADV-hEGF (Figure 7B). Figure 7C shows FAH IHC on humanized liver sections from FSRG-IL6HumInmice treated with c-MET antibody alone or c- MET antibody in combination with AAV8-hRSPO3 or ADV-hEGF.DEFINITIONS

[0085] The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any part of a protein or polypeptide having a particular function or structure.

[0086] The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0087] The term “genomically integrated” refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence integrates into the genome of the cell. Any protocol may be used for the stable incorporation of a nucleic acid into the genome of a cell.

[0088] The term “expression vector” or “expression construct” or “expression cassette” refers to a recombinant nucleic acid containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell or organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, as well as other sequences. Eukaryotic cells are generally known to utilize promoters, enhancers,and termination and polyadenylation signals, although some elements may be deleted and other elements added without sacrificing the necessary expression.

[0089] The term “viral vector” refers to a recombinant nucleic acid that includes at least one element of viral origin and includes elements sufficient for or permissive of packaging into a viral vector particle. The vector and / or particle can be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells either ex vivo or in vivo. Numerous forms of viral vectors are known.

[0090] The term “isolated” with respect to proteins, nucleic acids, and cells includes proteins, nucleic acids, and cells that are relatively purified with respect to other cellular or organism components that may normally be present in situ, up to and including a substantially pure preparation of the protein, nucleic acid, or cell. The term “isolated” may include proteins and nucleic acids that have no naturally occurring counterpart or proteins or nucleic acids that have been chemically synthesized and are thus substantially uncontaminated by other proteins or nucleic acids. The term “isolated” may include proteins, nucleic acids, or cells that have been separated or purified from most other cellular components or organism components with which they are naturally accompanied (e.g., but not limited to, other cellular proteins, nucleic acids, or cellular or extracellular components).

[0091] The term “wild type” includes entities having a structure and / or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0092] The term “endogenous sequence” refers to a nucleic acid sequence that occurs naturally within a cell or animal. For example, an endogenous IL6 sequence of an animal refers to a native IL6 sequence that naturally occurs at the IL6 locus in the animal.

[0093] “Exogenous” molecules or sequences include molecules or sequences that are not normally present in a cell in that form or that are introduced into a cell from an outside source. Normal presence includes presence with respect to the particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence, for example, can include a mutated version of a corresponding endogenous sequence within the cell, such as a humanized version of the endogenous sequence, or can include a sequence corresponding to an endogenous sequence within the cell but in a different form (i.e., not within a chromosome). In contrast, endogenous molecules or sequences include molecules or sequences that are normallypresent in that form in a particular cell at a particular developmental stage under particular environmental conditions.

[0094] The term “heterologous” when used in the context of a nucleic acid or a protein indicates that the nucleic acid or protein comprises at least two segments that do not naturally occur together in the same molecule. For example, the term “heterologous,” when used with reference to segments of a nucleic acid or segments of a protein, indicates that the nucleic acid or protein comprises two or more sub-sequences that are not found in the same relationship to each other (e.g., joined together) in nature. As one example, a “heterologous” region of a nucleic acid vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid vector could include a coding sequence flanked by a heterologous promoter not found in association with the coding sequence in nature. Likewise, a “heterologous” region of a protein is a segment of amino acids within or attached to another peptide molecule that is not found in association with the other peptide molecule in nature (e.g., a fusion protein, or a protein with a tag). Similarly, a nucleic acid or protein can comprise a heterologous label or a heterologous secretion or localization sequence.

[0095] Codon optimization” takes advantage of the degeneracy of codons, as exhibited by the multiplicity of three-base pair codon combinations that specify an amino acid, and generally includes a process of modifying a nucleic acid sequence for enhanced expression in particular host cells by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. For example, a nucleic acid encoding a TAR DNA-binding protein 43 (TDP-43) protein can be modified to substitute codons having a higher frequency of usage in a given prokaryotic or eukaryotic cell, including a bacterial cell, a yeast cell, a human cell, a nonhuman cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, or any other host cell, as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, at the “Codon Usage Database.” These tables can be adapted in a number of ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, herein incorporated by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (see, e.g., Gene Forge).

[0096] The term “locus” refers to a specific location of a gene (or significant sequence), DNA sequence, polypeptide-encoding sequence, or position on a chromosome of the genome of an organism. For example, an “ / / . / > locus” may refer to the specific location of an IL6 gene, IL6 DNA sequence, IL-6-encoding sequence, or IL6 position on a chromosome of the genome of an organism that has been identified as to where such a sequence resides. An “ / / .6 locus” may comprise a regulatory element of an IL6 gene, including, for example, an enhancer, a promoter, 5’ and / or 3’ untranslated region (UTR), or a combination thereof.

[0097] The term “gene” refers to DNA sequences in a chromosome that may contain, if naturally present, at least one coding and at least one non-coding region. The DNA sequence in a chromosome that codes for a product (e.g., but not limited to, an RNA product and / or a polypeptide product) can include the coding region interrupted with non-coding introns and sequence located adjacent to the coding region on both the 5’ and 3’ ends such that the gene corresponds to the full-length mRNA (including the 5’ and 3’ untranslated sequences).Additionally, other non-coding sequences including regulatory sequences (e.g., but not limited to, promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequence, and matrix attachment regions may be present in a gene. These sequences may be close to the coding region of the gene (e.g., but not limited to, within 10 kb) or at distant sites, and they influence the level or rate of transcription and translation of the gene.

[0098] The term “allele” refers to a variant form of a gene. Some genes have a variety of different forms, which are located at the same position, or genetic locus, on a chromosome. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ.

[0099] A “promoter” is a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other regions which influence the transcription initiation rate. The promoter sequences disclosed herein modulate transcription of an operably linked polynucleotide. A promoter can be active in one or more of the cell types disclosed herein (e.g., a eukaryotic cell, a non-human mammalian cell, a human cell, a rodent cell, a pluripotent cell, a one-cell stage embryo, a differentiated cell,or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013 / 176772, herein incorporated by reference in its entirety for all purposes.

[0100] A constitutive promoter is one that is active in all tissues or particular tissues at all developing stages. Examples of constitutive promoters include the human cytomegalovirus immediate early (hCMV), mouse cytomegalovirus immediate early (mCMV), human elongation factor 1 alpha (hEFla), mouse elongation factor 1 alpha (mEFla), mouse phosphoglycerate kinase (PGK), chicken beta actin hybrid (CAG or CBh), SV40 early, and beta 2 tubulin promoters.

[0101] Examples of inducible promoters include, for example, chemically regulated promoters and physically regulated promoters. Chemically regulated promoters include, for example, alcohol -regulated promoters (e.g., an alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., a tetracycline-responsive promoter, a tetracycline operator sequence (tetO), a tet-On promoter, or a tet-Off promoter), steroid regulated promoters (e.g., a rat glucocorticoid receptor, a promoter of an estrogen receptor, or a promoter of an ecdysone receptor), or metal-regulated promoters (e.g., a metalloprotein promoter). Physically regulated promoters include, for example temperature-regulated promoters (e.g., a heat shock promoter) and light-regulated promoters (e g., a light-inducible promoter or a light-repressible promoter).

[0102] Tissue-specific promoters can be, for example, liver-specific promoters or musclespecific promoters.

[0103] Developmentally regulated promoters include, for example, promoters active only during an embryonic stage of development, or only in an adult cell.

[0104] “Operable linkage” or being “operably linked” includes juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors.Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence).

[0105] The term “ / / / vitro" includes artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube or an isolated cell or cell line). The term “nz vivo" includes natural environments (e.g., a cell, organism, or body) and to processes or reactions that occur within a natural environment. The term “ex vivo" includes cells that have been removed from the body of an individual and processes or reactions that occur within such cells.

[0106] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients. The transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified elements recited in the claim and those that do not materially affect the basic and novel character! stic(s) of the claimed invention. Thus, the term “consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”

[0107] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not.

[0108] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range. For example, 5-10 nucleotides is understood as 5, 6, 7, 8, 9, or 10 nucleotides, whereas 5-10% is understood to contain 5% and all possible values through 10%.

[0109] When “at least,” “up to,” “more than,” “no more than,” “less than,” or other similar language modifies a number, it can be understood to modify each number in the series.

[0110] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. When “no more than” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified.

[0111] Unless otherwise apparent from the context, the term “about” encompasses values ± 5% of a stated value. In certain embodiments, the term “about” is understood to encompasstolerated variation or error within the art, e.g., 2 standard deviations from the mean, or the sensitivity of the method used to take a measurement, or a percent of a value as tolerated in the art, e.g., with age. When “about” is present before the first value of a series, it can be understood to modify each value in the series.

[0112] The term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0113] The term “or” refers to any one member of a particular list and also includes any combination of members of that list.

[0114] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof.

[0115] Statistically significant means p <0.05.

[0116] In the event of a conflict between a sequence in the application and an indicated accession number or position in an accession number, the sequence in the application predominates.DETAILED DESCRIPTIONI. Overview

[0117] Genetically modified non-human animals that are immunodeficient and optionally comprise xenotransplanted hepatocytes such as human hepatocytes, wherein the genetically modified non-human animal and / or the transplanted hepatocytes are modified to restore, activate, or increase interleukin-6 (IL-6) / interleukin-6 receptor (IL-6R) signaling pathway activity or interleukin-6 receptor subunit beta (GP130) signaling pathway activity and to restore, activate, or increase hepatocyte growth factor (HGF) / hepatocyte growth factor receptor (MET) signaling pathway activity in the transplanted hepatocytes, are provided. Also provided are methods of measuring or assessing the activity of human-liver-targeting reagents in such non-human animals and methods of making animals with a humanized liver (e.g., with reduced steatosis). Also provided are genetically modified, immunodeficient, non-human animals comprising a humanized HGF gene and optionally a humanized IL6 gene and methods of using and makingsuch animals. Also provided are methods of increasing or accelerating engraftment of xenotransplanted hepatocytes in non-human animals.

[0118] Humanized liver mouse models, in which donor human hepatocytes can repopulate recipient mouse or rat parenchyma, have been widely used in studying human liver biology, diseases and therapeutics. However, unlike rapid expansion of hepatocytes during liver regeneration, human hepatocytes engrafted in host mice or rats repopulate much slower, which greatly compromises the quality and usefulness of the models.

[0119] In many humanized liver mouse and rat models, two major liver regeneration pathways, (IL6-IL6R-GP130 and HGF-MET pathways) in engrafted human hepatocytes are compromised due to incompatibility between murine ligands expressed by host-derived non- parenchymal liver cells and their human receptors expressed on donor hepatocytes. However, restoration of either of these pathways alone showed no improvement of human hepatocyte engraftment speed. In the working examples below, we demonstrate that simultaneously restoration of both IL6 / GP130 and HGF / MET signaling (e.g., through supplementing with a Met agonist in recipient mice with humanized IL6 allele) resulted in robust acceleration of human hepatocyte expansion. The humanized liver also showed correction of fatty liver phenotype that occurs in conventional humanized liver models without humanized IL6 allele in host mice or rats due to defective IL6-GP130 pathway signaling. In addition, we show that supplementation with additional mitogen factors involved in human liver regeneration, such as RSPO3 and EGF, leads to further increase of repopulation speed of human hepatocytes in humanized liver mice.Moreover, we demonstrate that this approach resulted in successful engraftment of donor human hepatocytes considered to be “hard-to-engraff ’ by the current conventional methods. This results in a new class of humanized liver mouse and rat models which can be generated with a much shorter time-line, as well as with both increased liver humanization and corrected liver fattiness.II. Genetically Modified Non-Human Animals Comprising Xenotransplanted Hepatocytes

[0120] Provided herein are genetically modified non-human animals (e.g., mice or rats) suitable for xenotransplantation of hepatocytes / hepatocyte progenitors, such as human hepatocytes / hepatocyte progenitors, as well as genetically modified non-human animal cells and genomes.

[0121] The genetically modified non-human animals can comprise xenotransplanted hepatocytes / hepatocyte progenitors (e.g., xenotransplanted and expanded hepatocytes / hepatocyte progenitors). The xenotransplanted hepatocytes / hepatocyte progenitors can be from any species other than that of the recipient non-human animal. For example, the xenotransplanted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors. The transplanted hepatocytes / hepatocyte progenitors can be from a species whose IL-6R is incompatible with the endogenous non-human animal IL-6 (e.g., the transplanted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors, and the non-human animal can be a mouse or a rat). The transplanted hepatocytes / hepatocyte progenitors can be from a species whose MET is incompatible with the endogenous non-human animal HGF (e.g., the transplanted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors, and the non-human animal can be a mouse or a rat). The transplanted hepatocytes / hepatocyte progenitors can be from a species whose IL-6R is incompatible with the endogenous non-human animal IL-6 (e.g., the transplanted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors, and the non-human animal can be a mouse or a rat) and whose MET is incompatible with the endogenous non-human animal HGF (e.g., the transplanted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors, and the non-human animal can be a mouse or a rat). The transplanted hepatocytes / hepatocyte progenitors can be normal, healthy cells, or can be diseased or mutantbearing cells. For example, the transplanted hepatocytes / hepatocyte progenitors can be wild type hepatocytes / hepatocyte progenitors, or they can comprise one or more mutations. In a specific example, the transplanted hepatocytes / hepatocyte progenitors have a wild type Fah gene or a Fah gene that produces a functional protein.

[0122] Also provided are modifications to the genetically modified non-human animals and / or the xenotransplanted hepatocytes / hepatocyte progenitors to restore, activate, or increase IL-6 / IL-6R signaling pathway or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors and to restore, activate, or increase HGF / MET signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. Such modifications can result in accelerated or increased engraftment of the xenotransplanted hepatocytes / hepatocyte progenitors and reduced lipid droplet accumulation and reduced steatosis in the xenotransplanted hepatocytes / hepatocyte progenitors compared to non-human animals inwhich the genetically modified non-human animals and the xenotransplanted hepatocytes / hepatocyte progenitors do not have modifications to restore, activate, or increase IL- 6 / IL-6R signaling pathway or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors and to restore, activate, or increase HGF / MET signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors.

[0123] Also provided are modifications to the genetically modified non-human animals and / or the xenotransplanted hepatocytes / hepatocyte progenitors to restore, activate, or increase EGF / EGFR signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. This can be in combination, for example, with the restoration, activation, or increase in IL-6 / IL-6R signaling pathway or GP130 signaling pathway activity and the restoration, activation, or increase in HGF / MET signaling pathway activity. Such modifications can result in accelerated or increased engraftment of the xenotransplanted hepatocytes / hepatocyte progenitors compared to non-human animals in which the genetically modified non-human animals and the xenotransplanted hepatocytes / hepatocyte progenitors do not have modifications to activate or increase EGF / EGFR signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors.

[0124] Also provided are modifications to the genetically modified non-human animals and / or the xenotransplanted hepatocytes / hepatocyte progenitors to activate or increase RSPO3 / LGR4 or 5 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. This can be in combination, for example, with the restoration, activation, or increase in IL-6 / IL-6R signaling pathway or GP130 signaling pathway activity and the restoration, activation, or increase in HGF / MET signaling pathway activity. This can also be in combination, for example, with the restoration, activation, or increase in IL-6 / IL-6R signaling pathway or GP130 signaling pathway activity and the restoration, activation, or increase in HGF / MET signaling pathway activity and the restoration, activation, or increase in EGF / EGFR signaling pathway activity. Such modifications can result in accelerated or increased engraftment of the xenotransplanted hepatocytes / hepatocyte progenitors compared to non-human animals in which the genetically modified non-human animals and the xenotransplanted hepatocytes / hepatocyte progenitors do not have modifications to activate or increase RSPO3 / LGR4 or 5 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors.A. Genetically Modified Non-Human Animals for Xenotransplantation of Hepatocytes

[0125] Provided herein are genetically modified non-human animals suitable for xenotransplantation of hepatocytes / hepatocyte progenitors. Any suitable non-human animal can be used. In some cases, the animals are genetically modified non-human animals in which the non-human animal’s immune system has been modified such that it is unable or has reduced ability to mount an immune response to xenografted cells (e.g., human hepatocytes / hepatocyte progenitors). For example, the non-human animal can be immunodeficient. In some cases, the animals are genetically modified non-human animals in which (1) non-human (i.e., endogenous) hepatocytes in the liver can be selectively and conditionally ablated; and (2) the non-human animal’s immune system has been modified such that it is unable to mount an immune response to xenografted cells (e.g., human hepatocytes / hepatocyte progenitors) (e.g., the non-human animal is immunodeficient). Also provided are cells and genomes comprising the genetic modifications disclosed herein. The genetically modified non-human animals disclosed herein can be used for in vivo engraftment and expansion of xenotransplanted hepatocytes / hepatocyte progenitors (e.g., human hepatocytes / hepatocyte progenitors). Xenotransplantation refers to the transplantation of living cells, tissues, or organs from one species to another. Such cells, tissues, or organs are called xenografts or xenotransplants (e g., xenotransplanted cells).

[0126] Any suitable immunodeficient non-human animal can be used. See, e.g., Weber et al. (2009) Liver Transplantation 15:7-14, herein incorporated by reference in its entirety for all purposes. Such non-human animals can be immunocompromised such that T cells and B cells do not develop. For example, such immunodeficient non-human animals can lack functional T cells, B cells, and / or natural killer (NK) cells. Immunodeficient non-human animals refer to non- human animals lacking in at least one essential function of the immune system. For example, an immunodeficient non-human animal can be one lacking specific components of the immune system or lacking function of specific components of the immune system (such as, for example, B cells, T cells, orNK cells). In some cases, an immunodeficient animal lacks macrophages. In some cases, an immunodeficient animal comprises one or more genetic alterations that prevent or inhibit the development of functional immune cells (such as B cells, T cells, or NK cells). In some examples, the genetic alteration is in the Ragl, Rag2, or Il2rg gene (i.e., the immunodeficient non-human animal is Ragl Rag2~'~, and / or Il2rg~'~). In some cases, the non-human animal comprises an inactivated endogenous Il2rg gene. In some cases, the non-human animal comprises an inactivated endogenous Ragl gene and / or an inactivated endogenous Rag2 gene. In some cases, the non-human animal comprises an inactivated endogenous Ragl gene. In some cases, the non-human animal comprises an inactivated endogenous Rag2 gene. In some cases, the non-human animal comprises an inactivated endogenous Ragl gene and an inactivated endogenous Rag2 gene. In some cases, the immunodeficient non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene (i.e., is Rag2 and Il2rg~'~). In some cases, the immunodeficient non-human animal comprises an inactivated endogenous Ragl gene, an inactivated endogenous Rag2 gene, and an inactivated endogenous Il2rg gene (i.e., is Ragl , Rag2 , and Il2rg~'~). In some cases, the non-human animal comprises a severe combined immunodeficiency (SCID) mutation in the Prkdc gene (Prkdcscid). See, e.g., Mercer et al. (2001) Nat. Med. 7(8):927-933, herein incorporated by reference in its entirety for all purposes. Prkdc encodes DNA-dependent protein kinase catalytic subunit and is assigned NCBI GenelD 19090 in mice. The scid mutation is a mutation in the Prkdc gene, protein kinase, DNA activated, catalytic polypeptide. The specific genetic alteration in the Prkdcsc,dallele in mice is a T-to-A transversion point mutation to codon 4046 (codon 4095 in transcript ENSMUST00000023352.8) created a premature stop codon (p.Y4046*). Prkdc is involved in DNA repair via non-homologous end joining (NHEJ). V(D)J recombination, which rearranges the genetic components of antibodies and T cell receptors during B and T cell development, requires NHEJ for proper function. In some cases, the non-human animal comprises a severe combined immunodeficiency (SCID) mutation in the Prkdc gene Prkdcsc,d) and an inactivated endogenous Il2rg gene, such as the NOG background. See, e.g., Ito et al. (2002) Blood 100(9):3175-3182, herein incorporated by reference in its entirety for all purposes. For example, the Il2rg gene can comprise a mutation that produces a protein that is expressed and will bind cytokines but cannot signal, or it can comprise a mutation so that no IL2RG protein is expressed. In either case, the Z / 2 / g is considered inactivated. NOG mice are immunodeficient mice lacking mature T, B, and NK cells. Other examples of mutations causing immunodeficiency include X-linked SCID characterized by autosomal recessive SCID characterized by JAK3 mutations, ADA mutations, IL7R mutations, CD 3 mutations, ARTEMIS (DCLRE1C) mutations, and CD45 (PTPRC) mutations. Although certain non-limiting examplesof genetic alterations that result in immunodeficiency are provided above, other known immunodeficient non-human animals can also be used.

[0127] Any suitable genetic modification to allow for non-human (i.e., endogenous) hepatocytes in the liver to be selectively and conditionally ablated can be used. In some cases, the non-human animals comprise an inactivated endogenous Fah gene. This inactivation results in a toxic accumulation of tyrosine catabolites within hepatocytes. The compound 2-(2-nitro-4- trifluoro-methylbenzoyl)-l,3-cyclohexanedione (NTBC) can be used to block the enzyme hydroxyphenylpyruvate dioxygenase upstream of FAH and therefore prevents the accumulation of hepatotoxic metabolites. In some cases, the non-human animals comprise a urokinase type plasminogen activator (uPA) gene (Plair, NCBI GenelD 18792 in mice) in which the uPA coding sequence is operably linked to a liver-specific promoter, such as an albumin promoter. See, e.g., Mercer et al. (2001) Nat. Med. 7(8):927-933, herein incorporated by reference in its entirety for all purposes. Such non-human animals have hepatotoxicity leading to liver failure. In some cases, the non-human animals comprise a herpes simplex virus type 1 thymidine kinase (HSVtk) gene in which the HSVtk coding sequence is operably linked to a liver-specific promoter, such as an albumin promoter. See, e.g., Hasegawa et al. (2011) Biochem. Biophys. Res. Commun. 405(3):405-410, herein incorporated by reference in its entirety for all purposes. Administration of ganciclovir (GCV), a drug that is not toxic to human or mouse tissues, induces tissue-specific ablation of transgenic liver parenchymal cells. Because HSVtk catalyzes GCV phosphorylation, which is the rate-limiting step that cannot be performed in mammalian cells lacking this transgene, liver cells expressing the transgene are selectively destroyed. Although certain nonlimiting examples of genetic modifications to allow for non-human (i.e., endogenous) hepatocytes in the liver to be selectively and conditionally ablated are provided above, any other suitable genetic modifications can also be used.

[0128] In one example, genetically modified non-human animals for xenotransplantation of hepatocytes / hepatocyte progenitors (e.g., transplantation of human hepatocytes / hepatocyte progenitors) comprise SCID mutation in Prkdc gene (Prkdcsc,d) and comprise a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter such as an albumin enhancer / promoter (e.g., a PXB mouse). See, e.g., Meuleman et al. (2005) Hepatology 41(4):847-856, Tateno et al. (2004) Am. J. Pathol. 165(3):901-912, and Tateno et al. (2013) Lab.Invest. 93(1 ):54-71 , each of which is herein incorporated by reference in its entirety for all purposes.

[0129] In one example, genetically modified non-human animals for xenotransplantation of hepatocytes / hepatocyte progenitors (e.g., transplantation of human hepatocytes / hepatocyte progenitors) comprise a SCID mutation in Prkdc gene (Prkdcscid) and an inactivated endogenous Il2rg gene and comprise a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter (e.g., an Alb-HSVtk-NOG or albumin-TK-NOG mouse). See, e.g., Hasegawa et al. (2011) Biochem. Biophys. Res. Commim. 405(3):405-410, herein incorporated by reference in its entirety for all purposes.

[0130] In one example, genetically modified non-human animals for xenotransplantation of hepatocytes / hepatocyte progenitors (e.g., transplantation of human hepatocytes / hepatocyte progenitors) have the following genes inactivated (i.e., knocked out): Fah (encodes fumarylacetoacetase); Rag2 (encodes V(D)J recombination-activating protein 2); and Il2rg (encodes interleukin 2 receptor subunit gamma). Also provided are genetically modified non- human animal cells or genomes having the following genes inactivated (i.e., knocked out): Fah, Rag2,' and Il2rg. In one example, genetically modified non-human animals for xenotransplantation of hepatocytes / hepatocyte progenitors (e.g., transplantation of human hepatocytes / hepatocyte progenitors) have the following genes inactivated (i.e., knocked out): Fah (encodes fumarylacetoacetase); Ragl (encodes V(D)J recombination-activating protein 1); Rag2 (encodes V(D)J recombination-activating protein 2); and Il2rg (encodes interleukin 2 receptor subunit gamma). Also provided are genetically modified non-human animal cells or genomes having the following genes inactivated (i.e., knocked out): Fah, Ragl, Rag2 and Il2rg. In one example, the genetically modified non-human animal is a rat. See, e.g., Carbonaro et al. (2022) Sci. Rep. 12(1): 14079 and US 2016 / 0249591, each of which is herein incorporated by reference in its entirety for all purposes. In another example, the genetically modified non-human animal is a mouse. See, e.g., Strom et al. (2010) Methods Mol. Biol. 640:491-509, Azuma et al. (2007) Nat. Biotechnol. 25(8):903-910, and US 8,569,573, each of which is herein incorporated by reference in its entirety for all purposes. In another example, the genetically modified non- human animal is a pig. See, e.g., US 9,000,257, herein incorporated by reference in its entirety for all purposes.

[0131] Fah is an essential gene in the tyrosine catabolism pathway. When Fah is mutated in animals, toxic intermediate metabolites accumulate in the liver, causing hepatocyte loss and, ultimately, liver failure and death. This toxicity can be ameliorated by blocking the activity of another tyrosine catabolism enzyme, 4-hydroxyphenylpyruvate dioxygenase, which can be achieved by the administration of the small molecule nitisinone (NTBC). Fah mutant mice are healthy and viable when NTBC is administered. They quickly become moribund and die, however, when NTBC is withdrawn. Thus, NTBC administration and withdrawal allows precise temporal control of hepatocyte toxicity in Fah mutant non-human animals.

[0132] Ragl, Rag2, and Il2rg are essential components of the adaptive immune system. When these genes are mutated in animals, T-cells and B-cells do not mature, and the animals are severely compromised. When these animals are challenged with xenotransplanted cells, they are unable to mount an immune response to the foreign cells.

[0133] V(D)J recombination-activating protein 1 (also known as RAG1, recombinationactivating 1, recombination activating gene 1, and recombination activating protein 1) is encoded by the Ragl gene (also known as recombination activating 1). RAG1 is a catalytic component of the RAG complex, a multiprotein complex that mediates the DNA cleavage phase during V(D)J recombination. V(D)J recombination assembles a diverse repertoire of immunoglobulin and T- cell receptor genes in developing B and T-lymphocytes through rearrangement of different V (variable), in some cases D (diversity), and J (joining) gene segments. In the RAG complex, RAG1 mediates the DNA-binding to the conserved recombination signal sequences (RSS) and catalyzes the DNA cleavage activities by introducing a double-strand break between the RSS and the adjacent coding segment. RAG2 is not a catalytic component but is required for all known catalytic activities. RAG1 and RAG2 are essential to the generation of mature B cells and T cells, two types of lymphocytes that are crucial components of the adaptive immune system.

[0134] Mouse Ragl maps to 2 E2; 2 53.88 cM on chromosome 2 (NCBI RefSeq Gene ID 19373; Assembly GRCm39 (GCF_000001635.27); location NC_000068.8(101468597..101479877, complement). Reference to the mouse Ragl gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical, wild type mouse RAG1 protein has been assigned UniProt accession number P15919 and NCBI Accession No. NP_033045.2. Reference to mouse RAG1 proteins includes wild type forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBIAccession No. NM_009019.2. Reference to the mouse Ragl mRNA (cDNA) and coding sequence includes the canonical, wild type forms as well as all allelic forms and isoforms.

[0135] Rat Ragl maps to 3q31 on chromosome 3 (NCBI RefSeq Gene ID 84600; Assembly GRCr8 (GCF_036323735.1); location NC_086021.1 (108372087..108383184, complement). Reference to the rat Ragl gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical, wild type rat RAG1 protein has been assigned UniProt accession number G3V6K9 and NCBI Accession No. NP_445920.1. Reference to rat RAG1 proteins includes canonical, wild type forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_053468.1. Reference to the rat Ragl mRNA (cDNA) and coding sequence includes the canonical, wild type forms as well as all allelic forms and isoforms.

[0136] An inactivated endogenous Ragl gene is a Ragl gene that does not produce a RAG1 protein or does not produce a functional RAG1 protein. The non-human animal (or cell or genome) can comprise the inactivated Ragl gene in its germline. The non-human animal (or cell or genome) can be homozygous for an inactivating mutation in the Ragl gene. As one example, an inactivated endogenous Ragl gene can comprise an insertion, a deletion, or one or more point mutations in the endogenous Ragl gene resulting in loss of expression of functional RAG1 protein. Some inactivated endogenous Ragl genes can comprise a deletion or disruption of all of the endogenous Ragl gene or can comprise a deletion or disruption of a fragment of (i.e., a part of or portion of) the endogenous Ragl gene. For example, some, most, or all of the coding sequence in the endogenous Ragl gene can be deleted or disrupted. In one example, a 5’ fragment of the Ragl gene can be deleted or disrupted (e.g., including the start codon). As one example, an inactivated endogenous Ragl gene can be one in which the start codon of the endogenous Ragl gene has been deleted or has been disrupted or mutated such that the start codon is no longer functional. For example, the start codon can be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated by, for example, by a substitution of one or more nucleotides. In another example, a 3’ fragment of the Ragl gene can be deleted or disrupted (e.g., including the stop codon). In another example, an internal fragment of the Ragl gene (i.e., a fragment from the middle of the Ragl gene) can be deleted or disrupted. In another example, all of the coding sequence in the endogenous Ragl gene is deleted or disrupted.

[0137] V(D)J recombination-activating protein 2 (also known as RAG2, recombinationactivating 2, recombination activating gene 2, and recombination activating protein 2) is encoded by the Rag2 gene (also known as recombination activating 2). As mentioned above, RAG1 is a catalytic component of the RAG complex, a multiprotein complex that mediates the DNA cleavage phase during V(D)J recombination. RAG2 is not a catalytic component but is required for all known catalytic activities. RAG1 and RAG2 are essential to the generation of mature B cells and T cells, two types of lymphocytes that are crucial components of the adaptive immune system.

[0138] Mouse Rag2 maps to 2 E2; 2 53.87 cM on chromosome 2 (NCBI RefSeq Gene ID 19374; Assembly GRCm39 (GCF_000001635.27); location NC_000068.8(101455057..101462873). Reference to the mouse Rag2 gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical, wild type mouse RAG2 protein has been assigned UniProt accession number P21784 and NCBI Accession No. NP_033046.1.Reference to mouse RAG2 proteins includes canonical, wild type forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_009020.3. Reference to the mouse Rag2 mRNA (cDNA) and coding sequence includes the canonical, wild type forms as well as all allelic forms and isoforms.

[0139] Rat Rag2 maps to 3q31 on chromosome 3 (NCBI RefSeq Gene ID 295953; Assembly GRCr8 (GCF_036323735.1); location NC_086021.1 (108357399..108367186). Reference to the rat Rag2 gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical, wild type rat RAG2 protein has been assigned UniProt accession number G3V6K7 and NCBI Accession No. NP 001093998.1. Reference to rat RAG2 proteins includes canonical, wild type forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_001100528.1. Reference to the rat Rag2 mRNA (cDNA) and coding sequence includes the canonical, wild type forms as well as all allelic forms and isoforms.

[0140] An inactivated endogenous Rag2 gene is a Rag2 gene that does not produce a RAG2 protein or does not produce a functional RAG2 protein. The non-human animal (or cell or genome) can comprise the inactivated Rag2 gene in its germline. The non-human animal (or cell or genome) can be homozygous for an inactivating mutation in the Rag2 gene. As one example, an inactivated endogenous Rag2 gene can comprise an insertion, a deletion, or one or more pointmutations in the endogenous Rag2 gene resulting in loss of expression of functional RAG2 protein. Some inactivated endogenous Rag2 genes can comprise a deletion or disruption of all of the endogenous Rag2 gene or can comprise a deletion or disruption of a fragment of (i.e., a part of or portion of) the endogenous Rag2 gene. For example, some, most, or all of the coding sequence in the endogenous Rag2 gene can be deleted or disrupted. In one example, a 5’ fragment of the Rag2 gene can be deleted or disrupted (e.g., including the start codon). As one example, an inactivated endogenous Rag2 gene can be one in which the start codon of the endogenous Rag2 gene has been deleted or has been disrupted or mutated such that the start codon is no longer functional. For example, the start codon can be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated by, for example, by a substitution of one or more nucleotides. In another example, a 3’ fragment of the Rag2 gene can be deleted or disrupted (e.g., including the stop codon). In another example, an internal fragment of the Rag2 gene (i.e., a fragment from the middle of the Rag2 gene) can be deleted or disrupted. In another example, all of the coding sequence in the endogenous Rag2 gene is deleted or disrupted.

[0141] Interleukin 2 receptor subunit gamma (also known as interleukin 2 receptor, gamma; interleukin 2 receptor, gamma (severe combined immunodeficiency), isoform CRA a; cytokine receptor common subunit gamma precursor) is encoded by the Il2rg gene (also known as interleukin 2 receptor subunit gamma or IL2RG). IL2RG is a cytokine receptor subunit that is common to receptor complexes for several different interleukin receptors. IL2RG is located on the surface of immature blood-forming cells in bone marrow. IL2RG partners with other proteins to direct blood-forming cells to form lymphocytes. IL2RG also directs the growth and maturation of T cells, B cells, and natural killer cells. Mutations in Il2rg can cause X-linked severe combined immunodeficiency in which lymphocytes cannot develop normally. A lack of functional mature lymphocytes disrupts the immune system’s ability to protect the body from infection.

[0142] Mouse Il2rg maps to X D; X 43.9 cM on chromosome X (NCBI RefSeq Gene ID 16186; Assembly GRCm39 (GCF_000001635.27); location NC_000086.8(100307991..100311861, complement). Reference to the mouse Il2rg ene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical, wild type mouse IL2rG protein has been assigned UniProt accession number P34902 and NCBI AccessionNo. NP 038591 .1 . Reference to mouse IL2RG proteins includes canonical, wild type forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM 013563.4. Reference to the mouse Il2rg mRNA (cDNA) and coding sequence includes the canonical, wild type forms as well as all allelic forms and isoforms.

[0143] Rat Il2rg maps to Xq22 on chromosome X (NCBI RefSeq Gene ID 140924;Assembly GRCr8 (GCF_036323735. 1); location NC_086039.1 (70435340..70439052, complement). Reference to the va\.H2rg gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical, wild type rat IL2rG protein has been assigned UniProt accession number Q68FU6 and NCBI Accession No. NP 543165.1. Reference to rat IL2RG proteins includes canonical, wild type forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_080889.1.Reference to the rat Il2rg mRNA (cDNA) and coding sequence includes the canonical, wild type forms as well as all allelic forms and isoforms.

[0144] An inactivated endogenous Il2rg gene is an Il2rg gene that does not produce a IL2RG protein or does not produce a functional IL2RG protein. For example, the Il2rg gene can comprise a mutation that produces a protein that is expressed and will bind cytokines but cannot signal, or it can comprise a mutation so that no IL2RG protein is expressed. In either case, the Il2rg is considered inactivated. The non-human animal (or cell or genome) can comprise the inactivated Il2rg gene in its germline. The non-human animal (or cell or genome) can be homozygous for an inactivating mutation in the Il2rg gene. As one example, an inactivated endogenous Il2rg gene can comprise an insertion, a deletion, or one or more point mutations in the endogenous H2rg gene resulting in loss of expression of functional IL2RG protein. Some inactivated endogenous Il2rg genes can comprise a deletion or disruption of all of the endogenous Il2rg gene or can comprise a deletion or disruption of a fragment of (i.e., a part of or portion of) the endogenous Il2rg gene. For example, some, most, or all of the coding sequence in the endogenous Il2rg gene can be deleted or disrupted. In one example, a 5’ fragment of the Il2rg gene can be deleted or disrupted (e g., including the start codon). As one example, an inactivated endogenous H2rg gene can be one in which the start codon of the endogenous H2rg gene has been deleted or has been disrupted or mutated such that the start codon is no longer functional. For example, the start codon can be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated by, for example, by a substitution of one or morenucleotides. In another example, a 3’ fragment of the U2rg ene can be deleted or disrupted (e.g., including the stop codon). In another example, an internal fragment of the Il2rg gene (i.e., a fragment from the middle of the Il2rg gene) can be deleted or disrupted. In another example, all of the coding sequence in the endogenous Il2rg gene is deleted or disrupted.

[0145] Fumarylacetoacetase (also known as FAH, FAA, beta-diketonase, or fumarylacetoacetate hydrolase) is encoded by the Fah gene (also known as fumarylacetoacetate hydrolase). FAH is an enzyme required in the last step of the tyrosine catabolic pathway, which hydrolyzes fumarylacetoacetate into fumarate and acetoacetate. A deficiency in Fah leads to the accumulation of toxic metabolites, including fumarylacetoacetate and maleylacetoacetate. 2-(2- Nitro-4-trifluorom ethylbenzoyl)- 1,3 -cyclohexanedi one (nitisinone or NTBC) as an inhibitor of 4-hydroxyphenylpyruvate dioxygenase acts by blocking the accumulation of toxic metabolites such as fumarylacetoacetate and maleylacetoacetate and can be effective in ameliorating liver and kidney damage in human patients with Fah deficiency. Several Fah mutations have been found that cause tyrosinemia type I (type 1 hereditary tyrosinemia, or HT) in humans. This condition is characterized by severe liver and kidney disease, neurological problems, and other signs and symptoms that begin in infancy. The altered Fah gene that causes this condition produces an unstable or inactive enzyme, which results in reduced or absent fumarylacetoacetate hydrolase activity. Without sufficient fumarylacetoacetate hydrolase activity, tyrosine and its byproducts are not properly broken down. As a result, fumarylacetoacetate accumulates in the liver and kidneys. Elevated levels of fumarylacetoacetate are thought to be toxic to cells and accumulation of this substance likely causes the liver and kidney problems and other features that are characteristic of tyrosinemia type I.

[0146] Mouse Fah maps to 7 D3; 7 48.36 cM on chromosome 7 (NCBI RefSeq Gene ID 14085; Assembly GRCm39 (GCF_000001635.27); location NC_000073.7(84234367..84255150, complement). Reference to the mouse Fah gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical, wild type mouse FAH protein has been assigned UniProt accession number P35505 and NCBI Accession No. NP_034306.2. Reference to mouse FAH proteins includes canonical, wild type forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_010176.4. Reference to the mouse Fah mRNA (cDNA) and coding sequence includes the canonical, wild type forms as well as all allelic forms and isoforms.

[0147] Rat Fah maps to 1 q31 on chromosome 1 (NCBI RefSeq Gene ID 29383; Assembly GRCr8 (GCF_036323735.1); location NC_086019.1 (147957931..147980708, complement). Reference to the rat Fah gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical, wild type rat FAH protein has been assigned UniProt accession number P25093 and NCBI Accession No. NP_058877.1. Reference to rat FAH proteins includes canonical, wild type forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_017181.2. Reference to the rat Fah mRNA (cDNA) and coding sequence includes the canonical, wild type forms as well as all allelic forms and isoforms.

[0148] An inactivated endogenous Fah gene is a Fah gene that does not produce a FAH protein or does not produce a functional FAH protein. The non-human animal (or cell or genome) can comprise the inactivated Fah gene in its germline. The non-human animal (or cell or genome) can be homozygous for an inactivating mutation in the Fah gene. As one example, an inactivated endogenous Fah gene can comprise an insertion, a deletion, or one or more point mutations in the endogenous Fah gene resulting in loss of expression of functional FAH protein. Some inactivated endogenous Fah genes can comprise a deletion or disruption of all of the endogenous Fah gene or can comprise a deletion or disruption of a fragment of (i.e., a part of or portion of) the endogenous Fah gene. For example, some, most, or all of the coding sequence in the endogenous Fah gene can be deleted or disrupted. In one example, a 5’ fragment of the Fah gene can be deleted or disrupted (e g., including the start codon). As one example, an inactivated endogenous Fah gene can be one in which the start codon of the endogenous Fah gene has been deleted or has been disrupted or mutated such that the start codon is no longer functional. For example, the start codon can be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated by, for example, by a substitution of one or more nucleotides. In another example, a 3’ fragment of the Fah gene can be deleted or disrupted (e.g., including the stop codon). In another example, an internal fragment of the Fah gene (i.e., a fragment from the middle of the Fah gene) can be deleted or disrupted. In another example, all of the coding sequence in the endogenous Fah gene is deleted or disrupted.

[0149] Some genetically modified non-human animals described herein further comprise a humanized SIRPA gene, although this is not required. For example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and a humanizedSJRPA gene. For example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanized SIRPA gene. In one example, the non-human animal is a mouse. In another example, the non-human animal is a rat.

[0150] SIRPA (also known as BIT, MFR, MYD1, PTPNSI, SHPS1, SIRPa, and SIRP) encodes tyrosine-protein phosphatase non-receptor type substrate 1 (also known as brain Ig-like molecule with tyrosine-based activation motifs (Bit), CD 172 antigen-like family member A, CD 172a, inhibitory receptor SHPS-1, macrophage fusion receptor, MyD-1 antigen, SIRPA, SIRPa, and signal-regulatory protein alpha), which is an immunoglobulin-like cell surface receptor for CD47. It acts as docking protein and induces translocation of PTPN6, PTPN11 and other binding partners from the cytosol to the plasma membrane.

[0151] Mouse Sirpa maps to 2 F 1 ; 2 63.19 cM on chromosome 2 (NCBI RefSeq Gene ID19261; Assembly GRCm39 (GCF_000001635.27); location NC_000068.8(129432962..129474148). Reference to the mouse Sirpa gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical mouse SIRPA protein has been assigned UniProt accession number P97797 and NCBI Accession Nos. NP_001277948.1 and NP_001277949.1. Reference to mouse SIRPA proteins includes canonical forms as well as all allelic forms and isoforms. mRNAs (cDNAs) encoding the canonical isoform are assigned NCBI Accession Nos. NM_001291019.1 and NM_001291020.1. Reference to the mouse Sirpa mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0152] Rat Sirpa maps to 3q36 on chromosome 3 (NCBI RefSeq Gene ID 25528; Assembly GRCr8 (GCF 036323735.1); location NC 086021.1 (137272932..137311279). Reference to the rat Sirpa gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical rat SIRPA protein has been assigned UniProt accession number P97710 and NCBI Accession No. NP 037148.2. Reference to rat SIRPA proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_013016.2. Reference to the rat Sirpa mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0153] Human SIRPA maps to 20pl3 on chromosome 20 (NCBI RefSeq Gene ID 140885; Assembly GRCh38.pl4 (GCF_000001405.40); location NC_000020.11 (1894167..1940592). Reference to the human SIRPA gene includes the canonical, wild type form as well as all allelicforms and isoforms. The canonical human SIRPA protein has been assigned UniProt accession number P78324 and NCBI Accession Nos. NP_001035111.1, NP_001035112.1 , and NP 542970.1. Reference to human SIRPA proteins includes the canonical form as well as all allelic forms and isoforms. mRNAs (cDNAs) encoding the canonical isoform are assigned NCBI Accession Nos. NM_001040022.1, NM_001040023.1, and NM_080792.2. Reference to the human SIRPA mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0154] Examples of non-human animals with humanized SIRPA genes are provided, e.g., in US 9,901,083, herein incorporated by reference in its entirety for all purposes. The non-human animal (or cell or genome) can be, in some cases, homozygous for the humanized SIRPA gene. In other cases, the animal (or cell or genome) can be heterozygous for the humanized SIRPA gene. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ. The non-human animal can comprise the humanized SIRPA gene in its germline. The humanized SIRPA gene can comprise a human SIRPA nucleic acid encoding a portion of a human SIRPA protein, such as the extracellular domain of a human SIRPA protein. The human SIRPA nucleic acid can be a genomic nucleic acid, such as a genomic nucleic acid comprising a region of a human SIRPA gene comprising exons 2-4, or can comprise a corresponding part of a human SIRPA complementary DNA (cDNA). Alternatively, the human SIRPA can comprise the entire coding sequence for a human SIRPA protein such that a fully human SIRPA protein is encoded. The human SIRPA nucleic acid can be inserted into the non-human animal Sirpa genomic locus, or it can replace a corresponding region of the non-human animal Sirpa locus (e.g., a region of a human SIRPA gene comprising exons 2-4 can replace exons 2-4 of the non-human animal Sirpa gene). The humanized SIRPA gene (or the human SIRPA nucleic acid) can be operably linked to the endogenous non-human animal Sirpa promoter. In other words, expression of the humanized SIRPA gene can be driven by the endogenous non-human animal Sirpa promoter.

[0155] Some genetically modified non-human animals described herein further comprise a humanized IL6 gene. In one example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and a humanized IL6 gene. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2. and Il2rggenes and humanized SJRPA and IL6 genes. In one example, such genetically modified nonhuman animals can comprise inactivated / ah, Ragl, Rag2, and Il2rg genes and a humanized IL6 gene. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA and IL6 genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized IL6 gene. In one example, the non-human animal is a mouse. In another example, the non-human animal is a rat.

[0156] IL6 (also known as IL-6 and IFNB2 encodes interleukin-6 (also known as IL-6, IL6, B-cell stimulatory factor 2 (BSF-2), CTL differentiation factor (CDF), hybridoma growth factor, and interferon beta-2 (IFN-beta-2). IL-6 is a cytokine with a wide variety of biological functions in immunity, tissue regeneration, and metabolism. It binds to IL-6R, then the complex associates with the signaling subunit IL6ST / gpl30 to trigger the intracellular IL6-signaling pathway.

[0157] Mouse 116 maps to 5 Bl; 5 15.7 cM on chromosome 5 (NCBI RefSeq Gene ID 16193; Assembly GRCm39 (GCF_000001635.27); location NC_00007L7(30218112..30224973). Reference to the mouse 116 gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical mouse IL-6 protein has been assigned UniProt accession number P08505 and NCBI Accession Nos. NP_001300983.1 andNP_112445.1. Reference to mouse IL-6 proteins includes canonical forms as well as all allelic forms and isoforms. mRNAs (cDNAs) encoding the canonical isoform are assigned NCBI Accession Nos. NM_001314054.1 and NM_031168.2. Reference to the mouse / / 6 mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0158] Rat 116 maps to 4ql 1 on chromosome 4 (NCBI RefSeq Gene ID 24498; Assembly GRCr8 (GCF_036323735.1); location NC_086022.1 (5889999..5894575, complement).Reference to the rat 116 gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical rat IL-6 protein has been assigned UniProt accession number P20607 and NCBI Accession No. NP_036721.1. Reference to rat IL-6 proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_012589.2. Reference to the rat 116 mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0159] Human JL6 maps to 7pl 5.3 on chromosome 7 (NCBI RefSeq Gene ID 3569; Assembly GRCh38.pl4 (GCF_000001405.40); location NC_000007.14 (22727200. 22731998). Reference to the human IL6 gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical human IL-6 protein has been assigned UniProt accession number P05231 and NCBI Accession No. NP_000591.1 (SEQ ID NO: 6). Reference to human IL-6 proteins includes the canonical form as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_000600.4. A coding sequence for the canonical isoform is assigned CCDS Accession No. CCDS5375.1 (SEQ ID NO: 5). Reference to the human IL6 mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0160] Examples of non-human animals with humanized IL6 genes are provided, e.g., in US 9,622,460, herein incorporated by reference in its entirety for all purposes. The non-human animal (or cell or genome) can be, in some cases, homozygous for the humanized IL6 gene. In other cases, the animal (or cell or genome) can be heterozygous for the humanized IL6 gene. The non-human animal can comprise the humanized IL6 gene in its germline. The humanized IL6 gene can comprise a human IL6 nucleic acid encoding a human IL-6 protein (e.g., a fully human IL-6 protein). The human 1L6 nucleic acid can be a genomic nucleic acid, such as a genomic nucleic acid comprising a region of a human IL6 gene from the start codon to the stop codon, or can comprise a human IL6 complementary DNA (cDNA). The human IL6 nucleic acid can be inserted into the non-human animal 116 genomic locus, or it can replace a corresponding region of the non-human animal 116 locus (e.g., a region of a human 1L6 gene from the start codon to the stop codon can replace a region of the non-human animal 116 gene from the start codon to the stop codon). The humanized IL6 gene (or the human IL6 nucleic acid) can be operably linked to the endogenous non-human animal 116 promoter. In other words, expression of the humanized IL6 gene can be driven by the endogenous non-human animal 116 promoter.

[0161] Some genetically modified non-human animals described herein further comprise a humanized OSM gene. In one example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and a humanized OSM gene. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA and OSM genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanizedOS gene. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA and OSM genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized OSM gene. In another example, such genetically modified non- human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized IL6 and OSM genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA, IL6, and OSM enes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized IL6 and OSM genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA, IL6, and OSM genes. In another example, such genetically modified non- human animals can be immunodeficient non-human animals comprising humanized IL6 and OSM genes. In one example, the non-human animal is a mouse. In another example, the non- human animal is a rat.

[0162] OSM encodes oncostatin-M (also known as OSM). OSM is a pleiotropic cytokine that belongs to the interleukin 6 group of cytokines. Of these cytokines, it most closely resembles leukemia inhibitory factor (LIF) in both structure and function. It is important in liver development, hematopoiesis, inflammation and possibly CNS development. It is also associated with bone formation and destruction. OSM signals through cell surface receptors that contain the protein GP130. The type I receptor is composed of GP130 and LIFR, the type II receptor is composed of GP130 and OSMR.

[0163] Mouse Osm maps to 11 Al ; 11 2.94 cM on chromosome 11 (NCBI RefSeq Gene ID 18413; Assembly GRCm39 (GCF_000001635.27); location NC_000077.7 (4186785..4191026). Reference to the mouse Osm gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical mouse OSM protein has been assigned UniProt accession number P53347 and NCBI Accession No. NP_001013383.1. Reference to mouse OSM proteins includes canonical forms as well as all allelic forms and isoforms. mRNAs (cDNAs) encoding the canonical isoform are assigned NCBI Accession No. NM_001013365.3. Reference to the mouse Osm mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0164] Rat Osm maps to 14q21 on chromosome 14 (NCBI RefSeq Gene ID 289747;Assembly GRCr8 (GCF_036323735.1); location NC_086032.1 (83327202 .83332073).Reference to the rat Osm gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical rat OSM protein has been assigned UniProt accession number Q65Z15 and NCBI Accession No. NP_001006962.1. Reference to rat OSM proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_001006961.2. Reference to the rat Osm mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0165] Human OSM maps to 22ql2.2 on chromosome 22 (NCBI RefSeq Gene ID 5008; Assembly GRCh38.pl4 (GCF_000001405.40); location NC_000022.11 (30262829..30266851, complement). Reference to the human OSM gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical human OSM protein has been assigned UniProt accession number Pl 3725 and NCBI Accession No. NP 065391.1 (SEQ ID NO: 12). Reference to human OSM proteins includes the canonical form as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_020530.6. A coding sequence for the canonical isoform is assigned CCDS Accession No. CCDS13873.1 (SEQ ID NO: 11). Reference to the human OSM mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0166] Some genetically modified non-human animals described herein further comprise a humanized HGF gene. In one example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and a humanized HGF gene. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA and HGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanized HGF gene. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA and HGF genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized HGF gene. In one example, such genetically modified non- human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized IL6 and HGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA, IL6, and HGF genes. In oneexample, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanized IL6 and HGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA, IL6, and HGF genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized IL6 and HGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized OSM and HGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA, OSM, and HGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanized OSM and HGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA, OSM, and HGF genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized OSM and HGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized IL6, OSM, and HGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and H2rg genes and humanized SIRPA, IL6, OSM, and HGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanized IL6, OSM, and HOJF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and I12rg genes and humanized SIRPA, IL6, OSM, and HGF genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized IL6, OSM, and HGF genes. In one example, the non-human animal is a mouse. In another example, the non-human animal is a rat.

[0167] HGF (also known as HPTA) encodes hepatocyte growth factor (also known as HGF, hepatopoietin-A, and scatter factor (SF)). HGF is the activating ligand for the receptor tyrosine kinase MET by binding to it and promoting its dimerization.

[0168] Mouse 7 / / maps to 5 A3; 5 7.07 cM on chromosome 5 (NCBI RefSeq Gene ID 15234; Assembly GRCm39 (GCF_000001635.27); location NC_000071.7(16758493..16827448). Reference to the mouse Tfg ’gene includes the canonical, wild type formas well as all allelic forms and isoforms. The canonical mouse HGF protein has been assigned UniProt accession number Q08048 and NCBI Accession No. NP_001276387.1. Reference to mouse HGF proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_001289458.1. A coding sequence for the canonical isoform is assigned CCDS Accession No. CCDS 19097.1. Reference to the mouse 7 / q / mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0169] Rat Hgf maps to 4ql2 on chromosome 4 (NCBI RefSeq Gene ID 24446; AssemblyGRCr8 (GCF_036323735.1); location NC_086022.1 (19628902..19700467, complement). Reference to the rat Hgf gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical rat HGF protein has been assigned UniProt accession number P17945 and NCBI Accession No. NP_058713.1. Reference to rat HGF proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_017017.2. Reference to the rat Hgf mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0170] Human HG maps to 7q21.11on chromosome 7 (NCBI RefSeq Gene ID 3082; Assembly GRCh38.pl4 (GCF_000001405.40); location NC_000007.14 (81699010..81770047, complement). Reference to the human HGF gene includes all allelic forms and isoforms. The canonical human HGF protein has been assigned UniProt accession number Pl 4210 and NCBI Accession No. NP 000592.3 (SEQ ID NO: 18). Reference to human HGF proteins includes the canonical form as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_000601.5. A coding sequence for the canonical isoform is assigned CCDS Accession No. CCDS5597.1 (SEQ ID NO: 17). Reference to the human HGF mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0171] An example of a non-human animal with a humanized HGF gene is provided, e.g., in Brodeur et al. (2009) Cancer Res. 69(9_Supplement):305, herein incorporated by reference in its entirety for all purposes. The non-human animal (or cell or genome) can be, in some cases, homozygous for the humanized HGF gene. In other cases, the animal (or cell or genome) can be heterozygous for the humanized HGF gene. The non-human animal can comprise the humanizedHGF gene in its germline. The humanized H GF gene can comprise a human HGF nucleic acid encoding a human HGF protein (e.g., a fully human HGF protein). The human HGF nucleic acid can be a genomic nucleic acid, such as a genomic nucleic acid comprising a region of a human HGF gene from the start codon to the stop codon, or can comprise a human HGF complementary DNA (cDNA). The human HGF nucleic acid can be inserted into the non-human animal Hgf genomic locus, or it can replace a corresponding region of the non-human animal F / g locus (e.g., a region of a human HGF gene from the start codon to the stop codon can replace a region of the non-human animal Hgf gene from the start codon to the stop codon). The humanized HGF gene (or the human HGF nucleic acid) can be operably linked to the endogenous non-human animal / / g / promoter. In other words, expression of the humanized HGF gene can be driven by the endogenous non-human animal Hgf promoter.

[0172] Some genetically modified non-human animals described herein further comprise a humanized EGF gene. In one example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and H2rg genes and a humanized EGF gene. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA and EGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and H2rg genes and a humanized EGF gene. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA and EGF genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized EGF gene. In one example, such genetically modified non- human animals can comprise inactivated Fah, Rag2, and H2rg genes and humanized IL6 and EGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA, IL6, and EGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanized IL6 and EGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA, 1L6, and EGF genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized IL6 and EGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized OSM and EGF genes. Inanother example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA, OSM, and EGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanized OSM and EGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA, OSM, and EGF genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized OSM and EGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized IL6, OSM, and EGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA, IL6, OSM, and EG genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanized IL6, OSM, and EGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA, IL6, OSM, and EGF genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized IL6, OSM, and EGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized IL6, HGF, and EGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA, IL6, HGF, and EGF genes. In one example, such genetically modified non- human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanized IL6, HGF, and EGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA, IL6, HGF, and EGF genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized IL6, HGF, and EGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized OSM, HGF, and EGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA, OSM, HGF, and EGF genes. In one example, such genetically modified non- human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanized OSM, HGF, and EGF genes. In another example, such genetically modified non-human animalscan comprise inactivated ah, Rag I, Rag2, and U2rg genes and humanized SIRPA, OSM, HGF, and EGF genes. In another example, such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized OSM, HGF, and EGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and H2rg genes and humanized IL6, OSM, HGF, and EGF genes. In another example, such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA, IL6, OSM, HGF, and EGF genes. In one example, such genetically modified non-human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and a humanized 1L6, OSM, HGF, and EGF genes. In another example, such genetically modified non- human animals can comprise inactivated Fah, Ragl, Rag2, and Il2rg genes and humanized SIRPA, IL6, OSM, HGF, and EGF genes. In another example, such genetically modified non- human animals can be immunodeficient non-human animals comprising a humanized IL6, OSM, HGF, and EGF genes. In one example, the non-human animal is a mouse. In another example, the non-human animal is a rat.

[0173] EGF encodes epidermal growth factor (also known as EGF or pro-epidermal growth factor). EGF stimulates the growth of various epidermal and epithelial tissues in vivo and in vitro and of some fibroblasts in cell culture.

[0174] Mouse Eg maps 3 G3; 3 58.5 cM on chromosome 3 (NCBI RefSeq Gene ID 13645; Assembly GRCm39 (GCF_000001635.27); location NC_000069.7 (129471223..129548971, complement). Reference to the mouse Egf gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical mouse EGF protein has been assigned UniProt accession number P01132 and NCBI Accession No. NP 034243.2. Reference to mouse EGF proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_010113.4. A coding sequence for the canonical isoform is assigned CCDS Accession No. CCDS 17833.1. Reference to the mouse Eg mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0175] Rat Eg maps to 2q43 on chromosome 2 (NCBI RefSeq Gene ID 25313; Assembly GRCr8 (GCF_036323735.1); location NC_086020.1 (220893660..220976331, complement). Reference to the rat Egf gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical rat EGF protein has been assigned UniProt accession numberP07522 and NCBI Accession No. NP_036974.1 . Reference to rat EGF proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM 012842.1. Reference to the rat Egf mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0176] Human epidermal growth factor (also known as EGF or pro-epidermal growth factor) is encoded by the gene EGF. Human EGF maps to 4q25 on chromosome 4 (NCBI RefSeq Gene ID 1950; Assembly GRCh38.pl4 (GCF_000001405.40); location NC_000004.12 (109912883..110013766). Reference to the human EGF gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical human EGF protein has been assigned UniProt accession number P01133 and NCBI Accession No. NP_001954.2 (SEQ ID NO: 28). Reference to human EGF proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_001963.5. A coding sequence encoding the canonical isoform is assigned CCDS Accession No. CCDS3689.1 (SEQ ID NO: 27). Reference to the human EGF mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0177] The non-human animal (or cell or genome) can be, in some cases, homozygous for the humanized EGF gene. In other cases, the animal (or cell or genome) can be heterozygous for the humanized EGF gene. The non-human animal can comprise the humanized EGF gene in its germline. The humanized EGF gene can comprise a human EGiF nucleic acid encoding a human EGF protein (e.g., a fully human EGF protein). The human EGF nucleic acid can be a genomic nucleic acid, such as a genomic nucleic acid comprising a region of a human EGF gene from the start codon to the stop codon, or can comprise a human EGF complementary DNA (cDNA). The human EGF nucleic acid can be inserted into the non-human animal Egf genomic locus, or it can replace a corresponding region of the non-human animal Eg locus (e.g., a region of a human EGF gene from the start codon to the stop codon can replace a region of the non-human animal Eg gene from the start codon to the stop codon). The humanized EGF gene (or the human EGF nucleic acid) can be operably linked to the endogenous non-human animal Eq / promoter. In other words, expression of the humanized EGF gene can be driven by the endogenous non-human animal Egf promoter.

[0178] Some genetically modified non-human animals described herein further comprise ahumanized Growth Hormone gene. Similar to IL6, mGH shows species specificities in receptor binding, and administration of human Growth Hormone (GH) in humanized liver mice could correct fatty liver in humanized liver mice. Tateno et al. (2011) Endocrinology 152: 1479-1491, herein incorporated by reference in its entirety for all purposes.

[0179] The non-human animals (or cells or genomes) disclosed herein can be male or female. Non-human animal cells disclosed herein can be any type of undifferentiated or differentiated state. For example, a non-human animal cell can be a totipotent cell, a pluripotent cell (e.g., mouse or rat pluripotent cell such as a mouse or rat embryonic stem (ES) cell), or a non- pluripotent cell. Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that possess the ability to develop into more than one differentiated cell types. Such pluripotent and / or totipotent cells can be, for example, ES cells or ES-like cells, such as an induced pluripotent stem (iPS) cell. ES cells include embryo- derived totipotent or pluripotent cells that are capable of contributing to any tissue of the developing embryo upon introduction into an embryo. ES cells can be derived from the inner cell mass of a blastocyst and are capable of differentiating into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[0180] The cells provided herein can also be germ cells (e.g., sperm or oocytes). The cells can be mitotically competent cells or mitotically inactive cells, meiotically competent cells or meiotically-inactive cells. Similarly, the cells can also be primary somatic cells or cells that are not a primary somatic cell. Somatic cells include any cell that is not a gamete, germ cell, gametocyte, or undifferentiated stem cell. For example, the cells can be liver cells (e.g., hepatocytes / hepatocyte progenitors).

[0181] Suitable cells provided herein also include primary cells. Primary cells include cells or cultures of cells that have been isolated directly from an organism, organ, or tissue. Primary cells include cells that are neither transformed nor immortal. They include any cell obtained from an organism, organ, or tissue which was not previously passed in tissue culture or has been previously passed in tissue culture but is incapable of being indefinitely passed in tissue culture. Such cells can be isolated by conventional techniques and include, for example, liver cells (e.g., hepatocytes / hepatocyte progenitors) .

[0182] Other suitable cells provided herein include immortalized cells. Immortalized cells include cells from a multicellular organism that would normally not proliferate indefinitely but,due to mutation or alteration, have evaded normal cellular senescence and instead can keep undergoing division. Such mutations or alterations can occur naturally or be intentionally induced. Numerous types of immortalized cells are well known. Immortalized or primary cells include cells that are typically used for culturing or for expressing recombinant genes or proteins.

[0183] The cells provided herein also include one-cell stage embryos (i.e., fertilized oocytes or zygotes). Such one-cell stage embryos can be from any genetic background, can be fresh or frozen, and can be derived from natural breeding or in vitro fertilization. The cells provided herein can be normal, healthy cells, or can be diseased or mutant-bearing cells.

[0184] The non-human animal can be a eukaryote, which includes, for example, animals and mammals. The term “animal” includes any member of the animal kingdom, including, for example, mammals, fishes, reptiles, amphibians, and birds. A mammal can be, for example, a pig, a rodent, a rat, or a mouse. Other mammals include, for example, non-human primates, cats, dogs, rabbits, cows, sheep, goats, pigs, and boars, and so forth. Birds include, for example, chickens, turkeys, ostrich, geese, ducks, and so forth. The term “non-human” excludes humans.

[0185] The non-human animals can be from any genetic background. For example, suitable mice, mouse cells, or mouse genomes can be from a 129 strain, a C57BL / 6 strain, a mix of 129 and C57BL / 6, a BALB / c strain, or a Swiss Webster strain. Examples of 129 strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129Sl / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. See, e.g., Festing et al. (1999) Mamm. Genome 10(8): 836, herein incorporated by reference in its entirety for all purposes. Examples of C57BL strains include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / Kal wN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / 01a. Suitable mice can also be from a mix of an aforementioned 129 strain and an aforementioned C57BL / 6 strain (e.g., 50% 129 and 50% C57BL / 6). Likewise, suitable mice can be from a mix of aforementioned 129 strains or a mix of aforementioned BL / 6 strains (e.g., the 129S6 (129 / SvEvTac) strain).

[0186] Rats or rat cells or rat genomes can be from any rat strain, including, for example, an ACI rat strain, a Dark Agouti (DA) rat strain, a Wistar rat strain, a LEA rat strain, a Sprague Dawley (SD) rat strain, or a Fischer rat strain such as Fisher F344 or Fisher F6. Rats, rat cells, or rat genomes can also be obtained from a strain derived from a mix of two or more strains recited above. For example, a suitable rat can be from a DA strain or an ACI strain. The ACI rat strain ischaracterized as having black agouti, with white belly and feet and an RTlavlhaplotype. Such strains are available from a variety of sources including Harlan Laboratories. The Dark Agouti (DA) rat strain is characterized as having an agouti coat and an RTlavlhaplotype. Such rats are available from a variety of sources including Charles River and Harlan Laboratories. Some suitable rats, rat cells, and rat genomes can be from an inbred rat strain. See, e.g., US 2014 / 0235933, herein incorporated by reference in its entirety for all purposes.

[0187] The non-human animals described herein can also exhibit decreased liver function, such as phenotypic and biochemical manifestations of human hereditary tyrosinemia type I (HT1). Procedures for testing liver function are well known. See, e.g., Grompe et al. (1993) Genes Dev. 7:2298-2307 and Manning et al. (1999) Proc. Natl. Acad. Set. U.S.A. 96:11928- 11933. As an example, the non-human animals can exhibit one or more of the following (e.g., in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency and / or in the absence of transplantation of hepatocytes / hepatocyte progenitors with a functional copy of Fah): hypertyrosinemia; liver fibrosis; cirrhosis; liver failure; and renal tubular damage or dysfunction. For example, the non-human animals described herein can exhibit hypertyrosinemia, liver failure, and renal tubular damage or dysfunction. Likewise, the non-human animals can exhibit liver fibrosis and cirrhosis. The non-human animals described herein can comprise an accumulation of toxic metabolites such as fumarylacetoacetate and maleylacetoacetate (e.g., in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency and / or in the absence of transplantation of hepatocytes / hepatocyte progenitors with a functional copy of Fah). This accumulation can lead to hepatocyte loss, liver failure, and death (e.g., in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency and / or in the absence of transplantation of hepatocytes / hepatocyte progenitors with a functional copy of Fah).

[0188] Some non-human animals described herein can further comprise nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency. Some non-human animals described herein can also comprise transplanted (i.e., xenotransplanted) hepatocytes / hepatocyte progenitors or transplanted and expanded hepatocytes / hepatocyte progenitors (e.g., hepatocytes / hepatocyte progenitors from a different species than the non-human animal, such as human hepatocytes / hepatocyte progenitors). The transplanted hepatocytes / hepatocyte progenitors can repopulate the liver of the non-human animals disclosed herein and can restore liver function(e g., that was lost or decreased due to the inactivated Fah gene). As one example, the nonhuman animals described herein can comprise xenotransplanted human hepatocytes / hepatocyte progenitors. The transplanted hepatocytes / hepatocyte progenitors can be wild type hepatocytes / hepatocyte progenitors, or they can comprise one or more mutations. In a specific example, the transplanted hepatocytes / hepatocyte progenitors have a wild type FAH gene or an FAH gene that produces a functional FAH protein.B. Modifications to Restore, Activate, or Increase IL-6 / IL-6R Signaling Pathway Activity or GP130 Signaling Pathway Activity in Xenotransplanted Hepatocytes

[0189] The genetically modified non-human animals (e.g., mice or rats) comprising xenotransplanted hepatocytes / hepatocyte progenitors (e.g., human hepatocytes / hepatocyte progenitors) disclosed herein can comprise modifications to the genetically modified non-human animals and / or the xenotransplanted hepatocytes / hepatocyte progenitors to restore, activate, or increase interleukin-6 (IL-6) / interleukin-6 receptor (IL-6R) signaling pathway or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells that are species-matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells in the liver that are species-matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non- human animal does not comprise (and / or is not modified to comprise) a reconstituted immune system that is species-matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) human Kupffer cells. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) human Kupffer cells in the liver. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) a reconstituted human immune system. In some cases, the genetically modified non- human animal does not comprise (and / or is not modified to comprise) Kupffer cells that are species-compatible to the transplanted hepatocytes / hepatocyte progenitors (i.e., that produce IL-6 compatible with the IL-6R in the transplanted hepatocytes / hepatocyte progenitors). In some cases, the genetically modified non-human animal does not comprise (and / or is not modified tocomprise) Kupffer cells in the liver that are species-compatible to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) a reconstituted immune system that is species-compatible to the transplanted hepatocytes / hepatocyte progenitors.

[0190] The genetically modified non-human animals can comprise xenotransplanted hepatocytes / hepatocyte progenitors (e.g., xenotransplanted and expanded hepatocytes / hepatocyte progenitors). The xenotransplanted hepatocytes / hepatocyte progenitors can be from any species other than that of the recipient non-human animal. For example, the xenotransplanted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors. In another example, the xenotransplanted hepatocytes / hepatocyte progenitors can be non-human primate (NHP) hepatocytes / hepatocyte progenitors, such as cynomolgus hepatocytes / hepatocyte progenitors. The transplanted hepatocytes / hepatocyte progenitors can be from a species whose IL-6R is incompatible with the endogenous non-human animal IL-6 (e.g., the transplanted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors, and the non-human animal can be a mouse or a rat). The cross-species incompatibility between the transplanted hepatocytes / hepatocyte progenitors and the recipient non-human animals (i.e., the suboptimal interaction or lack of reactivity between the recipient non-human animal IL-6 ligands and the IL-6R on the xenotransplanted hepatocytes / hepatocyte progenitors) can result in a steatosis-like phenotype and lipid droplet accumulation. The cross-species incompatibility between the transplanted hepatocytes / hepatocyte progenitors and the recipient non-human animals (i.e., the suboptimal interaction or lack of reactivity between the recipient non-human animal IL-6 ligands and the IL-6R on the xenotransplanted hepatocytes / hepatocyte progenitors) can also result in a slower or impaired engraftment of the transplanted hepatocytes / hepatocyte progenitors.

[0191] The modifications described herein can result in reduced lipid droplet accumulation and reduced steatosis in the xenotransplanted hepatocytes / hepatocyte progenitors compared to non-human animals in which the genetically modified non-human animals and the xenotransplanted hepatocytes / hepatocyte progenitors do not have modifications to restore, activate, or increase IL-6 / IL-6R signaling pathway or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. The modifications described herein can also accelerate or improve engraftment of the xenotransplanted hepatocytes / hepatocyteprogenitors compared to non-human animals in which the genetically modified non-human animals and the xenotransplanted hepatocytes / hepatocyte progenitors do not have modifications to restore, activate, or increase IL-6 / IL-6R signaling pathway or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. The modifications can, for example, restore, activate, or increase species-matched hepatic IL-6 / IL-6R pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors, or can simply restore, activate, or increase IL-6 / IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. Likewise, the modifications can, for example, restore, activate, or increase species-compatible hepatic IL- 6 / IL-6R pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors.

[0192] In some cases, the xenotransplanted hepatocytes / hepatocyte progenitors are modified to restore, activate, or increase IL-6 / IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. As one example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) non-human animal IL-6R (i.e., IL-6R from the same species as the recipient non-human animal — in other words, IL-6R species-matched to the recipient non-human animal). As another example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) species-compatible IL-6R (i.e., IL-6R from a species that is compatible with the IL-6 produced by the recipient non-human animal, such that the recipient non-human animal IL-6 can bind to and activate IL-6R signaling). For example, if the hepatocytes / hepatocyte progenitors are being transplanted into a mouse or a rat, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to express mouse IL-6R or rat IL-6R, respectively. In some cases, the transplanted hepatocytes / hepatocyte progenitors can be modified to comprise a vector comprising an expression construct for the non-human animal IL-6R comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. Alternatively, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to comprise in their genome a non-human animal IL-6Rexpression construct comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a liver-specific promoter or a promoter active in liver cells (e.g., hepatocytes) can be used. Examples of such promoters include TTR, ALB, and HBV promoters. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFl alpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0193] Mouse interleukin-6 receptor subunit alpha (also known as IL-6 receptor subunit alpha, IL-6R subunit alpha, IL-6R-alpha, IL-6RA, IL-6R, IL-6R 1, and CD 126) is encoded by the gene Il6ra (also known as 7Z6r). IL-6R is part of the receptor for interleukin 6. It binds to IL- 6 with low affinity but does not transduce a signal. Signal activation necessitate an association with IL6ST. Activation leads to the regulation of the immune response, acute-phase reactions and hematopoiesis. The interaction with membrane-bound IL-6R and IL6ST stimulates classic signaling, and the restricted expression of the IL-6R limits classic IL-6 signaling to only a few tissues such as the liver and some cells of the immune system. Mouse H6ra maps to 3 Fl; 3 39.19 cM on chromosome 3 (NCBI RefSeq Gene ID 16194; Assembly GRCm39 (GCF_000001635.27); location NC_000069.7 (89776631..89820503, complement). Reference to the mouse Il6ra gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical mouse IL-6R protein has been assigned UniProt accession number P22272 and NCBI Accession No. NP 034689.2 (SEQ ID NO: 2). Reference to mouse IL-6R proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_010559.3. The coding sequence for the canonical isoform is assigned CCDS Accession No. CCDS38496. 1 (SEQ ID NO: 1). Reference to the mouse Il6ra mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0194] Rat U6ra maps to 2q34 on chromosome 2 (NCBI RefSeq Gene ID 24499; Assembly GRCr8 (GCF_036323735.1); location NC_086020.1 (177582020..177646705, complement). Reference to the rat Il6ra gene includes the canonical, wild type form as well as all allelic forms and isoforms. Rat IL-6R protein has been assigned UniProt accession number P22273. Thecanonical rat IL-6R protein has been assigned NCBI Accession No. NP 058716.2 (SEQ ID NO: 4). Reference to rat IL-6R proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_017020.3. A coding sequence for the canonical isoform is set forth in SEQ ID NO: 3. Reference to the rat Il6ra mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0195] As another example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) non-human animal OSMR (i.e., OSMR from the same species as the recipient non-human animal — in other words, OSMR species-matched to the recipient non-human animal). As another example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) species-compatible OSMR (i.e., OSMR from a species that is compatible with the OSM produced by the recipient non-human animal, such that the recipient non-human animal OSM can bind to and activate OSMR signaling). For example, if the hepatocytes / hepatocyte progenitors are being transplanted into a mouse or a rat, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to express mouse OSMR or rat OSMR, respectively. In some cases, the transplanted hepatocytes / hepatocyte progenitors can be modified to comprise a vector comprising an expression construct for the non-human animal OSMR comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. Alternatively, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to comprise in their genome a non-human animal OSMR expression construct comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a liver-specific promoter or a promoter active in liver cells (e.g., hepatocytes) can be used. Examples of such promoters include 1TR, ALB, and HBB promoters. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFl alpha). As another example, an inducible promoter can be used. In the case ofgenomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0196] Mouse oncostatin-M-specific receptor subunit beta (also known as oncostatin M receptor or OSMR or OSMRB) is encoded by the gene Osmr (also known as Osmrb or oncostatin M receptor). It is capable of transducing OSM-specific signaling events through association with GP130 and activating STAT3 downstream. Mouse Osmr maps to 15 Al; 15 3.3 cM on chromosome 15 (NCBI RefSeq Gene ID 18414; Assembly GRCm39 (GCF_000001635.27); location NC_000081.7 (6843049..6904434, complement). Reference to the mouse Osmr gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical mouse OSMR protein has been assigned UniProt accession number 070458 and NCBI Accession No. NP_O35149.2 (SEQ ID NO: 14). Reference to mouse OSMR proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_011019.4. The coding sequence for the canonical isoform is assigned CCDS Accession No. CCDS27368. 1 (SEQ ID NO: 13). Reference to the mouse Osmr mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0197] Rat Osmr maps to 2ql6 on chromosome 2 (NCBI RefSeq Gene ID 310132; Assembly GRCr8 (GCF_036323735.1); location NC_086020.1 (57634517..57688802, complement). Reference to the rat Osmr gene includes the canonical, wild type form as well as all allelic forms and isoforms. Rat OSMR protein has been assigned UniProt accession number Q65Z14. The canonical rat OSMR protein has been assigned NCBI Accession No.NP 001005384.1 (SEQ ID NO: 16). Reference to rat OSMR proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_001005384.1. A coding sequence for the canonical isoform is set forth in SEQ ID NO: 15. Reference to the rat Osmr mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0198] As another example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) non-human animal IL-6R and non-human animal OSMR (i.e., IL-6R and OSMR from the same species as the recipient non-human animal — in other words, IL-6R and OSMR species-matched to the recipient non-human animal). As another example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e.,ectopically express) species-compatible IL-6R and OSMR (i.e., IL-6R and OSMR from a species that is compatible with the IL-6 and OSM, respectively, produced by the recipient non-human animal, such that the recipient non-human animal IL-6 and OSM can bind to and activate IL-6R and OSMR signaling, respectively). As another example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) the non- human animal receptors for IL-6, LIF, OSM, CNTF, IL-11, CTF1, BSF3, or any combination thereof. As another example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) species-compatible receptors for IL-6, LIF, OSM, CNTF, IL-11, CTF1, BSF3, or any combination thereof.

[0199] As another example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) non-human animal Growth Hormone Receptor (GHR) (i.e., GHR from the same species as the recipient non-human animal — in other words, GHR species-matched to the recipient non-human animal). As another example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) speciescompatible GHR (i.e., GHR from a species that is compatible with the GH produced by the recipient non-human animal, such that the recipient non-human animal GH can bind to and activate GHR signaling). For example, if the hepatocytes / hepatocyte progenitors are being transplanted into a mouse or a rat, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to express mouse GHR or rat GHR, respectively. In some cases, the transplanted hepatocytes / hepatocyte progenitors can be modified to comprise a vector comprising an expression construct for the non-human animal GHR comprising a nucleic acid encoding the non-human animal GHR operably linked to a promoter. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. Alternatively, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to comprise in their genome a non-human animal GHR expression construct comprising a nucleic acid encoding the non-human animal GHR operably linked to a promoter. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a liver-specific promoter or a promoter active in liver cells (e.g., hepatocytes) can be used. Examples of such promoters include TTR, ALB, and HB V promoters. In anotherexample, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFlalpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0200] As another example of modifying the transplanted hepatocytes / hepatocyte progenitors, they can be modified to express (i.e., ectopically express) a ligand-independent, constitutively active form of IL-6R co-receptor glycoprotein 130 (GP130). For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the constitutively active form of GP130 can be a human GP130. In some cases, the transplanted hepatocytes / hepatocyte progenitors can be modified to comprise a vector comprising an expression construct for the constitutively active form of GP130 comprising a nucleic acid encoding the constitutively active form of GP130 operably linked to a promoter. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. Alternatively, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to comprise in their genome a GP130 expression construct comprising a nucleic acid encoding the constitutively active form of GP130 operably linked to a promoter. For example, the expression construct could be at a safe harbor locus in the non-human animal. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a liver-specific promoter or a promoter active in liver cells (e.g., hepatocytes) can be used. Examples of such promoters include TTR, ALB, and HBV promoters. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFl alpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0201] Human interleukin-6 receptor subunit beta (also known as IL6ST, IL-6 receptor subunit beta, IL-6R subunit beta, IL-6R-beta, IL-6RB, CDwl30, interleukin-6 signal transducer, membrane glycoprotein 130 (gpl30), CD130, and oncostatin-M receptor subunit alpha) isencoded by the gene JL6ST. The receptor systems for IL-6, LIF, OSM, CNTF, IL-11, CTF1, and BSF3 can utilize IL6ST (GP130) for initiating signal transmission. Binding of IL-6 to IL-6R induces IL6ST (GP130) homodimerization and formation of a high-affinity receptor complex, which activates the intracellular JAK-MAPK and JAK-STAT3 signaling pathways. Human IL6ST maps to 5ql l.2 on chromosome 5 (NCBI RefSeq Gene ID 3572; Assembly GRCh38.pl4 (GCF_000001405.40); location NC_000005.10 (55935095..55994963, complement). Reference to the human IL6ST gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical human IL6ST (GP130) protein has been assigned UniProt accession number P40189 and NCBI Accession No. NP_002175.2 (SEQ ID NO: 10). Reference to human IL6ST (GP130) proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_002184.3. A coding sequence encoding the canonical isoform is assigned CCDS Accession No. CCDS3971.1 (SEQ ID NO: 9). Reference to the human IL6ST mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms. In one example, the constitutively active human GP130 comprises a deletion of the region of GP130 from Tyrl86 to Tyrl90 (GP130Y186-Y190del). An exemplary GP130Y186-Y190delprotein is set forth in SEQ ID NO: 8 and is encoded by the coding sequence set forth in SEQ ID NO: 7.

[0202] In other cases, the recipient non-human animal is modified to restore, activate, or increase IL-6 / IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. For example, the non-human animal can comprise a ligand that can activate IL-6 / IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., a ligand that can activate human IL-6R signaling in human xenotransplanted hepatocytes / hepatocyte progenitors). For example, the non-human animal can comprise an IL-6R agonist antigenbinding protein such as an IL-6R agonist antibody (e.g., a human IL-6R agonist antigen-binding protein such as a human IL-6R agonist antibody), and / or the non-human animal can comprise an OSMR agonist antigen-binding protein such as an OSMR agonist antibody (e.g., a human OSMR agonist antigen-binding protein such as a human OSMR agonist antibody). Alternatively, the non-human animal can comprise IL-6 from the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., IL-6 that is species-matched with the xenotransplanted hepatocytes / hepatocyte progenitors). Likewise, the non-human animal can comprise IL-6compatible with the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e g., IL- 6 that is species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors, such that the IL-6 can activate IL-6R signaling in the xenotransplanted hepatocytes / hepatocyte progenitors). For example, the non-human animal can comprise human IL-6 (or a human-IL-6R- compatible ligand (e.g., human-IL-6R-compatible IL-6), such as cynomolgus IL-6) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. For example, the non-human animal can comprise human IL-6 (or a human-IL-6R- compatible IL-6, such as cynomolgus IL-6) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. Human IL-6 is described above. The IL-6 can be, for example, in the serum of the non-human animal or the liver of the non-human animal. For example, the IL-6 can be present or expressed locally in the liver of the non-human animal. The IL-6 can be expressed by any suitable cell type in the non-human animal. In one example, the IL-6 is expressed in liver cells in the non-human animal. In one example, the IL-6 is expressed in muscle cells in the non-human animal. In one example, the non-human animal comprises a vector comprising an expression construct for species-matched IL-6 (e.g., human IL- 6) comprising a nucleic acid encoding the species-matched IL-6 (e.g., human IL-6) operably linked to a promoter. Likewise, the non-human animal can comprise a vector comprising an expression construct for species-compatible IL-6 (e.g., the IL-6 is compatible with the IL-6R expressed by the transplanted hepatocytes / hepatocyte progenitors such that the IL-6 can activate IL-6R signaling in the hepatocytes / hepatocyte progenitors) comprising a nucleic acid encoding the species-compatible IL-6 operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non- human animal can comprise a vector comprising an expression construct for human-IL-6R- compatible ligand (e.g., human-IL-6R-compatible IL-6) (e g., cynomolgus IL-6) comprising a nucleic acid encoding the human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6) operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise a vector comprising an expression construct for human-IL-6R-compatible IL-6 (e.g., cynomolgus IL-6) comprising a nucleic acid encoding the human-IL-6R-compatible IL-6 operably linked to a promoter. For example, the non-human animal can comprise the vector in liver cells. Alternatively, the non-human animal can comprise the vector in muscle cells. Any suitablevector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in muscle. In a specific example, a recombinant AAV9 vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in liver. In a specific example, a recombinant AAV8 vector is used. Alternatively, the non-human animal can comprise in its genome a species-matched IL-6 (e.g., human IL-6) expression construct comprising a nucleic acid encoding the species-matched IL-6 (e.g., human IL-6) operably linked to a promoter. Likewise, the non-human animal can comprise in its genome a species-compatible IL-6 (e.g., the IL-6 is compatible with the IL-6R expressed by the transplanted hepatocytes / hepatocyte progenitors such that the IL-6 can activate IL-6R signaling in the hepatocytes / hepatocyte progenitors) expression construct comprising a nucleic acid encoding the species-compatible IL-6 operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non- human animal can comprise in its genome a human-IL-6R-compatible ligand (e.g., human-IL- 6R-compatible IL-6) (e.g., cynomolgus IL-6) expression construct comprising a nucleic acid encoding the human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6) operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise in its genome a human-IL-6R-compatible IL-6 (e.g., cynomolgus IL-6) expression construct comprising a nucleic acid encoding the human-IL-6R-compatible IL-6 operably linked to a promoter. For example, the expression construct could be at a safe harbor locus in the non-human animal. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a tissue-specific promoter can be used. For example, a liver-specific promoter or a promoter active in liver cells can be used. Examples of liver-specific promoters or promoters active in liver cells include TTR, ALB, and HBV promoters. For example, a muscle-specific promoter or a promoter active in muscle cells can be used. An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor-1 alpha (EFlalpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0203] In another example, the non-human animal can comprise OSM from the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., OSM that is species-matched with the xenotransplanted hepatocytes / hepatocyte progenitors). Likewise, the non-human animal can comprise OSM compatible with the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., OSM that is species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors, such that the OSM can activate OSMR signaling in the xenotransplanted hepatocytes / hepatocyte progenitors). For example, the non-human animal can comprise human OSM (or a human-0 SMR-compatible ligand (e.g., human-OSMR-compatible OSM)) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. Human OSM is described above. The OSM can be, for example, in the serum of the non-human animal or the liver of the non-human animal. For example, the OSM can be present or expressed locally in the liver of the non-human animal. The OSM can be expressed by any suitable cell type in the non-human animal. In one example, the OSM is expressed in liver cells in the non-human animal. In one example, the OSM is expressed in muscle cells in the non-human animal. In one example, the non-human animal comprises a vector comprising an expression construct for species-matched OSM (e.g., human OSM) comprising a nucleic acid encoding the species-matched OSM (e.g., human OSM) operably linked to a promoter. Likewise, the non-human animal can comprise a vector comprising an expression construct for species-compatible OSM (e.g., the OSM is compatible with the OSMR expressed by the transplanted hepatocytes / hepatocyte progenitors such that the OSM can activate OSMR signaling in the hepatocytes / hepatocyte progenitors) comprising a nucleic acid encoding the species-compatible OSM operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non- human animal can comprise a vector comprising an expression construct for human-OSMR- compatible ligand (e.g., human-OSMR-compatible OSM) (e.g., cynomolgus OSM) comprising a nucleic acid encoding the human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) operably linked to a promoter. For example, the non-human animal can comprise thevector in liver cells. For example, the non-human animal can comprise the vector in muscle cells. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in muscle. In a specific example, a recombinant AAV9 vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in liver. In a specific example, a recombinant AAV8 vector is used. Alternatively, the non-human animal can comprise in its genome a species-matched OSM (e.g., human OSM) expression construct comprising a nucleic acid encoding the species-matched OSM (e.g., human OSM) operably linked to a promoter. Likewise, the non-human animal can comprise in its genome a species-compatible OSM (e.g., the OSM is compatible with the OSMR expressed by the transplanted hepatocytes / hepatocyte progenitors such that the OSM can activate OSMR signaling in the hepatocytes / hepatocyte progenitors) expression construct comprising a nucleic acid encoding the species-compatible OSM operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise in its genome a human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) (e.g., cynomolgus OSM) expression construct comprising a nucleic acid encoding the human-OSMR-compatible ligand (e g., human-OSMR-compatible OSM) operably linked to a promoter. For example, the expression construct could be at a safe harbor locus in the non-human animal. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a tissue-specific promoter can be used. For example, a liver-specific promoter or a promoter active in liver cells can be used. Examples of liver-specific promoters or promoters active in liver cells include TTR, ALB, and HBV promoters. For example, a muscle-specific promoter or a promoter active in muscle cells can be used. An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavychain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFlalpha). As another example, an inducible promoter can be used. In the case ofgenomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0204] In other cases, the recipient non-human animal is modified to restore, activate, or increase IL-6 / IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. For example, the non-human animal can comprise a GP130 activator (e.g., human GP130 activator), such as a GP130-activating ligand (e.g., human-GP130-activating ligand). In one example, the non-human animal can comprise one or more GP130-activating ligands. In another example, the non-human animal can comprise two or more GP130-activating ligands. In another example, the non-human animal can comprise three or more GP130-activating ligands. In another example, the non-human animal can comprise four or more GP130-activating ligands. Ligands that activate GP130 are known. For example, IL-6, LIF, OSM, CNTF, IL-11, CTF1, and BSF3 are all ligands that can activate GP130. In one example, the non-human animals can comprise a ligand (e.g., IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), interleukin- 11 (IL-11), cardiotrophin-1 (CTF1), or cardiotrophin-like cytokine factor 1 (BSF3)) from the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., IL-6, LIF, OSM, CNTF, IL-11, CTF1, or BSF3 that is species-matched or species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors). In one example, the non-human animals can comprise IL-6 (e.g., species-matched or species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors). In another example, the non-human animals can comprise OSM (e.g., species-matched or species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors). In another example, the non-human animals can comprise IL-6 and OSM (e.g., species-matched or species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors). In another example, the non-human animals can comprise IL-6, LIF, OSM, CNTF, IL-11, CTF1, BSF3, or any combination thereof (e.g., species-matched or species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors). For example, the non-human animal can comprise human IL-6, LIF, OSM, CNTF, IL-11, CTF1, or BSF3 if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. Another example of a GP130 activator is a GP130 agonist antibody or antigen-binding protein (e.g., human GP130 agonist antibody or antigen-binding protein). GP130-activating antibodies are known. See, e.g., Autissier et al. (1997) Eur. J.Immunol. 27(3):794-797, herein incorporated by reference in its entirety for all purposes. Another example of a GP130 activator is a chimeric GP130 ligand, termed IC7Fc, where one GP130 binding site has been removed from IL-6 and replaced with the leukemia inhibitory factor receptor (LIFR) binding site from CNTF and then fused with the fragment crystallizable (Fc) domain of immunoglobulin G (IgG). See, e.g., Findeisen et al. (2019) Nature 574:63-68, herein incorporated by reference in its entirety for all purposes. The GP130 activator (e.g., ligand) can be, for example, in the serum of the non-human animal or the liver of the non-human animal. For example, the GP130 activator (e.g., ligand) can be present or expressed locally in the liver of the non-human animal. The GP130 activator (e.g., ligand) can be expressed by any suitable cell type in the non-human animal. In one example, the GP130 activator (e.g., ligand) is expressed in liver cells in the non-human animal. In one example, the GP130 activator (e.g., ligand) is expressed in muscle cells in the non-human animal. In one example, the non-human animal comprises a vector comprising an expression construct for the GP130 activator (e.g., ligand) comprising a nucleic acid encoding the GP130 activator operably linked to a promoter. For example, the non- human animal can comprise the vector in liver cells. For example, the non-human animal can comprise the vector in muscle cells. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in muscle. In a specific example, a recombinant AAV9 vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in liver. In a specific example, a recombinant AAV8 vector is used. Alternatively, the non-human animal can comprise in its genome a GP130 activator (e.g., ligand) expression construct comprising a nucleic acid encoding the GP130 activator (e.g., ligand) operably linked to a promoter. For example, the expression construct could be at a safe harbor locus in the non-human animal. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a tissue-specific promoter can be used. For example, a liver-specific promoter or a promoter active in liver cells can be used. Examples of liver-specific promoters or promoters active in liver cells include TTR, ALB, and HBV promoters. For example, a muscle-specific promoter or a promoter active in muscle cells can be used. An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFl alpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0205] In another example, the non-human animal can comprise Growth Hormone (GH) from the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., GH that is species-matched with the xenotransplanted hepatocytes / hepatocyte progenitors). Likewise, the non-human animal can comprise GH compatible with the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., GH that is species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors, such that the GH can activate GHR signaling in the xenotransplanted hepatocytes / hepatocyte progenitors). For example, the non- human animal can comprise human GH (or a human-GHR-compatible GH) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. The GH can be, for example, in the serum of the non-human animal or the liver of the non-human animal. For example, the GH can be present or expressed locally in the liver of the non-human animal. The GH can be expressed by any suitable cell type in the non-human animal. In one example, the GH is expressed in liver cells in the non-human animal. In one example, the GH is expressed in muscle cells in the non-human animal. In one example, the non- human animal comprises a vector comprising an expression construct for species-matched GH (e.g., human GH) comprising a nucleic acid encoding the species-matched GH (e.g., human GH) operably linked to a promoter. Likewise, the non-human animal can comprise a vector comprising an expression construct for species-compatible GH (e.g., the GH is compatible with the GHR expressed by the transplanted hepatocytes / hepatocyte progenitors such that the GH can activate GHR signaling in the hepatocytes / hepatocyte progenitors) comprising a nucleic acid encoding the species-compatible GH operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise a vector comprising an expression construct for human- GHR-compatible GH (e.g., cynomolgus GH) comprising a nucleic acid encoding the human- GHR-compatible GH operably linked to a promoter. For example, the non-human animal cancomprise the vector in liver cells. For example, the non-human animal can comprise the vector in muscle cells. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in muscle. In a specific example, a recombinant AAV9 vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in liver. In a specific example, a recombinant AAV8 vector is used. Alternatively, the non-human animal can comprise in its genome a species-matched GH (e.g., human GH) expression construct comprising a nucleic acid encoding the species-matched GH (e.g., human GH) operably linked to a promoter. Likewise, the non-human animal can comprise in its genome a species-compatible GH (e.g., the GH is compatible with the GHR expressed by the transplanted hepatocytes / hepatocyte progenitors such that the GH can activate GHR signaling in the hepatocytes / hepatocyte progenitors) expression construct comprising a nucleic acid encoding the species-compatible GH operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non- human animal can comprise in its genome a human-GHR-compatible GH (e.g., cynomolgus GH) expression construct comprising a nucleic acid encoding the human-GHR-compatible GH operably linked to a promoter. For example, the expression construct could be at a safe harbor locus in the non-human animal. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a tissue-specific promoter can be used. For example, a liver-specific promoter or a promoter active in liver cells can be used. Examples of liver-specific promoters or promoters active in liver cells include TTR, ALB, and HBV promoters. For example, a muscle-specific promoter or a promoter active in muscle cells can be used. An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor-1 alpha (EFlalpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0206] In another example, the non-human animal can comprise a humanized IL6 locus as described elsewhere herein. Examples of non-human animals with humanized IL6 genes are provided, e.g., in US 9,622,460, herein incorporated by reference in its entirety for all purposes. The non-human animal can be, in some cases, homozygous for the humanized IL6 gene. In other cases, the non-human animal can be heterozygous for the humanized IL6 gene. The non-human animal can comprise the humanized IL6 gene in its germline. The humanized IL6 gene can comprise a human IL6 nucleic acid encoding a human IL-6 protein (e g., a fully human IL-6 protein). The human IL6 nucleic acid can be a genomic nucleic acid, such as a genomic nucleic acid comprising a region of a human IL6 gene from the start codon to the stop codon, or can comprise a human IL6 complementary DNA (cDNA). The human IL6 nucleic acid can be inserted into the non-human animal 116 genomic locus, or it can replace a corresponding region of the non-human animal 116 locus (e.g., a region of a human IL6 gene from the start codon to the stop codon can replace a region of the non-human animal 116 gene from the start codon to the stop codon). The humanized IL6 gene (or the human IL6 nucleic acid) can be operably linked to the endogenous non-human animal 116 promoter. In other words, expression of the humanized IL6 gene can be driven by the endogenous non-human animal 116 promoter.

[0207] In another example, the non-human animal can comprise a humanized OSM locus as described elsewhere herein. The non-human animal can be, in some cases, homozygous for the humanized OSM gene. In other cases, the non-human animal can be heterozygous for the humanized OSM gene. The non-human animal can comprise the humanized OSM gene in its germline. The humanized OSM gene can comprise a human OSM nucleic acid encoding a human OSM protein (e.g., a fully human OSM protein). The human OSM nucleic acid can be a genomic nucleic acid, such as a genomic nucleic acid comprising a region of a human OSM gene from the start codon to the stop codon, or can comprise a human OSM complementary DNA (cDNA). The human OSM nucleic acid can be inserted into the non-human animal Osm genomic locus, or it can replace a corresponding region of the non-human animal Osm locus (e.g., a region of a human OSM gene from the start codon to the stop codon can replace a region of the non-human animal Osm gene from the start codon to the stop codon). The humanized OSM gene (or the human OSM nucleic acid) can be operably linked to the endogenous non-human animal Osm promoter. In other words, expression of the humanized OSM gene can be driven by the endogenous non-human animal Osm promoter.

[0208] In another example, the non-human animal can comprise a humanized IL6 locus as described elsewhere herein and a humanized OSM locus as described elsewhere herein.

[0209] In another example, the non-human animal can comprise a humanized GH locus. Similar to IL6, mGH shows species specificities in receptor binding, and administration of human Growth Hormone (GH) in humanized liver mice could correct fatty liver in humanized liver mice. Tateno et aL (2011) Endocrinolog) ' 152: 1479-1491, herein incorporated by reference in its entirety for all purposes.

[0210] In another example, the non-human animal can comprise a humanized IL6 locus as described elsewhere herein and a humanized GH locus as described elsewhere herein.

[0211] In another example, the non-human animal can comprise a humanized OSM locus as described elsewhere herein and a humanized GH locus as described elsewhere herein.

[0212] In another example, the non-human animal can comprise a humanized IL6 locus as described elsewhere herein and a humanized OSM locus as described elsewhere herein and a humanized GH locus as described elsewhere herein.C. Modifications to Restore, Activate, or Increase HGF / Met Signaling Pathway Activity in Xenotransplanted Hepatocytes

[0213] The genetically modified non-human animals (e g., mice or rats) comprising xenotransplanted hepatocytes / hepatocyte progenitors (e.g., human hepatocytes / hepatocyte progenitors) disclosed herein can comprise modifications to the genetically modified non-human animals and / or the xenotransplanted hepatocytes / hepatocyte progenitors to restore, activate, or increase hepatocyte growth factor (HGF) / hepatocyte growth factor receptor (MET) signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells that are species-matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells in the liver that are species-matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) a reconstituted immune system that is species-matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise)human Kupffer cells. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) human Kupffer cells in the liver. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) a reconstituted human immune system. In some cases, the genetically modified non- human animal does not comprise (and / or is not modified to comprise) Kupffer cells that are species-compatible to the transplanted hepatocytes / hepatocyte progenitors (i.e., that produce HGF compatible with the MET in the transplanted hepatocytes / hepatocyte progenitors). In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells in the liver that are species-compatible to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) a reconstituted immune system that is species-compatible to the transplanted hepatocytes / hepatocyte progenitors.

[0214] The genetically modified non-human animals can comprise xenotransplanted hepatocytes / hepatocyte progenitors (e.g., xenotransplanted and expanded hepatocytes / hepatocyte progenitors). The xenotransplanted hepatocytes / hepatocyte progenitors can be from any species other than that of the recipient non-human animal. For example, the xenotransplanted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors. In another example, the xenotransplanted hepatocytes / hepatocyte progenitors can be non-human primate (NHP) hepatocytes / hepatocyte progenitors, such as cynomolgus hepatocytes / hepatocyte progenitors. The transplanted hepatocytes / hepatocyte progenitors can be from a species whose MET is incompatible with the endogenous non-human animal HGF (e.g., the transplanted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors, and the non-human animal can be a mouse or a rat). The cross-species incompatibility between the transplanted hepatocytes / hepatocyte progenitors and the recipient non-human animals (i.e., the suboptimal interaction or lack of reactivity between the recipient non-human animal HGF ligands and the MET on the xenotransplanted hepatocytes / hepatocyte progenitors) can result in a slower or impaired engraftment of the transplanted hepatocytes / hepatocyte progenitors.

[0215] The modifications described herein can result in accelerated or improved engraftment of the xenotransplanted hepatocytes / hepatocyte progenitors compared to non-human animals in which the genetically modified non-human animals and the xenotransplanted hepatocytes / hepatocyte progenitors do not have modifications to restore, activate, or increaseHGF / MET signaling pathway activity in the xenotranspl anted hepatocytes / hepatocyte progenitors. The modifications can, for example, restore, activate, or increase species-matched hepatic HGF / MET signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors, or can simply restore, activate, or increase HGF / MET signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. Likewise, the modifications can, for example, restore, activate, or increase species-compatible hepatic HGF / MET signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors.

[0216] In some cases, the xenotransplanted hepatocytes / hepatocyte progenitors are modified to restore, activate, or increase HGF / MET signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. As one example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) non-human animal MET (i.e., MET from the same species as the recipient non-human animal — in other words, MET species- matched to the recipient non-human animal). As another example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) speciescompatible MET (i.e., MET from a species that is compatible with the HGF produced by the recipient non-human animal, such that the recipient non-human animal HGF can bind to and activate MET signaling). For example, if the hepatocytes / hepatocyte progenitors are being transplanted into a mouse or a rat, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to express mouse MET or rat MET, respectively. In some cases, the transplanted hepatocytes / hepatocyte progenitors can be modified to comprise a vector comprising an expression construct for the non-human animal MET comprising a nucleic acid encoding the non-human animal MET operably linked to a promoter. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. Alternatively, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to comprise in their genome a non-human animal MET expression construct comprising a nucleic acid encoding the non-human animal MET operably linked to a promoter. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a liver-specific promoter or a promoter active in liver cells (e.g., hepatocytes) can be used. Examples of such promoters include TTR, ALB, and HB V promoters. In anotherexample, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFlalpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0217] Mouse hepatocyte growth factor receptor (also known as MET, c-MET, HGF receptor, HGF / SF receptor, proto-oncogene c-Met, scatter factor receptor (SF receptor), or tyrosine-protein kinase Met) is encoded by the gene Met (also known as met proto-oncogene). MET is a receptor tyrosine kinase that transduces signals from the extracellular matrix into the cytoplasm by binding to hepatocyte growth factor / HGF ligand. It regulates many physiological processes including proliferation, scattering, morphogenesis and survival. Ligand binding at the cell surface induces autophosphorylation of MET on its intracellular domain that provides docking sites for downstream signaling molecules. Following activation by ligand, MET interacts with the PI3 -kinase subunit PIK3R1, PLCG1, SRC, GRB2, STAT3 or the adapter GAB 1. Recruitment of these downstream effectors by MET leads to the activation of several signaling cascades including the RAS-ERK, PI3 kinase- AKT, or PLCgamma-PKC. Mouse Met maps to 6 A2; 6 7.83 cM on chromosome 6 (NCBI RefSeq Gene ID 17295; Assembly GRCm39 (GCF_000001635.27); location NC_000072.7 (17463351..17573979). Reference to the mouse Met gene includes the canonical, wild type form as well as all allelic forms and isoforms. The mouse MET protein has been assigned UniProt accession number P16056, and isoform P16056- 1 is set forth in SEQ ID NO: 21. Another isoform has been assigned NCBI Accession No.NP 001397049.1 (SEQ ID NO: 20). Reference to mouse MET proteins includes all allelic forms and isoforms. An mRNA (cDNA) encoding the NP_001397049.1 isoform is assigned NCBI Accession No. NM_001410120.1. The coding sequence for this isoform is assigned CCDS Accession No. CCDS 19925. 1 (SEQ ID NO: 19). Reference to the mouse Met mRNA (cDNA) and coding sequence includes all allelic forms and isoforms.

[0218] Rat Met maps to 4q22 on chromosome 4 (NCBI RefSeq Gene ID 24553; AssemblyGRCr8 (GCF_036323735.1); location NC_086022.1 (46756823..46864041). Reference to the rat Met gene includes all allelic forms and isoforms. Rat MET protein has been assigned UniProt accession number P97523, and isoform P97523-1 is set forth in SEQ ID NO: 24. Another isoform has been assigned NCBI Accession No. NP_113705.1 (SEQ ID NO: 23). Reference torat MET proteins includes allelic forms and isoforms. An mRNA (cDNA) encoding the NP_113705.1 isoform is assigned NCBI Accession No. NM_031517.2. A coding sequence for the canonical isoform is set forth in SEQ ID NO: 22. Reference to the rat Met mRNA (cDNA) and coding sequence includes all allelic forms and isoforms.

[0219] As another example of modifying the transplanted hepatocytes / hepatocyte progenitors, they can be modified to overexpress MET. Overexpression of wild-type MET in hepatocytes can enable ligand-independent activation of the receptor. See, e.g., Wang et al. (2001) J. Cell. Biol. 153(5): 1023-1034, herein incorporated by reference in its entirety for all purposes. For example, the MET can be species-matched with the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., human MET). Alternatively, the MET can be compatible with the species of the xenotransplanted hepatocytes / hepatocyte progenitors such that it can activate downstream signaling in the xenotransplanted hepatocytes / hepatocyte progenitors. For example, if the xenotransplanted hepatocytes / hepatocyte progenitors, the MET can be human MET.

[0220] In some cases, the transplanted hepatocytes / hepatocyte progenitors can be modified to comprise a vector comprising an expression construct for the MET comprising a nucleic acid encoding the MET operably linked to a promoter (i.e., for overexpression). Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. Alternatively, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to comprise in their genome a MET expression construct comprising a nucleic acid encoding the MET operably linked to a promoter (i.e., for overexpression). In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a liver-specific promoter or a promoter active in liver cells (e.g., hepatocytes) can be used. Examples of such promoters include TTR, ALB, and HBV promoters. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFl alpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0221] Human hepatocyte growth factor receptor (also known as MET, c-MET, HGF receptor, HGF / SF receptor, proto-oncogene c-Met, scatter factor receptor (SF receptor), or tyrosine-protein kinase Met) is encoded by the gene Met (also known as met proto-oncogene). MET is a receptor tyrosine kinase that transduces signals from the extracellular matrix into the cytoplasm by binding to hepatocyte growth factor / HGF ligand. It regulates many physiological processes including proliferation, scattering, morphogenesis and survival. Ligand binding at the cell surface induces autophosphorylation of MET on its intracellular domain that provides docking sites for downstream signaling molecules. Following activation by ligand, MET interacts with the PI3-kinase subunit PIK3R1, PLCG1, SRC, GRB2, STAT3 or the adapter GAB 1. Recruitment of these downstream effectors by MET leads to the activation of several signaling cascades including the RAS-ERK, PI3 kinase-AKT, or PLCgamma-PKC. Human MET maps to 7q31.2 on chromosome 7 (NCBI RefSeq Gene ID 4233; Assembly GRCh38.pl4 (GCF_000001405.40); location NC_000007.14 (116672196 .116798377). Reference to the human MET gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical human MET protein has been assigned UniProt accession number P08581 and NCBI Accession No. NP_000236.2 (SEQ ID NO: 30). Reference to human MET proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_000245.4. A coding sequence encoding the canonical isoform is assigned CCDS Accession No. CCDS43636.1 (SEQ ID NO: 29). Reference to the human MET mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0222] In other cases, the recipient non-human animal is modified to restore HGF / MET signaling pathway activity in the xenotranspl anted hepatocytes / hepatocyte progenitors. For example, the non-human animal can comprise a ligand that can activate MET signaling in the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., a ligand that can activate human MET signaling in human xenotransplanted hepatocytes / hepatocyte progenitors). For example, the non-human animal can comprise a MET agonist antigen-binding protein such as a MET agonist antibody (e.g., a human MET agonist antigen-binding protein such as a human MET agonist antibody). An example of such an agonist antibody is META4. See, e.g., Ma et al. (2022) Cell. Mol. Gastroenterol. Hepatol., 13(2):565-582, herein incorporated by reference in its entirety for all purposes. Alternatively, the non-human animal can comprise HGF from thespecies of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., HGF that is species- matched with the xenotransplanted hepatocytes / hepatocyte progenitors). Likewise, the nonhuman animal can comprise HGF compatible with the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., HGF that is species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors, such that the HGF can activate MET signaling in the xenotransplanted hepatocytes / hepatocyte progenitors). For example, the nonhuman animal can comprise human HGF (or a human-MET-compatible ligand (e.g., human- MET-compatible HGF), such as cynomolgus HGF) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. For example, the non-human animal can comprise human HGF (or a human-MET-compatible HGF, such as cynomolgus HGF) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. Human HGF is described above. The HGF can be, for example, in the serum of the non-human animal or the liver of the non-human animal. For example, the HGF can be present or expressed locally in the liver of the non-human animal. The HGF can be expressed by any suitable cell type in the non-human animal. In one example, the HGF is expressed in liver cells in the non-human animal. In one example, the HGF is expressed in liver cells in the non-human animal. In one example, the HGF is expressed in muscle cells in the non-human animal. In one example, the non-human animal comprises a vector comprising an expression construct for species-matched HGF (e g., human HGF) comprising a nucleic acid encoding the species-matched HGF (e.g., human HGF) operably linked to a promoter. Likewise, the non-human animal can comprise a vector comprising an expression construct for speciescompatible HGF (e.g., the HGF is compatible with the MET expressed by the transplanted hepatocytes / hepatocyte progenitors such that the HGF can activate MET signaling in the hepatocytes / hepatocyte progenitors) comprising a nucleic acid encoding the species-compatible HGF operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise a vector comprising an expression construct for human-MET-compatible ligand (e.g., human- MET-compatible HGF) (e.g., cynomolgus HGF) comprising a nucleic acid encoding the human- MET-compatible ligand (e.g., human-MET-compatible HGF) operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise a vector comprising anexpression construct for human-MET-compatible HGF (e.g., cynomolgus HGF) comprising a nucleic acid encoding the human-MET-compatible HGF operably linked to a promoter. For example, the non-human animal can comprise the vector in liver cells. For example, the nonhuman animal can comprise the vector in muscle cells. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in muscle. In a specific example, a recombinant AAV9 vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in liver. In a specific example, a recombinant AAV8 vector is used. Alternatively, the non-human animal can comprise in its genome a species-matched HGF (e.g., human HGF) expression construct comprising a nucleic acid encoding the species-matched HGF (e.g., human HGF) operably linked to a promoter. Likewise, the non-human animal can comprise in its genome a speciescompatible HGF (e.g., the HGF is compatible with the MET expressed by the transplanted hepatocytes / hepatocyte progenitors such that the HGF can activate MET signaling in the hepatocytes / hepatocyte progenitors) expression construct comprising a nucleic acid encoding the species-compatible HGF operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non- human animal can comprise in its genome a human-MET-compatible ligand (e.g., human-MET- compatible HGF) (e g., cynomolgus HGF) expression construct comprising a nucleic acid encoding the human-MET-compatible ligand (e.g., human-MET-compatible HGF) operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise in its genome a human-MET-compatible HGF (e.g., cynomolgus HGF) expression construct comprising a nucleic acid encoding the human-MET-compatible HGF operably linked to a promoter. For example, the expression construct could be at a safe harbor locus in the non-human animal. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a tissue-specific promoter can be used. For example, a liver-specific promoter or a promoter active in liver cells can be used. Examples of liver-specific promoters or promoters active in liver cells include TTR, ALB, and HBV promoters. For example, a muscle-specificpromoter or a promoter active in muscle cells can be used. An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta- actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFl alpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0223] In another example, the non-human animal can comprise a humanized HGF locus as described elsewhere herein. The non-human animal can be, in some cases, homozygous for the humanized HGF gene. In other cases, the non-human animal can be heterozygous for the humanized HGF gene. The non-human animal can comprise the humanized HGF gene in its germline. The humanized HGF gene can comprise a human HGF nucleic acid encoding a human HGF protein (e.g., a fully human HGF protein). The human HGF nucleic acid can be a genomic nucleic acid, such as a genomic nucleic acid comprising a region of a human HGF gene from the start codon to the stop codon, or can comprise a human HGF complementary DNA (cDNA). The human HGF nucleic acid can be inserted into the non-human animal Hgf genomic locus, or it can replace a corresponding region of the non-human animal Hgf locus (e.g., a region of a human HGF gene from the start codon to the stop codon can replace a region of the non-human animal Hgf gene from the start codon to the stop codon). The humanized HGF gene (or the human HCTF nucleic acid) can be operably linked to the endogenous non-human animal / / / ' pro oter. In other words, expression of the humanized HGF gene can be driven by the endogenous non- human animal / / ' pro oter.D. Modifications to Activate or Increase RSPO3 Signaling Pathway Activity in Xenotransplanted Hepatocytes

[0224] The genetically modified non-human animals (e.g., mice or rats) comprising xenotransplanted hepatocytes / hepatocyte progenitors (e.g., human hepatocytes / hepatocyte progenitors) disclosed herein can comprise modifications to the genetically modified non-human animals and / or the xenotransplanted hepatocytes / hepatocyte progenitors to activate or increase R-spondin-3 (RSPO3) / leucine-rich repeat-containing G-protein coupled receptor 4 or 5(LGR4 / 5) signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells that are species-matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells in the liver that are species- matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) a reconstituted immune system that is species-matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) human Kupffer cells. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) human Kupffer cells in the liver. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) a reconstituted human immune system. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells that are species-compatible to the transplanted hepatocytes / hepatocyte progenitors (i.e., that produce RSPO3 compatible with the LGR4 or LGR5 in the transplanted hepatocytes / hepatocyte progenitors). In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells in the liver that are speciescompatible to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) a reconstituted immune system that is species-compatible to the transplanted hepatocytes / hepatocyte progenitors.

[0225] The genetically modified non-human animals can comprise xenotransplanted hepatocytes / hepatocyte progenitors (e.g., xenotransplanted and expanded hepatocytes / hepatocyte progenitors). The xenotransplanted hepatocytes / hepatocyte progenitors can be from any species other than that of the recipient non-human animal. For example, the xenotransplanted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors. In another example, the xenotransplanted hepatocytes / hepatocyte progenitors can be non-human primate (NHP) hepatocytes / hepatocyte progenitors, such as cynomolgus hepatocytes / hepatocyte progenitors.

[0226] The modifications described herein can result in accelerated or improved engraftment of the xenotransplanted hepatocytes / hepatocyte progenitors compared to non-human animals in which the genetically modified non-human animals and the xenotransplanted hepatocytes / hepatocyte progenitors do not have modifications to activate or increase RSPO3 / LGR4 / 5 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors.

[0227] In some cases, the recipient non-human animal is modified to activate or increase RSPO3 / LGR4 / 5 signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. For example, the non-human animal can comprise a ligand that can activate LGR4 or LGR5 signaling in the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., a ligand that can activate human LGR4 or LGR5 signaling in human xenotransplanted hepatocytes / hepatocyte progenitors). For example, the non-human animal can comprise an LGR4 or LGR5 agonist antigen-binding protein such as an LGR4 or LGR5 agonist antibody (e.g., a human LGR4 or LGR5 agonist antigen-binding protein such as a human LGR4 or LGR5 agonist antibody). RSPO3 can activate Wnt pathway through binding to both LGR4 / 5 and RNF43 and in turn inhibiting RNF43 from negatively regulating Wnt pathway activity. In some embodiments, the LGR4 or LGR5 agonist antigen-binding protein can be an LGR5-RNF43 tethering molecule (such as bispecific antigen-binding protein (e.g., antibody) or a protein having binding activity to bind both LGR5 and RNF43). Alternatively, the non-human animal can comprise RSPO3 from the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., RSPO3 that is species-matched with the xenotransplanted hepatocytes / hepatocyte progenitors). Likewise, the non-human animal can comprise RSPO3 compatible with the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., RSPO3 that is species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors, such that the RSPO3 can activate RSPO3 signaling in the xenotransplanted hepatocytes / hepatocyte progenitors). For example, the non- human animal can comprise human RSPO3 (or a human-LGR4 / 5-compatible ligand (e.g., human human-LGR4 / 5-compatible RSPO3), such as cynomolgus RSPO3) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. For example, the non-human animal can comprise human RSPO3 (or a human-LGR4 / 5 -compatible RSPO3, such as cynomolgus RSPO3) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. Human RSPO3 is described below. The RSPO3 canbe, for example, in the serum of the non-human animal or the liver of the non-human animal. For example, the RSPO3 can be present or expressed locally in the liver of the non-human animal. The RSPO3 can be expressed by any suitable cell type in the non-human animal. In one example, the RSPO3 is expressed in liver cells in the non-human animal. In one example, the RSPO3 is expressed in liver cells in the non-human animal. In one example, the RSPO3 is expressed in muscle cells in the non-human animal. In one example, the non-human animal comprises a vector comprising an expression construct for species-matched RSPO3 (e.g., human RSPO3) comprising a nucleic acid encoding the species-matched RSPO3 (e.g., human RSPO3) operably linked to a promoter. Likewise, the non-human animal can comprise a vector comprising an expression construct for species-compatible RSPO3 (e.g., the RSPO3 is compatible with the LGR4 / 5 expressed by the transplanted hepatocytes / hepatocyte progenitors such that the RSPO3 can activate RSPO3 signaling in the hepatocytes / hepatocyte progenitors) comprising a nucleic acid encoding the species-compatible RSPO3 operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise a vector comprising an expression construct for human- LGR4 / 5-compatible ligand (e.g., human-LGR4 / 5-compatible RSPO3) (e.g., cynomolgus RSPO3) comprising a nucleic acid encoding the human-LGR4 / 5-compatible ligand (e.g., human- LGR4 / 5-compatible RSPO3) operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non- human animal can comprise a vector comprising an expression construct for human-LGR4 / 5- compatible RSPO3 (e.g., cynomolgus RSPO3) comprising a nucleic acid encoding the human- LGR4 / 5-compatible RSPO3 operably linked to a promoter. For example, the non-human animal can comprise the vector in liver cells. For example, the non-human animal can comprise the vector in muscle cells. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in muscle. In a specific example, a recombinant AAV9 vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in liver. In a specific example, a recombinant AAV8 vector is used. Alternatively, the non-human animal can comprise in its genome aspecies-matched RSPO3 (e.g., human RSPO3) expression construct comprising a nucleic acid encoding the species-matched RSPO3 (e.g., human RSPO3) operably linked to a promoter. Likewise, the non-human animal can comprise in its genome a species-compatible RSPO3 (e.g., the RSPO3 is compatible with the LGR4 / 5 expressed by the transplanted hepatocytes / hepatocyte progenitors such that the RSPO3 can activate RSPO3 signaling in the hepatocytes / hepatocyte progenitors) expression construct comprising a nucleic acid encoding the species-compatible RSPO3 operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise in its genome a human-LGR4 / 5-compatible ligand (e.g., human-LGR4 / 5-compatible RSPO3) (e.g., cynomolgus RSPO3) expression construct comprising a nucleic acid encoding the human- LGR4 / 5-compatible ligand (e.g., human human-LGR4 / 5-compatible RSPO3) operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise in its genome a human- LGR4 / 5-compatible RSPO3 (e.g., cynomolgus RSPO3) expression construct comprising a nucleic acid encoding the human-LGR4 / 5-compatible RSPO3 operably linked to a promoter. For example, the expression construct could be at a safe harbor locus in the non-human animal. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a tissue-specific promoter can be used. For example, a liver-specific promoter or a promoter active in liver cells can be used. Examples of liver-specific promoters or promoters active in liver cells include TTR, ALB, and HBV promoters. For example, a muscle-specific promoter or a promoter active in muscle cells can be used. An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta- actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EF l alpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e g., a safe harbor locus) can be used.

[0228] Human R-spondin-3 (also known as RSPO3) is encoded by the gene RSPO3. RSPO3 can activate Wnt pathway through binding to both LGR4 / 5 and RNF43 and in turn inhibiting RNF43 from negatively regulating Wnt pathway activity. In some embodiments, the LGR4 orLGR5 agonist antigen-binding protein can be an LGR5-RNF4.3 tethering molecule (such as bispecific antigen-binding protein (e.g., antibody) or a protein having binding activity to bind both LGR5 and RNF43). Human RSPO3 maps to 6q22.33 on chromosome 6 (NCBI RefSeq Gene ID 84870; Assembly GRCh38.pl4 (GCF_000001405.40); location NC_000006.12 (127118671..127199481). Reference to the human RSPO3 gene includes the canonical, wild type form as well as all allelic forms and isoforms. The canonical human RSPO3 protein has been assigned UniProt accession number Q9BXY4 and NCBI Accession No. NP_116173.2 (SEQ ID NO: 26). Reference to human RSPO3 proteins includes canonical forms as well as all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_032784.4. A coding sequence encoding the canonical isoform is assigned CCDS Accession No. CCDS5135.1 (SEQ ID NO: 25). Reference to the human RSPO3 mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.

[0229] Other ways to mimic RSPO3 activity could also include using other RSPO family members, such as RSPO1 or RSPO2, in place of RSPO3. In some embodiments, the non-human animal can comprise RSPO1 or RSPO2 from the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., RSPO1 or RSPO2 that is species-matched with the xenotransplanted hepatocytes / hepatocyte progenitors). Likewise, the non-human animal can comprise RSPO1 or RSPO2 compatible with the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., RSPO1 or RSPO2 that is species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors, such that the RSPO1 or RSPO2 can activate LGR4 / 5 signaling in the xenotransplanted hepatocytes / hepatocyte progenitors). For example, the non-human animal can comprise human RSPO1 or RSPO2 (or a human-LGR4 / 5- compatible ligand (e.g., human-LGR4 / 5-compatible RSPO1 or RSPO2), such as cynomolgus RSPO1 or RSPO2) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. For example, the non-human animal can comprise human RSPO1 or RSPO2 (or a human-LGR4 / 5-compatible RSPO1 or RSPO2, such as cynomolgus RSPO1 or RSPO2) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. The RSPO1 or RSPO2 can be, for example, in the serum of the non-human animal or the liver of the non-human animal. For example, the RSPO1 or RSPO2 can be present or expressed locally in the liver of the non-human animal. The RSPO1 or RSPO2can be expressed by any suitable cell type in the non-human animal. In one example, the RSPO1 or RSPO2 is expressed in liver cells in the non-human animal. In one example, the RSPO1 or RSPO2 is expressed in liver cells in the non-human animal. In one example, the RSPO1 or RSPO2 is expressed in muscle cells in the non-human animal. In one example, the non-human animal comprises a vector comprising an expression construct for species-matched RSPO1 or RSPO2 (e.g., human RSPO1 or RSPO2) comprising a nucleic acid encoding the species-matched RSPO1 or RSPO2 (e.g., human RSPO1 or RSPO2) operably linked to a promoter. Likewise, the non-human animal can comprise a vector comprising an expression construct for speciescompatible RSPO1 or RSPO2 (e.g., the RSPO1 or RSPO2 is compatible with the LGR4 / 5 expressed by the transplanted hepatocytes / hepatocyte progenitors such that the RSPO1 or RSPO2 can activate RSPO1 or RSPO2 signaling in the hepatocytes / hepatocyte progenitors) comprising a nucleic acid encoding the species-compatible RSPO1 or RSPO2 operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise a vector comprising an expression construct for human-LGR4 / 5-compatible ligand (e.g., human-LGR4 / 5-compatible RSPO1 or RSPO2) (e.g., cynomolgus RSPO1 or RSPO2) comprising a nucleic acid encoding the human-LGR4 / 5-compatible ligand (e.g., human-LGR4 / 5 -compatible RSPO1 or RSPO2) operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise a vector comprising an expression construct for human -LGR4 / 5 -compatible RSPO1 or RSPO2 (e.g., cynomolgus RSPO1 or RSPO2) comprising a nucleic acid encoding the human-LGR4 / 5- compatible RSPO1 or RSPO2 operably linked to a promoter. For example, the non-human animal can comprise the vector in liver cells. For example, the non-human animal can comprise the vector in muscle cells. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in muscle. In a specific example, a recombinant AAV9 vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in liver. In a specific example, a recombinant AAV8 vector is used. Alternatively, the non-human animal can comprise in its genome aspecies-matched RSPO1 or RSPO2 (e.g., human RSPO1 or RSPO2) expression construct comprising a nucleic acid encoding the species-matched RSPO1 or RSPO2 (e.g., human RSPO1 or RSPO2) operably linked to a promoter. Likewise, the non-human animal can comprise in its genome a species-compatible RSPO1 or RSPO2 (e.g., the RSPO1 or RSPO2 is compatible with the LGR4 / 5 expressed by the transplanted hepatocytes / hepatocyte progenitors such that the RSPO1 or RSPO2 can activate LGR4 / 5 signaling in the hepatocytes / hepatocyte progenitors) expression construct comprising a nucleic acid encoding the species-compatible RSPO1 or RSPO2 ha operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non- human animal can comprise in its genome a human-LGR4 / 5-compatible ligand (e.g., human- LGR4 / 5-compatible RSPOl or RSPO2) (e.g., cynomolgus RSPO1 or RSPO2) expression construct comprising a nucleic acid encoding the human-LGR4 / 5-compatible ligand (e g., human-LGR4 / 5-compatible RSPO1 or RSPO2) operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise in its genome a human-LGR4 / 5-compatible RSPO1 or RSPO2 (e.g., cynomolgus RSPO1 or RSPO2) expression construct comprising a nucleic acid encoding the human-LGR4 / 5-compatible RSPO1 or RSPO2 operably linked to a promoter. For example, the expression construct could be at a safe harbor locus in the non- human animal. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a tissue-specific promoter can be used. For example, a liver-specific promoter or a promoter active in liver cells can be used. Examples of liver-specific promoters or promoters active in liver cells include TTR, ALB, and HBV promoters. For example, a muscle-specific promoter or a promoter active in muscle cells can be used. An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFl alpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.E. Modifications to Restore, Activate, or Increase EGF Signaling Pathway Activity in Xenotransplanted Hepatocytes

[0230] The genetically modified non-human animals (e.g., mice or rats) comprising xenotransplanted hepatocytes / hepatocyte progenitors (e.g., human hepatocytes / hepatocyte progenitors) disclosed herein can comprise modifications to the genetically modified non-human animals and / or the xenotransplanted hepatocytes / hepatocyte progenitors to restore, activate, or increase epidermal growth factor (EGF) / epi dermal growth factor receptor (EGFR) signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells that are species-matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells in the liver that are species-matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) a reconstituted immune system that is species-matched to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) human Kupffer cells. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) human Kupffer cells in the liver. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) a reconstituted human immune system. In some cases, the genetically modified non- human animal does not comprise (and / or is not modified to comprise) Kupffer cells that are species-compatible to the transplanted hepatocytes / hepatocyte progenitors (i.e., that produce EGF compatible with the EGFR in the transplanted hepatocytes / hepatocyte progenitors). In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) Kupffer cells in the liver that are species-compatible to the transplanted hepatocytes / hepatocyte progenitors. In some cases, the genetically modified non-human animal does not comprise (and / or is not modified to comprise) a reconstituted immune system that is species-compatible to the transplanted hepatocytes / hepatocyte progenitors.

[0231] The genetically modified non-human animals can comprise xenotransplanted hepatocytes / hepatocyte progenitors (e.g., xenotransplanted and expanded hepatocytes / hepatocyte progenitors). The xenotransplanted hepatocytes / hepatocyte progenitors can be from any speciesother than that of the recipient non-human animal. For example, the xenotranspl anted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors. In another example, the xenotransplanted hepatocytes / hepatocyte progenitors can be non-human primate (NHP) hepatocytes / hepatocyte progenitors, such as cynomolgus hepatocytes / hepatocyte progenitors. The transplanted hepatocytes / hepatocyte progenitors can be from a species whose EGFR is incompatible with the endogenous non-human animal EGF (e.g., the transplanted hepatocytes / hepatocyte progenitors can be human hepatocytes / hepatocyte progenitors, and the non-human animal can be a mouse or a rat). The cross-species incompatibility between the transplanted hepatocytes / hepatocyte progenitors and the recipient non-human animals (i.e., the suboptimal interaction or lack of reactivity between the recipient non-human animal EGF ligands and the EGFR on the xenotransplanted hepatocytes / hepatocyte progenitors) can result in a slower or impaired engraftment of the transplanted hepatocytes / hepatocyte progenitors.

[0232] The modifications described herein can result in accelerated or improved engraftment of the xenotransplanted hepatocytes / hepatocyte progenitors compared to non-human animals in which the genetically modified non-human animals and the xenotransplanted hepatocytes / hepatocyte progenitors do not have modifications to restore, activate, or increase EGF / EGFR signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. The modifications can, for example, restore, activate, or increase species-matched hepatic EGF / EGFR signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors, or can simply restore, activate, or increase EGF / EGFR signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. Likewise, the modifications can, for example, restore, activate, or increase species-compatible hepatic EGF / EGFR signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors.

[0233] In some cases, the xenotransplanted hepatocytes / hepatocyte progenitors are modified to restore, activate, or increase EGF / EGFR signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. As one example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) non-human animal EGFR (i.e., EGFR from the same species as the recipient non-human animal — in other words, EGFR species-matched to the recipient non-human animal). As another example, the transplanted hepatocytes / hepatocyte progenitors can be modified to express (i.e., ectopically express) speciescompatible EGFR (i.e., EGFR from a species that is compatible with the EGF produced by therecipient non-human animal, such that the recipient non-human animal EGF can bind to and activate EGFR signaling). For example, if the hepatocytes / hepatocyte progenitors are being transplanted into a mouse or a rat, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to express mouse EGFR or rat EGFR, respectively. In some cases, the transplanted hepatocytes / hepatocyte progenitors can be modified to comprise a vector comprising an expression construct for the non-human animal EGFR comprising a nucleic acid encoding the non-human animal EGFR operably linked to a promoter. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. Alternatively, the transplanted hepatocytes / hepatocyte progenitors can be genetically modified to comprise in their genome a non-human animal EGFR expression construct comprising a nucleic acid encoding the non-human animal EGFR operably linked to a promoter. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a liver-specific promoter or a promoter active in liver cells (e.g., hepatocytes) can be used. Examples of such promoters include TTR, ALB, and HBV promoters. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFl alpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.

[0234] Mouse epidermal growth factor receptor (also known as EGFR) is encoded by the gene Egfr. EGFR is a receptor tyrosine kinase binding ligands of the EGF family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses. Mouse Egfr maps to 11 A2; 11 9.41 cM on chromosome 11 (NCBI RefSeq Gene ID 13649; Assembly GRCm39 (GCF_000001635.27); location NC_000077.7 (16700153..16868158). Reference to the mouse Egfr gene includes the canonical, wild type form as well as all allelic forms and isoforms. The mouse EGFR protein has been assigned UniProt accession number Q01279, and the canonical isoform (NCBI Accession No. NP_997538.1) is set forth in SEQ ID NO: 32. Reference to mouse EGFR proteins includes all allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_207655.2. Thecoding sequence for this isoform is assigned CCDS Accession No. CCDS24443.1 (SEQ ID NO: 31). Reference to the mouse Egfr mRNA (cDNA) and coding sequence includes all allelic forms and isoforms.

[0235] Rat Egfr maps to 14q22 on chromosome 14 (NCBI RefSeq Gene ID 24329; Assembly GRCr8 (GCF_036323735.1); location NC_086032.1 (95378626 .95551358). Reference to the rat Egfr gene includes all allelic forms and isoforms. Rat EGFR protein (canonical isoform) has been assigned NCBI Accession No. NP_113695.2 (SEQ ID NO: 34). Reference to rat EGFR proteins includes allelic forms and isoforms. An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_031507.2. A coding sequence for the canonical isoform is set forth in SEQ ID NO: 33. Reference to the rat Egfr mRNA (cDNA) and coding sequence includes all allelic forms and isoforms.

[0236] In some cases, the recipient non-human animal is modified to restore, activate, or increase EGF / EGFR signaling pathway activity in the xenotransplanted hepatocytes / hepatocyte progenitors. For example, the non-human animal can comprise a ligand that can activate EGF / epidermal growth factor receptor (EGFR) signaling in the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., a ligand that can activate human EGFR signaling in human xenotransplanted hepatocytes / hepatocyte progenitors). For example, the non-human animal can comprise an EGFR agonist antigen-binding protein such as an EGFR agonist antibody (e.g., a human EGFR agonist antigen-binding protein such as a human EGFR agonist antibody). Alternatively, the non-human animal can comprise EGF from the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., EGF that is species-matched with the xenotransplanted hepatocytes / hepatocyte progenitors). Likewise, the non-human animal can comprise EGF compatible with the species of the xenotransplanted hepatocytes / hepatocyte progenitors (e.g., EGF that is species-compatible with the xenotransplanted hepatocytes / hepatocyte progenitors, such that the EGF can activate EGF signaling in the xenotransplanted hepatocytes / hepatocyte progenitors). For example, the non-human animal can comprise human EGF (or a human-EGFR-compatible ligand (e.g., human-EGFR-compatible EGF), such as cynomolgus EGF) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors. For example, the non-human animal can comprise human EGF (or a human-EGFR-compatible EGF, such as cynomolgus EGF) if the xenotransplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyteprogenitors. Human EGF is described below. The EGF can be, for example, in the serum of the non-human animal or the liver of the non-human animal. For example, the EGF can be present or expressed locally in the liver of the non-human animal. The EGF can be expressed by any suitable cell type in the non-human animal. In one example, the EGF is expressed in liver cells in the non-human animal. In one example, the EGF is expressed in liver cells in the non-human animal. In one example, the EGF is expressed in muscle cells in the non-human animal. In one example, the non-human animal comprises a vector comprising an expression construct for species-matched EGF (e.g., human EGF) comprising a nucleic acid encoding the species- matched EGF (e.g., human EGF) operably linked to a promoter. Likewise, the non-human animal can comprise a vector comprising an expression construct for species-compatible EGF (e.g., the EGF is compatible with the EGFR expressed by the transplanted hepatocytes / hepatocyte progenitors such that the EGF can activate EGF signaling in the hepatocytes / hepatocyte progenitors) comprising a nucleic acid encoding the species-compatible EGF operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise a vector comprising an expression construct for human-EGFR-compatible ligand (e.g., human- EGFR-compatible EGF) (e.g., cynomolgus EGF) comprising a nucleic acid encoding the human- EGFR-compatible ligand (e.g., human-EGFR-compatible EGF) operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise a vector comprising an expression construct for human-EGFR-compatible EGF (e.g., cynomolgus EGF) comprising a nucleic acid encoding the human-EGFR-compatible EGF operably linked to a promoter. For example, the non-human animal can comprise the vector in liver cells. For example, the non- human animal can comprise the vector in muscle cells. Any suitable vector can be used. For example, the vector can be a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In a specific example, a lentiviral vector is used. In another specific example, an adenoviral vector is used. In another specific example, an AAV vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in muscle. In a specific example, a recombinant AAV9 vector is used. In a specific example, an AAV vector is used, such as an AAV serotype for expression in liver. In a specific example, a recombinant AAV8 vector is used. Alternatively, the non-human animal canI l lcomprise in its genome a species-matched EGF (e.g., human EGF) expression construct comprising a nucleic acid encoding the species-matched EGF (e.g., human EGF) operably linked to a promoter. Likewise, the non-human animal can comprise in its genome a species-compatible EGF (e.g., the EGF is compatible with the EGFR expressed by the transplanted hepatocytes / hepatocyte progenitors such that the EGF can activate EGF signaling in the hepatocytes / hepatocyte progenitors) expression construct comprising a nucleic acid encoding the species-compatible EGF operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non- human animal can comprise in its genome a human -EGFR-compatible ligand (e.g., human- EGFR-compatible EGF) (e.g., cynomolgus EGF) expression construct comprising a nucleic acid encoding the human-EGFR-compatible ligand (e.g., human-EGFR-compatible EGF) operably linked to a promoter. For example, if the transplanted hepatocytes / hepatocyte progenitors are human hepatocytes / hepatocyte progenitors, the non-human animal can comprise in its genome a human-EGFR-compatible EGF (e.g., cynomolgus EGF) expression construct comprising a nucleic acid encoding the human-EGFR-compatible EGF operably linked to a promoter. For example, the expression construct could be at a safe harbor locus in the non-human animal. In the case of genomic modification or in the case of a vector, any suitable promoter can be used. In one example, a tissue-specific promoter can be used. For example, a liver-specific promoter or a promoter active in liver cells can be used. Examples of liver-specific promoters or promoters active in liver cells include TTR, ALB, and HBV promoters. For example, a muscle-specific promoter or a promoter active in muscle cells can be used. An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein. In another example, a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta- actin / CMV enhancer (CAG), and elongation factor- 1 alpha (EFl alpha). As another example, an inducible promoter can be used. In the case of genomic modification, an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e g., a safe harbor locus) can be used.

[0237] Human epidermal growth factor is described in more detail above. Transforming growth factor alpha is another mitogenic polypeptide that is able to bind to EGFR and activate EGFR signaling. Human transforming growth factor alpha (also known as TGF alpha or TGFA)is encoded by the gene TGFA. Human TGFA maps to 2p 13.3 on chromosome 2 (NCBI RefSeq Gene ID 7039; Assembly GRCh38.pl4 (GCF_000001405.40); location NC_000002.12 (70447284..70553826, complement). Reference to the human TGFA gene includes the canonical, wild type form ...

Claims

We claim:

1. A genetically modified non-human animal, wherein the genetically modified non-human animal comprises transplanted hepatocytes from a different species than the non-human animal, optionally wherein the hepatocyte are human hepatocytes, wherein the genetically modified non-human animal and / or the transplanted hepatocytes are modified to restore or increase interleukin-6 (IL-6) / interleukin-6 receptor (IL- 6R) signaling pathway activity or interleukin-6 receptor subunit beta (GP130) signaling pathway activity in the transplanted hepatocytes, and wherein the genetically modified non-human animal and / or the transplanted hepatocytes are modified to restore or increase hepatocyte growth factor (HGF) / hepatocyte growth factor receptor (MET) signaling pathway activity in the transplanted hepatocytes.

2. The genetically modified non-human animal of claim 1, wherein the genetically modified non-human animal is immunodeficient.

3. The genetically modified non-human animal of claim 1 or 2, wherein:(I) the genetically modified non-human animal comprises an inactivated endogenous Il2rg gene;(II) the genetically modified non-human animal comprises an inactivated endogenous Ragl gene or an inactivated endogenous Rag2 gene, optionally wherein the genetically modified non-human animal comprises the inactivated endogenous Rag2 gene;(III) the genetically modified non-human animal comprises an inactivated endogenous Ragl gene and an inactivated endogenous Rag2 gene;(IV) the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the genetically modified non-human animal further comprises an inactivated endogenous Ragl gene;(V) the genetically modified non-human animal comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcsad, or(VI) the genetically modified non-human animal comprises a SCID mutation in a Prkdc gene (Prkdcsc,d) and an inactivated endogenous Il2rg gene.

4. The genetically modified non-human animal of any one of claims 1-3, wherein the genetically modified non-human animal is genetically modified so that endogenous non-human animal hepatocytes in the liver can be selectively and conditionally ablated.

5. The genetically modified non-human animal of claim 4, wherein:(I) the genetically modified non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter;(II) the genetically modified non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter; or(III) the genetically modified non-human animal comprises an inactivated endogenous Fah gene.

6. The genetically modified non-human animal of any one of claims 1-5, wherein:(I) the genetically modified non-human animal comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcscid'), and the genetically modified non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter;(II) the genetically modified non-human animal comprises a SCID mutation in a Prkdc gene (Prkdcsc,d) and an inactivated endogenous Il2rg gene, and the genetically modified non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter; or(III) the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the genetically modified non-human animal further comprises an inactivated endogenous Ragl gene, and the genetically modified non-human animal comprises an inactivated endogenous Fah gene.

7. The genetically modified non-human animal of any one of claims 1-6, wherein the transplanted hepatocytes are human hepatocytes.

8. The genetically modified non-human animal of any one of claims 1-7, wherein the genetically modified non-human animal comprises an inactivated endogenous Rag2gene, an inactivated endogenous U2rg gene, and an inactivated endogenous Fah gene, optionally wherein the genetically modified non-human animal comprises an inactivated endogenous Ragl gene.

9. The genetically modified non-human animal of any one of claims 1-8, wherein:(I) the transplanted hepatocytes express non-human animal IL-6R, wherein the non-human animal IL-6R is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non- human animal IL-6R is mouse IL-6R, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal IL-6R is rat IL-6R;(II) the transplanted hepatocytes express non-human animal oncostatin-M- specific receptor subunit beta (OSMR), wherein the non-human animal OSMR is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal OSMR is mouse OSMR, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal OSMR is rat OSMR; or(III) the transplanted hepatocytes express non-human animal IL-6R and non- human animal OSMR, wherein the non-human animal IL-6R and non-human animal OSMR are from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse, the non-human animal IL-6R is mouse IL- 6R, and the non-human animal OSMR is mouse OSMR, or optionally wherein the genetically modified non-human animal is a rat, the non-human animal IL-6R is rat IL-6R, and the non- human animal OSMR is rat OSMR.

10. The genetically modified non-human animal of claim 9, wherein:(I) the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal IL-6R comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter; or(II) the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal OSMR comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter.11 . The genetically modified non-human animal of claim 10, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector.

12. The genetically modified non-human animal of claim 9, wherein:(I) the transplanted hepatocytes comprise in their genome a non-human animal IL-6R expression construct comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter; or(II) the transplanted hepatocytes comprise in their genome a non-human animal OSMR expression construct comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter.

13. The genetically modified non-human animal of any one of claims 10-12, wherein the promoter is a liver-specific promoter or a constitutive promoter.

14. The genetically modified non-human animal of any one of claims 1-8, wherein the transplanted hepatocytes express a ligand-independent, constitutively active form of GP130.

15. The genetically modified non-human animal of claim 14, wherein the transplanted hepatocytes comprise a vector comprising an expression construct for the constitutively active GP130 comprising a nucleic acid encoding the constitutively active GP130 operably linked to a promoter.

16. The genetically modified non-human animal of claim 15, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an AAV vector, optionally wherein the viral vector is the lentivirus vector.

17. The genetically modified non-human animal of claim 14, wherein the transplanted hepatocytes comprise in their genome an expression construct for the constitutively active GP130 comprising a nucleic acid encoding the constitutively active GP130 operably linked to a promoter.

18. The genetically modified non-human animal of any one of claims 15-17, wherein the promoter is a liver-specific promoter or a constitutive promoter.

19. The genetically modified non-human animal of any one of claims 14-18, wherein the constitutively active GP130 is a constitutively active human GP130, optionally wherein the constitutively active human GP130 comprises a deletion of a region of GP130 from Tyrl86 to Tyrl90 (GP130Y186-Y190del).

20. The genetically modified non-human animal of any one of claims 1-8, wherein the genetically modified non-human animal further comprises a GP130-activating ligand.

21. The genetically modified non-human animal of claim 20, wherein the GP130-activating ligand comprises a human IL-6R agonist antigen-binding protein or a human OSMR agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody.

22. The genetically modified non-human animal of claim 20, wherein:(I) the GP130-activating ligand comprises human IL-6 or a human-IL-6R- compatible ligand or a human-IL-6R-compatible IL-6; or(II) the GP130-activating ligand comprises human oncostatin-M (OSM) or a human-OSMR-compatible ligand or a human-0 SMR-compatible OSM.

23. The genetically modified non-human animal of any one of claims 20-22, wherein the genetically modified non-human animal further comprises one or more additional GP130-activating ligands, optionally wherein the GP130-activating ligands comprise: (1) human IL-6 or a human-IL-6R-compatible ligand or a human-IL-6R-compatible IL-6; and (2) human OSM or a human-OSMR-compatible ligand or a human-OSMR-compatible OSM.

24. The genetically modified non-human animal of any one of claims 20-23, wherein the genetically modified non-human animal comprises a vector comprising an expression construct for the GP130-activating ligand comprising a nucleic acid encoding the GP130-activating ligand operably linked to a promoter.

25. The genetically modified non-human animal of claim 24, wherein the genetically modified non-human animal comprises the vector in muscle cells or in liver cells.

26. The genetically modified non-human animal of claim 24 or 25, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

27. The genetically modified non-human animal of any one of claims 24-26, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

28. The genetically modified non-human animal of claim any one of claims 20-23, wherein the genetically modified non-human animal comprises in its genome a GP130- activating ligand expression construct comprising a nucleic acid encoding the GP130-activating ligand operably linked to a promoter.

29. The genetically modified non-human animal of claim 28, wherein:(I) the GP130-activating ligand comprises human IL-6 or a human-IL-6R- compatible ligand or a human-IL-6R-compatible IL-6; or(II) the GP130-activating ligand comprises human OSM or a human-0 SMR- compatible ligand or a human-OSMR-compatible OSM.

30. The genetically modified non-human animal of claim 28 or 29, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7);(II) the promoter is a constitutive promoter;(III) the promoter is an exogenous promoter; or(IV) the promoter is an endogenous promoter.

31. The genetically modified non-human animal of any one of claims 20-23, wherein:(I) the genetically modified non-human animal comprises a humanized non- human animal IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein;(II) the genetically modified non-human animal comprises a humanized non- human animal OSM gene comprising a human OSM nucleic acid encoding a human OSM protein; or(III) the genetically modified non-human animal comprises a humanized non- human animal IL 6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein, and wherein the genetically modified non-human animal comprises a humanized non-human animal OSM gene comprising a human OSM nucleic acid encoding a human OSM protein.

32. The genetically modified non-human animal of claim 31, wherein:(I) the human nucleic acid comprises a region of human IL6 genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human IL6 complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal IL6 gene; and / or(III) the human nucleic acid is inserted into the non-human animal IL6 gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal IL6 promoter; and / or(V) the genetically modified non-human animal is heterozygous for the humanized IL6 gene; and / or(VI) the genetically modified non-human animal is homozygous for the humanized IL6 gene; and / or(VII) the genetically modified non-human animal comprises the humanized IL6 gene in its germline.

33. The genetically modified non-human animal of claim 31 or 32, wherein:(I) the human nucleic acid comprises a region of human OSM genomic sequence from the start codon to the stop codon or the human nucleic acid comprises a human OSM complementary DNA(cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal OSA gene; and / or(III) the human nucleic acid is inserted into the non-human animal OSM gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal OSM promoter; and / or(V) the genetically modified non-human animal is heterozygous for the humanized OSM gene; and / or(VI) the genetically modified non-human animal is homozygous for the humanized OSM gene; and / or(VII) the genetically modified non-human animal comprises the humanized OSM gene in its germline.

34. The genetically modified non-human animal of any one of claims 1-33, wherein the transplanted hepatocytes express non-human animal MET, wherein the non-human animal MET is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal MET is mouse MET, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal MET is rat MET.

35. The genetically modified non-human animal of claim 34, wherein the transplanted hepatocytes comprise a vector comprising an expression construct for the non- human animal MET comprising a nucleic acid encoding the non-human animal MET operably linked to a promoter.

36. The genetically modified non-human animal of claim 35, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector.

37. The genetically modified non-human animal of claim 34, wherein the transplanted hepatocytes comprise in their genome a non-human animal MET expression construct comprising a nucleic acid encoding the non-human animal MET operably linked to a promoter.

38. The genetically modified non-human animal of any one of claims 35-37, wherein the promoter is a liver-specific promoter or a constitutive promoter.

39. The genetically modified non-human animal of any one of claims 1-33, wherein the genetically modified non-human animal further comprises a MET-activating ligand.

40. The genetically modified non-human animal of claim 39, wherein the MET-activating ligand comprises a human MET agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody.

41. The genetically modified non-human animal of claim 39, wherein the MET-activating ligand comprises human HGF or a human-MET-compatible ligand or a human- MET-compatible HGF.

42. The genetically modified non-human animal of any one of claims 39-41, wherein the genetically modified non-human animal comprises a vector comprising an expression construct for the MET-activating ligand comprising a nucleic acid encoding the MET- activating ligand operably linked to a promoter.

43. The genetically modified non-human animal of claim 42, wherein the genetically modified non-human animal comprises the vector in muscle cells or in liver cells.

44. The genetically modified non-human animal of claim 42 or 43, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

45. The genetically modified non-human animal of any one of claims 42-44, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

46. The genetically modified non-human animal of claim any one of claims 39-41, wherein the genetically modified non-human animal comprises in its genome aMET- activating ligand expression construct comprising a nucleic acid encoding the MET-activating ligand operably linked to a promoter.

47. The genetically modified non-human animal of claim 46, wherein the MET-activating ligand comprises human HGF or a human-MET-compatible ligand or a human- MET-compatible HGF.

48. The genetically modified non-human animal of claim 46 or 47, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7);(II) the promoter is a constitutive promoter;(III) the promoter is an exogenous promoter; or(IV) the promoter is an endogenous promoter.

49. The genetically modified non-human animal of any one of claims 39-41, wherein the genetically modified non-human animal comprises a humanized non-human animal HGF gene comprising a human HGF nucleic acid encoding a human HGF protein.

50. The genetically modified non-human animal of claim 49, wherein:(I) the human nucleic acid comprises a region of human HGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human HGF complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal HGF gene; and / or(Ill) the human nucleic acid is inserted into the non-human animal HGF gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal HGF promoter; and / or(V) the genetically modified non-human animal is heterozygous for the humanized HGF gene; and / or(VI) the genetically modified non-human animal is homozygous for the humanized HGF gene; and / or(VII) the genetically modified non-human animal comprises the humanized HGF gene in its germline.

51. The genetically modified non-human animal of any one of claims 1-50, wherein the transplanted hepatocytes express non-human animal EGFR, wherein the non-human animal EGFR is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal EGFR is mouse EGFR, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal EGFR is rat EGFR.

52. The genetically modified non-human animal of claim 51, wherein the transplanted hepatocytes comprise a vector comprising an expression construct for the non- human animal EGFR comprising a nucleic acid encoding the non-human animal EGFR operably linked to a promoter.

53. The genetically modified non-human animal of claim 52, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector.

54. The genetically modified non-human animal of claim 51, wherein the transplanted hepatocytes comprise in their genome a non-human animal EGFR expression construct comprising a nucleic acid encoding the non-human animal EGFR operably linked to a promoter.

55. The genetically modified non-human animal of any one of claims 52-54, wherein the promoter is a liver-specific promoter or a constitutive promoter.

56. The genetically modified non-human animal of any one of claims 1-50, wherein the genetically modified non-human animal further comprises an EGFR-activating ligand.

57. The genetically modified non-human animal of claim 56, wherein the EGFR-activating ligand comprises a human EGFR agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody.

58. The genetically modified non-human animal of claim 56, wherein the EGFR-activating ligand comprises human EGF or a human-EGFR-compatible ligand or a human-EGFR-compatible EGF.

59. The genetically modified non-human animal of any one of claims 56-58, wherein the genetically modified non-human animal comprises a vector comprising an expression construct for the EGFR-activating ligand comprising a nucleic acid encoding the EGFR-activating ligand operably linked to a promoter.

60. The genetically modified non-human animal of claim 59, wherein the genetically modified non-human animal comprises the vector in muscle cells or in liver cells.

61. The genetically modified non-human animal of claim 59 or 60, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

62. The genetically modified non-human animal of any one of claims 59-61, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

63. The genetically modified non-human animal of claim any one of claims 56-58, wherein the genetically modified non-human animal comprises in its genome an EGFR- activating ligand expression construct comprising a nucleic acid encoding the EGFR-activating ligand operably linked to a promoter.

64. The genetically modified non-human animal of claim 63, wherein the EGFR-activating ligand comprises human EGF or a human-EGFR-compatible ligand or a human-EGFR-compatible EGF.

65. The genetically modified non-human animal of claim 63 or 67, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7);(II) the promoter is a constitutive promoter;(III) the promoter is an exogenous promoter; or(IV) the promoter is an endogenous promoter.

66. The genetically modified non-human animal of any one of claims 56-58, wherein the genetically modified non-human animal comprises a humanized non-human animal EGF gene comprising a human EGF nucleic acid encoding a human EGF protein.

67. The genetically modified non-human animal of claim 66, wherein:(I) the human nucleic acid comprises a region of human EGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human EGF complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal EGF gene; and / or(III) the human nucleic acid is inserted into the non-human animal EGF gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal EGF promoter; and / or(V) the genetically modified non-human animal is heterozygous for the humanized EGF gene; and / or(VI) the genetically modified non-human animal is homozygous for the humanized EGF gene; and / or(VII) the genetically modified non-human animal comprises the humanized EGF gene in its germline.

68. The genetically modified non-human animal of any one of claims 1-67, wherein the genetically modified non-human animal further comprises human R-spondin-3 (RSPO3).

69. The genetically modified non-human animal of claim 68, wherein the genetically modified non-human animal comprises a vector comprising an expression construct for the human RSPO3 comprising a nucleic acid encoding the human RSPO3 operably linked to a promoter.

70. The genetically modified non-human animal of claim 69, wherein the genetically modified non-human animal comprises the vector in muscle cells or in liver cells.

71. The genetically modified non-human animal of claim 69 or 70, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

72. The genetically modified non-human animal of any one of claims 69-71, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

73. The genetically modified non-human animal of claim 68, wherein the genetically modified non-human animal comprises in its genome a human RSPO3 expression construct comprising a nucleic acid encoding the human RSPO3 operably linked to a promoter.

74. The genetically modified non-human animal of claim 73, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7);(II) the promoter is a constitutive promoter;(III) the promoter is an exogenous promoter; or(IV) the promoter is an endogenous promoter.

75. The genetically modified non-human animal of any one of claims 1-74, wherein the genetically modified non-human animal does not comprise human Kupffer cells in the liver.

76. The genetically modified non-human animal of any one of claims 1-75, wherein the genetically modified non-human animal does not comprise a reconstituted human immune system.

77. The genetically modified non-human animal of any one of claims 1-76, further comprising a humanized non-human animal SIRPA gene.

78. The genetically modified non-human animal of claim 77, wherein:(I) the humanized non-human animal SIRPA gene comprises a replacement of exons 2-4 of the non-human animal SIRPA gene with exons 2-4 of human SIRPA, wherein the humanized non-human animal SIRPA gene encodes a chimeric SIRPA protein comprising an extracellular portion of a human SIRPA protein and an intracellular portion of a non-human animal SIRPA protein; and / or(II) the humanized non-human animal SIRPA gene is operably linked to an endogenous non-human animal SIRPA promoter; and / or(III) the genetically modified non-human animal is heterozygous for the humanized SIRPA gene; and / or(IV) the genetically modified non-human animal is homozygous for the humanized SIRPA gene; and / or(V) the genetically modified non-human animal comprises the humanized SIRPA gene in its germline.

79. The genetically modified non-human animal of any one of claims 1-78, wherein the genetically modified non-human animal is a male.

80. The genetically modified non-human animal of any one of claims 1-78, wherein the genetically modified non-human animal is a female.

81. The genetically modified non-human animal of any one of claims 1-80, wherein the genetically modified non-human animal is a mammal.

82. The genetically modified non-human animal of claim 81, wherein the mammal is a rodent.

83. The genetically modified non-human animal of claim 82, wherein the rodent is a rat or a mouse.

84. The genetically modified non-human animal of claim 83, wherein the rodent is the rat.

85. The genetically modified non-human animal of claim 83, wherein the rodent is the mouse.

86. The genetically modified non-human animal of any one of claims 1-85, wherein the transplanted hepatocytes have reduced lipid droplet accumulation compared to transplanted hepatocytes in genetically modified non-human animals in which the genetically modified non-human animal and the transplanted hepatocytes are not modified to restore or increase IL-6 / IL-6R signaling pathway activity or GP130 signaling pathway activity in the transplanted hepatocytes.

87. A method of measuring the activity of a human-liver-targeting reagent in vivo, comprising:(a) administering the human-liver-targeting reagent to the genetically modified non-human animal of any one of claims 1-86; and(b) measuring the activity of the human-liver-targeting reagent in the liver of the genetically modified non-human animal.

88. The method of claim 87, wherein step (a) comprises AAV-mediated delivery, lipid nanoparticle (LNP)-mediated delivery, hydrodynamic delivery (HDD), or injection.

89. The method of claim 87 or 88, wherein the human-liver-targeting reagent targets a target gene expressed in a human liver.

90. The method of claim 89, wherein:(I) step (b) comprises measuring expression of a messenger RNA or a protein encoded by the target gene; and / or(II) step (b) comprises measuring modification of a genomic locus comprising the target gene, optionally wherein step (b) comprises measuring the frequency of insertions or deletions within the genomic locus comprising the target gene; and / or(III) the human-liver-targeting reagent comprises a nuclease agent designed to target a region of the target gene, optionally wherein the nuclease agent comprises a Cas protein and a guide RNA designed to target a guide RNA target sequence in the target gene, optionally wherein the Cas protein is a Cas9 protein; and / or(IV) the human-liver-targeting reagent comprises an exogenous donor nucleic acid, wherein the exogenous donor nucleic acid is designed to target the target gene, and optionally wherein the exogenous donor nucleic acid is delivered via AAV.

91. The method of claim 87 or 88, wherein the human-liver-targeting reagent targets a target RNA expressed in a human liver, optionally wherein the human-liver-targeting reagent is an RNAi agent or an antisense oligonucleotide.

92. The method of claim 87 or 88, wherein the human-liver-targeting reagent targets a target protein expressed in a human liver, optionally wherein the human-liver-targeting reagent is an antigen-binding protein or a small molecule.

93. A method of making a non-human animal with a humanized liver, comprising:(a) transplanting human hepatocytes or human hepatocyte progenitors into a genetically modified non-human animal; and(b) allowing the human hepatocytes or human hepatocyte progenitors to expand, wherein the genetically modified non-human animal and / or the transplanted human hepatocytes or human hepatocyte progenitors are modified to restore or increase interleukin-6 (IL-6) / interleukin-6 receptor (IL-6R) signaling pathway activity or interleukin-6 receptor subunit beta (GP130) signaling pathway activity in the transplanted human hepatocytes or human hepatocyte progenitors, and wherein the genetically modified non-human animal and / or the transplanted hepatocytes are modified to restore or increase hepatocyte growth factor (HGF) / hepatocyte growth factor receptor (MET) signaling pathway activity in the transplanted hepatocytes.

94. The method of claim 93, wherein the genetically modified non-human animal is immunodeficient.

95. The method of claim 93 or 94, wherein:(I) the genetically modified non-human animal comprises an inactivated endogenous Il2rg gene;(II) the genetically modified non-human animal comprises an inactivated endogenous Ragl gene or an inactivated endogenous Rag2 gene, optionally wherein the genetically modified non-human animal comprises the inactivated endogenous Rag2 gene;(III) the genetically modified non-human animal comprises an inactivated endogenous Ragl gene and an inactivated endogenous Rag2 gene;(IV) the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the genetically modified non-human animal further comprises an inactivated endogenous Ragl gene;(V) the genetically modified non-human animal comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcsad, or(VI) the genetically modified non-human animal comprises a SCID mutation in a Prkdc gene (Prkdcsc,d) and an inactivated endogenous Il2rg gene.

96. The method of any one of claims 93-95, wherein the genetically modified non-human animal is genetically modified so that endogenous non-human animal hepatocytes in the liver can be selectively and conditionally ablated.

97. The method of claim 96, wherein:(I) the genetically modified non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter;(II) the genetically modified non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter; or(III) the genetically modified non-human animal comprises an inactivated endogenous Fah gene.

98. The method of any one of claims 93-97, wherein:(I) the genetically modified non-human animal comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcsadand the genetically modified non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter;(II) the genetically modified non-human animal comprises a SCID mutation in a Prkdc gene (Prkdcscld) and an inactivated endogenous Jl2rg gene, and the genetically modified non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter; or(III) the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the genetically modified non-human animal further comprises an inactivated endogenous Ragl gene, and the genetically modified non-human animal comprises an inactivated endogenous Fah gene.

99. The method of any one of claims 93-98, wherein the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, an inactivated endogenous Fah gene, and optionally an inactivated endogenous Ragl gene.

100. The method of any one of claims 93-99, wherein:(I) the transplanted hepatocytes express non-human animal IL-6R, wherein the non-human animal IL-6R is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non- human animal IL-6R is mouse IL-6R, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal IL-6R is rat IL-6R;(II) the transplanted hepatocytes express non-human animal oncostatin-M- specific receptor subunit beta (OSMR), wherein the non-human animal OSMR is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal OSMR is mouse OSMR, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal OSMR is rat OSMR; or(III) the transplanted hepatocytes express non-human animal IL-6R and non- human animal OSMR, wherein the non-human animal IL-6R and non-human animal OSMR are from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse, the non-human animal IL-6R is mouse IL- 6R, and the non-human animal OSMR is mouse OSMR, or optionally wherein the genetically modified non-human animal is a rat, the non-human animal IL-6R is rat IL-6R, and the non- human animal OSMR is rat OSMR.101 . The method of claim 100, wherein:(I) the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal IL-6R comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter; or(II) the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal OSMR comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter.

102. The method of claim 101, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector.

103. The method of claim 100, wherein:(I) the transplanted hepatocytes comprise in their genome a non-human animal IL-6R expression construct comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter; or(II) the transplanted hepatocytes comprise in their genome a non-human animal OSMR expression construct comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter.

104. The method of any one of claims 101-103, wherein the promoter is a liverspecific promoter or a constitutive promoter.

105. The method of any one of claims 93-99, wherein the transplanted hepatocytes express a ligand-independent, constitutively active form of GP130.

106. The method of claim 105, wherein the transplanted hepatocytes comprise a vector comprising an expression construct for the constitutively active GP130 comprising a nucleic acid encoding the constitutively active GP130 operably linked to a promoter.

107. The method of claim 106, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an AAV vector, optionally wherein the viral vector is the lentivirus vector.

108. The method of claim 105, wherein the transplanted hepatocytes comprise in their genome an expression construct for the constitutively active GP130 comprising a nucleic acid encoding the constitutively active GP130 operably linked to a promoter.

109. The method of any one of claims 106-108, wherein the promoter is a liverspecific promoter or a constitutive promoter.

110. The method of any one of claims 105-109, wherein the constitutively active GP130 is a constitutively active human GP130, optionally wherein the constitutively active human GP130 comprises a deletion of a region of GP130 from Tyrl86 to Tyrl90 (GP130Y186'Y190del)111. The method of any one of claims 93-99, wherein the genetically modified non-human animal further comprises a GP130-activating ligand.

112. The method of claim 111 , wherein the GP130-activating ligand comprises a human IL-6R agonist antigen-binding protein or a human OSMR agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody.

113. The method of claim 111, wherein:(I) the GP130-activating ligand comprises human IL-6 or a human-IL-6R- compatible ligand or a human-IL-6R-compatible IL-6; or(II) the GP130-activating ligand comprises human oncostatin-M (OSM) or a human-OSMR-compatible ligand or a human-OSMR-compatible OSM.

114. The method of any one of claims 111-113, wherein the genetically modified non-human animal further comprises one or more additional GP130-activating ligands, optionally wherein the GP130-activating ligands comprise: (1) human IL-6 or a human-IL-6R- compatible ligand or a human-IL-6R-compatible IL-6; and (2) human OSM or a human-OSMR- compatible ligand or a human-OSMR-compatible OSM.

115. The method of any one of claims 111-114, wherein the genetically modified non-human animal comprises a vector comprising an expression construct for the GP130-activating ligand comprising a nucleic acid encoding the GP130-activating ligand operably linked to a promoter.

116. The method of claim 115, wherein the genetically modified non-human animal comprises the vector in muscle cells or in liver cells.

117. The method of claim 115 or 116, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

118. The method of any one of claims 115-117, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or anadeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

119. The method of any one of claims 111-114, wherein the genetically modified non-human animal comprises in its genome a GP130-activating ligand expression construct comprising a nucleic acid encoding the GP130-activating ligand operably linked to a promoter.

120. The method of claim 119, wherein:(I) the GP130-activating ligand comprises human IL-6 or a human-IL-6R- compatible ligand or a human-IL-6R-compatible IL-6; or(II) the GP130-activating ligand comprises human OSM or a human-OSMR- compatible ligand or a human-OSMR-compatible OSM.

121. The method of claim 119 or 120, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7);(II) the promoter is a constitutive promoter;(III) the promoter is an exogenous promoter; or(IV) the promoter is an endogenous promoter.

122. The method of any one of claims 111-114, wherein:(I) the genetically modified non-human animal comprises a humanized non- human animal IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein;(II) the genetically modified non-human animal comprises a humanized non- human animal OSM gene comprising a human OSM nucleic acid encoding a human OSM protein; or(III) the genetically modified non-human animal comprises a humanized non- human animal IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein, and wherein the genetically modified non-human animal comprises a humanized non-human animal OSM gene comprising a human OSM nucleic acid encoding a human OSM protein.

123. The method of claim 122, wherein:(I) the human nucleic acid comprises a region of human IL6 genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human IL6 complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal IL6 gene; and / or(III) the human nucleic acid is inserted into the non-human animal IL6 gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal IL6 promoter; and / or(V) the genetically modified non-human animal is heterozygous for the humanized IL6 gene; and / or(VI) the genetically modified non-human animal is homozygous for the humanized IL6 gene; and / or(VII) the genetically modified non-human animal comprises the humanized IL6 gene in its germline.

124. The method of claim 122 or 123, wherein:(I) the human nucleic acid comprises a region of human OSM genomic sequence from the start codon to the stop codon or the human nucleic acid comprises a human OSM complementary DNA(cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal OSM gene; and / or(III) the human nucleic acid is inserted into the non-human animal OSM gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal OSM promoter; and / or(V) the genetically modified non-human animal is heterozygous for the humanized (AW gene; and / or(VI) the genetically modified non-human animal is homozygous for the humanized OSM gene; and / or(VII) the genetically modified non-human animal comprises the humanized OSM gene in its germline.

125. The method of any one of claims 93-124, wherein the transplanted hepatocytes express non-human animal MET, wherein the non-human animal MET is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal MET is mouse MET, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal MET is rat MET.

126. The method of claim 125, wherein the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal MET comprising a nucleic acid encoding the non-human animal MET operably linked to a promoter.

127. The method of claim 126, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector.

128. The method of claim 125, wherein the transplanted hepatocytes comprise in their genome a non-human animal MET expression construct comprising a nucleic acid encoding the non-human animal MET operably linked to a promoter.

129. The method of any one of claims 126-128, wherein the promoter is a liverspecific promoter or a constitutive promoter.

130. The method of any one of claims 93-124, wherein the genetically modified non-human animal further comprises a MET-activating ligand.

131. The method of claim 130, wherein the MET-activating ligand comprises a human MET agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody.

132. The method of claim 130, wherein the MET-activating ligand comprises human HGF or a human-MET-compatible ligand or a human-MET-compatible HGF.

133. The method of any one of claims 130-132, wherein the genetically modified non-human animal comprises a vector comprising an expression construct for the MET-activating ligand comprising a nucleic acid encoding the MET-activating ligand operably linked to a promoter.

134. The method of claim 133, wherein the genetically modified non-human animal comprises the vector in muscle cells or in liver cells.

135. The method of claim 133 or 134, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

136. The method of any one of claims 133-135, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

137. The method of any one of claims 130-132, wherein the genetically modified non-human animal comprises in its genome a MET-activating ligand expression construct comprising a nucleic acid encoding the MET-activating ligand operably linked to a promoter.

138. The method of claim 137, wherein the MET-activating ligand comprises human HGF or a human-MET-compatible ligand or a human-MET-compatible HGF.

139. The method of claim 137 or 138, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7);(II) the promoter is a constitutive promoter;(ITT) the promoter is an exogenous promoter; or(IV) the promoter is an endogenous promoter.

140. The method of any one of claims 130-132, wherein the genetically modified non-human animal comprises a humanized non-human animal HGF gene comprising a human HGF nucleic acid encoding a human HGF protein.

141. The method of claim 140, wherein:(I) the human nucleic acid comprises a region of human HGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human HGF complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal HGF gene; and / or(III) the human nucleic acid is inserted into the non-human animal HGF gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal HGF promoter; and / or(V) the genetically modified non-human animal is heterozygous for the humanized HGF gene; and / or(VI) the genetically modified non-human animal is homozygous for the humanized HGF gene; and / or(VII) the genetically modified non-human animal comprises the humanized HGF gene in its germline.

142. The method of any one of claims 93-141, wherein the transplanted hepatocytes express non-human animal EGFR, wherein the non-human animal EGFR is from the same species as the genetically modified non-human animal, optionally wherein the genetically modified non-human animal is a mouse and the non-human animal EGFR is mouse EGFR, or optionally wherein the genetically modified non-human animal is a rat and the non-human animal EGFR is rat EGFR.

143. The method of claim 142, wherein the transplanted hepatocytes comprise a vector comprising an expression construct for the non-human animal EGFR comprising a nucleic acid encoding the non-human animal EGFR operably linked to a promoter.

144. The method of claim 143, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the viral vector is the lentivirus vector.

145. The method of claim 142, wherein the transplanted hepatocytes comprise in their genome a non-human animal EGFR expression construct comprising a nucleic acid encoding the non-human animal EGFR operably linked to a promoter.

146. The method of any one of claims 143-145, wherein the promoter is a liverspecific promoter or a constitutive promoter.

147. The method of any one of claims 93-141, wherein the genetically modified non-human animal further comprises an EGFR-activating ligand.

148. The method of claim 147, wherein the EGFR-activating ligand comprises a human EGFR agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody.

149. The method of claim 147, wherein the EGFR-activating ligand comprises human EGF or a human-EGFR-compatible ligand or a human-EGFR-compatible EGF.

150. The method of any one of claims 147-149, wherein the genetically modified non-human animal comprises a vector comprising an expression construct for the EGFR-activating ligand comprising a nucleic acid encoding the EGFR-activating ligand operably linked to a promoter.

151. The method of claim 150, wherein the genetically modified non-human animal comprises the vector in muscle cells or in liver cells.

152. The method of claim 150 or 151, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

153. The method of any one of claims 150-152, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

154. The method of any one of claims 147-149, wherein the genetically modified non-human animal comprises in its genome an EGFR-activating ligand expression construct comprising a nucleic acid encoding the EGFR-activating ligand operably linked to a promoter.

155. The method of claim 154, wherein the EGFR-activating ligand comprises human EGF or a human-EGFR-compatible ligand or a human-EGFR-compatible EGF.

156. The method of claim 154 or 155, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7);(II) the promoter is a constitutive promoter;(III) the promoter is an exogenous promoter; or(IV) the promoter is an endogenous promoter.

157. The method of any one of claims 147-149, wherein the genetically modified non-human animal comprises a humanized non-human animal EGF gene comprising a human EGF nucleic acid encoding a human EGF protein.

158. The method of claim 157, wherein:(I) the human nucleic acid comprises a region of human EGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human EGF complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal EGF gene; and / or(III) the human nucleic acid is inserted into the non-human animal EGF gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal EGF promoter; and / or(V) the genetically modified non-human animal is heterozygous for the humanized EGF gene; and / or(VI) the genetically modified non-human animal is homozygous for the humanized EGF gene; and / or(VII) the genetically modified non-human animal comprises the humanized EGF gene in its germline.

159. The method of any one of claims 93-158, wherein the genetically modified non-human animal further comprises human R-spondin-3 (RSPO3).

160. The method of claim 159, wherein the genetically modified non-human animal comprises a vector comprising an expression construct for the human RSPO3 comprising a nucleic acid encoding the human RSPO3 operably linked to a promoter.

161. The method of claim 160, wherein the genetically modified non-human animal comprises the vector in muscle cells or in liver cells.

162. The method of claim 160 or 161, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

163. The method of any one of claims 160-162, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

164. The method of claim any claim 159, wherein the genetically modified non-human animal comprises in its genome a human RSPO3 expression construct comprising a nucleic acid encoding the human RSPO3 operably linked to a promoter.

165. The method of claim 164, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7);(II) the promoter is a constitutive promoter;(III) the promoter is an exogenous promoter; or(IV) the promoter is an endogenous promoter.

166. The method of any one of claims 93-165, wherein the genetically modified non-human animal does not comprise human Kupffer cells in the liver.

167. The method of any one of claims 93-166, wherein the genetically modified non-human animal does not comprise a reconstituted human immune system.

168. The method of any one of claims 93-167, wherein the genetically modified non-human animal further comprises a humanized non-human animal SIRPA gene.

169. The method of claim 168, wherein:(I) the humanized non-human animal SIRPA gene comprises a replacement of exons 2-4 of the non-human animal SIRPA gene with exons 2-4 of human SIRPA, wherein the humanized non-human animal SIRPA gene encodes a chimeric SIRPA protein comprising an extracellular portion of a human SIRPA protein and an intracellular portion of a non-human animal SIRPA protein; and / or(II) the humanized non-human animal SIRPA gene is operably linked to an endogenous non-human animal SIRPA promoter; and / or(III) the genetically modified non-human animal is heterozygous for the humanized SIRPA gene; and / or(IV) the genetically modified non-human animal is homozygous for the humanized SIRPA gene; and / or(V) the genetically modified non-human animal comprises the humanized SIRPA gene in its germline.

170. The method of any one of claims 93-169, wherein the genetically modified non-human animal is a male.

171. The method of any one of claims 93-169, wherein the genetically modified non-human animal is a female.

172. The method of any one of claims 93-171, wherein the genetically modified non-human animal is a mammal.

173. The method of claim 172, wherein the mammal is a rodent.

174. The method of claim 173, wherein the rodent is a rat or a mouse.

175. The method of claim 174, wherein the rodent is the rat.

176. The method of claim 174, wherein the rodent is the mouse.

177. The method of any one of claims 93-176, wherein exogenous urokinase plasminogen activator or an exogenous nucleic acid encoding urokinase plasminogen activator is administered to the genetically modified non-human animal prior to step (a) to prime the liver for improved repopulation by human hepatocytes, optionally wherein the exogenous nucleic acid is an adenovirus or adeno-associated virus (AAV) encoding urokinase plasminogen activator.

178. The method of any one of claims 93-177, wherein the human hepatocytes or human hepatocyte progenitors are injected into the genetically modified non-human animal intrasplenically in step (a).

179. The method of any one of claims 93-178, wherein at least about ten million human hepatocytes or human hepatocyte progenitors are transplanted into the genetically modified non-human animal in step (a).

180. The method of any one of claims 93-179, wherein step (a) is done in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency.

181. The method of any one of claims 93-180, wherein some or all of step (b) is done in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency.

182. The method of claim 181, wherein nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency is administered to the genetically modified non- human animal in step (b) in an on / off cycle to promote human hepatocyte repopulation.

183. The method of claim 182, wherein the on / off cycle comprises about 5 to about 7 days off and about 3 days on.

184. The method of any one of claims 93-183, wherein the humanized liver generated by the method has reduced lipid droplet accumulation or reduced steatosis compared to methods in which the genetically modified non-human animal and the transplanted human hepatocytes or human hepatocyte progenitors are not modified to restore or increase IL-6 / IL-6R signaling pathway activity or GP130 signaling pathway activity in the transplanted human hepatocytes or human hepatocyte progenitors.

185. A genetically modified non-human animal, non-human animal cell, or non-human animal genome comprising a humanized HGF gene comprising a human HGF nucleic acid encoding a human HGF protein, wherein the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises one or more genetic modifications causing immunodeficiency.

186. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of claim 185, further comprising a humanized IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein.

187. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of claim 185 or 186, further comprising a humanized OSM gene comprising a human OSM nucleic acid encoding a human OSM protein.

188. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 185-187, wherein:(I) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Il2rg gene;(II) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Ragl gene or an inactivated endogenous Rag2 gene, optionally wherein the genetically modified non-human animal, non- human animal cell, or non-human animal genome comprises the inactivated endogenous Rag2 gene;(III) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Ragl gene and an inactivated endogenous Rag2 gene;(IV) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the genetically modified non-human animal, non- human animal cell, or non-human animal genome further comprises an inactivated endogenous Ragl gene;(V) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcsady, or(VI) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a SCID mutation in a Prkdc gene (Prkdcscld) and an inactivated endogenous Il2rg gene.

189. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 185-188, wherein the genetically modified non- human animal, non-human animal cell, or non-human animal genome comprises a geneticmodification enabling selective and conditional ablation of endogenous non-human animal hepatocytes in the liver.

190. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of claim 189, wherein:(I) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter;(II) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter; or(III) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Fah gene.

191. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 185-190, wherein:(I) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcsad), and the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter;(II) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a SCID mutation in a Prkdc gene (Prkdcsc,d) and an inactivated endogenous Il2rg gene, and the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter; or(III) the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, an inactivated endogenous Fah gene, and optionally an inactivated endogenous Ragl gene.

192. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 185-191, wherein the genetically modified non- human animal, non-human animal cell, or non-human animal genome comprises an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, an inactivated endogenous Fah gene, and optionally an inactivated endogenous Ragl gene.

193. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 186 and 188-192, wherein:(I) the human nucleic acid comprises a region of human IL6 genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human IL6 complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal IL6 gene; and / or(III) the human nucleic acid is inserted into the non-human animal IL6 gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal IL6 promoter; and / or(V) the genetically modified non-human animal, non-human animal cell, or non-human animal genome is heterozygous for the humanized IL6 gene; and / or(VI) the genetically modified non-human animal, non-human animal cell, or non-human animal genome is homozygous for the humanized IL6 gene; and / or(VII) the genetically modified non-human animal comprises the humanized IL6 gene in its germline.

194. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 187-193, wherein:(I) the human nucleic acid comprises a region of human OSM genomic sequence from the start codon to the stop codon or the human nucleic acid comprises a human OSM complementary DNA(cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal 0571 / gene; and / or(Ill) the human nucleic acid is inserted into the non-human animal OSM ene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal OSM promoter; and / or(V) the genetically modified non-human animal, non-human animal cell, or non-human animal genome is heterozygous for the humanized OSM gene; and / or(VI) the genetically modified non-human animal, non-human animal cell, or non-human animal genome is homozygous for the humanized OSM gene; and / or(VII) the genetically modified non-human animal comprises the humanized OSM gene in its germline.

195. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 185-194, wherein:(I) the human nucleic acid comprises a region of human HGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human HGF complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal HGF gene; and / or(III) the human nucleic acid is inserted into the non-human animal HGF gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal HGF promoter; and / or(V) the genetically modified non-human animal, non-human animal cell, or non-human animal genome is heterozygous for the humanized HGF gene; and / or(VI) the genetically modified non-human animal, non-human animal cell, or non-human animal genome is homozygous for the humanized HGF gene; and / or(VII) the genetically modified non-human animal comprises the humanized HGF gene in its germline.

196. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 185-195, further comprising a humanized EGF gene comprising a human EGF nucleic acid encoding a human EGF protein.

197. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of claim 196, wherein:(I) the human nucleic acid comprises a region of human EGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human EGF complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal EGF gene; and / or(III) the human nucleic acid is inserted into the non-human animal EGF gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal EGF promoter; and / or(V) the genetically modified non-human animal, non-human animal cell, or non-human animal genome is heterozygous for the humanized EGF gene; and / or(VI) the genetically modified non-human animal, non-human animal cell, or non-human animal genome is homozygous for the humanized EGF gene; and / or(VII) the genetically modified non-human animal comprises the humanized EGF gene in its germline.

198. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of claim any one of claims 185-197, wherein the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises in its genome a human RSPO3 expression construct comprising a nucleic acid encoding the human RSPO3 operably linked to a promoter, and / or wherein the genetically modified non-human animal, non-human animal cell, or non-human animal genome comprises in its genome a human EGF expression construct comprising a nucleic acid encoding the human EGF operably linked to a promoter.

199. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of claim 198, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally whereinthe muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7);(II) the promoter is a constitutive promoter;(III) the promoter is an exogenous promoter; or(IV) the promoter is an endogenous promoter.

200. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 185-199, further comprising a humanized non- human animal SIRPA gene, optionally wherein:(I) the humanized non-human animal SIRPA gene comprises a replacement of exons 2-4 of the non-human animal SIRPA gene with exons 2-4 of human SIRPA, wherein the humanized non-human animal SIRPA gene encodes a chimeric SIRPA protein comprising an extracellular portion of a human SIRPA protein and an intracellular portion of a non-human animal SIRPA protein; and / or(II) the humanized non-human animal SIRPA gene is operably linked to an endogenous non-human animal SIRPA promoter; and / or(III) the genetically modified non-human animal, non-human animal cell, or non-human animal genome is heterozygous for the humanized SIRPA gene; and / or(IV) the genetically modified non-human animal, non-human animal cell, or non-human animal genome is homozygous for the humanized SIRPA gene; and / or(V) the genetically modified non-human animal comprises the humanized SIRPA gene in its germline.

201. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 185-200, wherein the genetically modified non- human animal, non-human animal cell, or non-human animal genome is male.

202. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 185-200, wherein the genetically modified non- human animal, non-human animal cell, or non-human animal genome is female.

203. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of any one of claims 185-202, wherein the non-human animal is a mammal.

204. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of claim 203, wherein the mammal is a rodent.

205. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of claim 204, wherein the rodent is a rat or a mouse.

206. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of claim 205, wherein the rodent is the rat.

207. The genetically modified non-human animal, non-human animal cell, or non-human animal genome of claim 205, wherein the rodent is the mouse.

208. A method of making the genetically modified non-human animal of any one of claims 185-207, comprising:(a) introducing a genetically modified non-human animal embryonic stem (ES) cell comprising: (1) a humanized HGF gene comprising a human HGF nucleic acid encoding a human HGF protein; and (2) one or more genetic modifications causing immunodeficiency into a non-human animal host embryo; and(b) implanting and gestating the non-human animal host embryo in a non- human animal surrogate mother, wherein the non-human animal surrogate mother produces an F0 progeny genetically modified non-human animal comprising: (1) the humanized HGF gene; and (2) the one or more genetic modifications causing immunodeficiency.

209. The method of claim 208, further comprising modifying a non-human animal ES cell to generate the genetically modified non-human animal ES cell comprising: (1) the humanized HGF gene; and (2) the one or more genetic modifications causing immunodeficiency prior to step (a).

210. A method of making the genetically modified non-human animal of any one of claims 185-207, comprising implanting and gestating a genetically modified non-humananimal one-cell stage embryo comprising: (1) a humanized HGF gene comprising a human HGF nucleic acid encoding a human HGF protein; and (2) one or more genetic modifications causing immunodeficiency in a non-human animal surrogate mother, wherein the non-human animal surrogate mother produces an FO progeny genetically modified non-human animal comprising: (1) the humanized HGF gene; and (2) the one or more genetic modifications causing immunodeficiency.

211. The method of claim 210, further comprising modifying a non-human animal one-cell stage embryo to generate the genetically modified non-human animal one-cell stage embryo comprising: (1) the humanized HGF gene; and (2) the one or more genetic modifications causing immunodeficiency gene prior to gestating the genetically modified non- human animal one-cell stage embryo in the non-human animal surrogate mother.

212. A method of making a non-human animal with a humanized liver, comprising:(a) transplanting human hepatocytes or human hepatocyte progenitors into the genetically modified non-human animal of any one of claims 185-207; and(b) allowing the human hepatocytes or human hepatocyte progenitors to expand.

213. The method of claim 212, wherein exogenous urokinase plasminogen activator or an exogenous nucleic acid encoding urokinase plasminogen activator is administered to the genetically modified non-human animal prior to step (a) to prime the liver for improved repopulation by human hepatocytes, optionally wherein the exogenous nucleic acid is an adenovirus or adeno-associated virus (AAV) encoding urokinase plasminogen activator.

214. The method of claim 212 or 213, wherein the human hepatocytes or human hepatocyte progenitors are injected into the genetically modified non-human animal intrasplenically in step (a).

215. The method of any one of claims 212-214, wherein at least about ten million human hepatocytes or human hepatocyte progenitors are transplanted into the genetically modified non-human animal in step (a).

216. The method of any one of claims 212-21 , wherein step (a) is done in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency.

217. The method of any one of claims 212-216, wherein some or all of step (b) is done in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency.

218. The method of claim 217, wherein nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency is administered to the genetically modified nonhuman animal in step (b) in an on / off cycle to promote human hepatocyte repopulation.

219. The method of claim 218, wherein the on / off cycle comprises about 5 to about 7 days off and about 3 days on.

220. A method of increasing or accelerating engraftment of transplanted human hepatocytes in a non-human animal, comprising: (1) administering a GP130-activating ligand or a nucleic acid encoding the GP130-activating ligand to the non-human animal, wherein the GP130-activating ligand restores or increases interleukin-6 (IL-6) / interleukin-6 receptor (IL-6R) signaling pathway activity or interleukin-6 receptor subunit beta (GP130) signaling pathway activity in the transplanted human hepatocytes; and / or (2) administering a hepatocyte growth factor receptor (MET)-activating ligand or a nucleic acid encoding the MET-activating ligand to the non-human animal, wherein the MET-activating ligand restores or increases hepatocyte growth factor (HGF) / MET signaling pathway activity in the transplanted hepatocytes.

221. The method of claim 220, comprising: (1) administering the GP130- activating ligand or the nucleic acid encoding the GP130-activating ligand to the non-human animal; and (2) administering the MET-activating ligand or the nucleic acid encoding the MET- activating ligand to the non-human animal.

222. The method of claim 220 or 221, wherein the GP130-activating ligand comprises a human IL-6R agonist antigen-binding protein or a human OSMR agonist antigenbinding protein, optionally wherein the antigen-binding protein is an antibody.

223. The method of claim 220 or 221, wherein:(I) the GP130-activating ligand comprises human IL-6 or a human-IL-6R- compatible ligand or a human-IL-6R-compatible IL-6; or(II) the GP130-activating ligand comprises human oncostatin-M (OSM) or a human-OSMR-compatible ligand or a human-0 SMR-compatible OSM.

224. The method of any one of claims 220-223, wherein the method further comprises administering one or more additional GP130-activating ligands or one or more nucleic acids encoding the one or more additional GP130-activating ligands, optionally wherein the GP130-activating ligands comprise: (1) human IL-6 or a human-IL-6R-compatible ligand or a human-IL-6R-compatible IL-6; and (2) human OSM or a human-OSMR-compatible ligand or a human-OSMR-compatible OSM.

225. The method of any one of claims 220-224, wherein the method comprises administering a vector comprising an expression construct for the GP130-activating ligand comprising a nucleic acid encoding the GP130-activating ligand operably linked to a promoter.

226. The method of claim 225, wherein the vector is administered to muscle cells or liver cells.

227. The method of claim 225 or 226, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

228. The method of any one of claims 225-227, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

229. The method of any one of claims 220-224, wherein the method comprises administering the GP130-activating ligand, optionally wherein the GP130-activating ligand is administered to the liver of the non-human animal.

230. The method of any one of claims 220-229, wherein the MET-activating ligand comprises a human MET agonist antigen-binding protein, optionally wherein the antigenbinding protein is an antibody.

231. The method of any one of claims 220-229, wherein the MET-activating ligand comprises human HGF or a human-MET-compatible ligand or a human-MET-compatible HGF.

232. The method of any one of claims 220-231, wherein the method comprises administering a vector comprising an expression construct for the MET-activating ligand comprising a nucleic acid encoding the MET-activating ligand operably linked to a promoter.

233. The method of claim 232, wherein the vector is administered to muscle cells or liver cells.

234. The method of claim 232 or 233, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

235. The method of any one of claims 232-234, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

236. The method of any one of claims 220-231, wherein the method comprises administering the MET-activating ligand, optionally wherein the MET-activating ligand is administered to the liver of the non-human animal.

237. The method of any one of claims 220-236, wherein the non-human animal is a male.

238. The method of any one of claims 220-236, wherein the non-human animal is a female.

239. The method of any one of claims 220-238, wherein the non-human animal is a mammal.

240. The method of claim 239, wherein the mammal is a rodent.

241. The method of claim 240, wherein the rodent is a rat or a mouse.

242. The method of claim 241, wherein the rodent is the rat.

243. The method of claim 241, wherein the rodent is the mouse.

244. The method of any one of claims 220-243, wherein the non-human animal is immunodeficient.

245. The method of any one of claims 220-244, wherein:(I) the non-human animal comprises an inactivated endogenous Il2rg gene;(II) the non-human animal comprises an inactivated endogenous Ragl gene or an inactivated endogenous Rag2 gene, optionally wherein the non-human animal comprises the inactivated endogenous Rag2 gene;(III) the non-human animal comprises an inactivated endogenous Ragl gene and an inactivated endogenous Rag2 gene;(IV) the non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the non-human animal further comprises an inactivated endogenous Ragl gene;(V) the non-human animal comprises a severe combined immunodeficiency (SCID) mutation in a Prkdc gene (Prkdcsc’dy, or(VI) the non-human animal comprises a SCID mutation in a Prkdc gene (Prkdcsad) and an inactivated endogenous Il2rg gene.

246. The method of any one of claims 220-245, wherein the non-human animal is genetically modified so that endogenous non-human animal hepatocytes in the liver can be selectively and conditionally ablated.

247. The method of claim 246, wherein:(I) the non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter;(II) the non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter; or(III) the non-human animal comprises an inactivated endogenous Fah gene.

248. The method of any one of claims 220-247, wherein:(I) the non-human animal comprises a severe combined immunodeficiency (SCID) mutation in zv Prkdc gene (Prkdcsc,dand the non-human animal comprises a urokinase type plasminogen activator coding sequence operably linked to a liver-specific promoter;(II) the non-human animal comprises a SCID mutation in a Prkdc gene (Prkdcscld)' and an inactivated endogenous Il2rg gene, and the non-human animal comprises a herpes simplex virus type 1 thymidine kinase (HSVtk) coding sequence operably linked to a liver-specific promoter; or(III) the non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene, optionally wherein the non-human animal further comprises an inactivated endogenous Ragl gene, and the non-human animal comprises an inactivated endogenous Fah gene.

249. The method of any one of claims 220-248, wherein the non-human animal comprises an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, and an inactivated endogenous Fah gene, optionally wherein the non-human animal comprises an inactivated endogenous Ragl gene.

250. The method of any one of claims 220-249, wherein:(I) the non-human animal comprises a humanized non-human animal IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein;(II) the non-human animal comprises a humanized non-human animal OSM gene comprising a human OSM nucleic acid encoding a human OSM protein; or(III) the non-human animal comprises a humanized non-human animal IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein, and wherein the non-human animal comprises a humanized non-human animal OS gene comprising a human OSM nucleic acid encoding a human OSM protein.

251. The method of claim 250, wherein:(I) the human nucleic acid comprises a region of human IL6 genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human IL6 complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal IL6 gene; and / or(III) the human nucleic acid is inserted into the non-human animal IL6 gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal IL6 promoter; and / or(V) the non-human animal is heterozygous for the humanized IL6 gene; and / or(VI) the non-human animal is homozygous for the humanized IL6 gene; and / or(VII) the non-human animal comprises the humanized IL6 gene in its germline.

252. The method of claim 250 or 251, wherein:(I) the human nucleic acid comprises a region of human OSM genomic sequence from the start codon to the stop codon or the human nucleic acid comprises a human OSM complementary DNA(cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal (AS gene; and / or(III) the human nucleic acid is inserted into the non-human animal OSM gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal OSM promoter; and / or(V) the non-human animal is heterozygous for the humanized OSM gene; and / or(VI) the non-human animal is homozygous for the humanized OSM gene; and / or(VII) the non-human animal comprises the humanized OSM gene in its germline.

253. The method of any one of claims 220-252, wherein the non-human animal comprises a humanized non-human animal HGF gene comprising a human HGF nucleic acid encoding a human HGF protein.

254. The method of claim 253, wherein:(I) the human nucleic acid comprises a region of human HGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human HGF complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal HGF gene; and / or(III) the human nucleic acid is inserted into the non-human animal HGF gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal HGF promoter; and / or(V) the non-human animal is heterozygous for the humanized HGF gene; and / or(VI) the non-human animal is homozygous for the humanized HGF gene; and / or(VII) the non-human animal comprises the humanized HGF gene in its germline.

255. The method of any one of claims 220-254, wherein the non-human animal comprises a humanized non-human animal SIRPA gene.

256. The method of claim 255, wherein:(I) the humanized non-human animal SIRPA gene comprises a replacement of exons 2-4 of the non-human animal SIRPA gene with exons 2-4 of human SIRPA, wherein the humanized non-human animal SIRPA gene encodes a chimeric SIRPA protein comprising an extracellular portion of a human SIRPA protein and an intracellular portion of a non-human animal SIRPA protein; and / or(II) the humanized non-human animal SIRPA gene is operably linked to an endogenous non-human animal SIRPA promoter; and / or(III) the non-human animal is heterozygous for the humanized SIRPA gene; and / or(IV) the non-human animal is homozygous for the humanized SIRPA gene; and / or(V) the non-human animal comprises the humanized SIRPA gene in its germline.

257. The method of any one of claims 220-256, further comprising administering an EGFR-activating ligand or a nucleic acid encoding the EGFR-activating ligand to the non-human animal.

258. The method of claim 257, wherein the EGFR-activating ligand comprises a human EGFR agonist antigen-binding protein, optionally wherein the antigen-binding protein is an antibody.

259. The method of claim 257, wherein the EGFR-activating ligand comprises human EGF or a human-EGFR-compatible ligand or a human-EGFR-compatible EGF.

260. The method of any one of claims 257-259, wherein the method comprises administering a vector comprising an expression construct for the EGFR-activating ligand comprising a nucleic acid encoding the EGFR-activating ligand operably linked to a promoter.

261. The method of claim 260, wherein the vector is administered to muscle cells or liver cells.

262. The method of claim 260 or 261, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

263. The method of any one of claims 260-262, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

264. The method of any one of claims 257-259, wherein the method comprises administering the EGFR-activating ligand, optionally wherein the EGFR-activating ligand is administered to the liver of the non-human animal.

265. The method of any one of claims 220-264, wherein the non-human animal comprises a humanized non-human animal EGF gene comprising a human EGF nucleic acid encoding a human EGF protein.

266. The method of claim 265, wherein:(I) the human nucleic acid comprises a region of human EGF genomic sequence from the start codon to the stop codon or wherein the human nucleic acid comprises a human EGF complementary DNA (cDNA); and / or(II) the human nucleic acid replaces a corresponding region of the non-human animal EGF gene; and / or(III) the human nucleic acid is inserted into the non-human animal EGF gene; and / or(IV) the human nucleic acid is operably linked to an endogenous non-human animal EGF promoter; and / or(V) the non-human animal is heterozygous for the humanized EGF gene; and / or(VI) the non-human animal is homozygous for the humanized EGF gene; and / or(VII) the non-human animal comprises the humanized EGF gene in its germline.

267. The method of any one of claims 220-266, further comprising administering to the non-human animal human R-spondin-3 (RSPO3) or a nucleic acid encoding human RSPO3.

268. The method of claim 267, comprising administering a vector comprising an expression construct for the human RSPO3 comprising a nucleic acid encoding the human RSPO3 operably linked to a promoter.

269. The method of claim 268, wherein the vector is administered to muscle cells or liver cells.

270. The method of claim 268 or 269, wherein:(I) the promoter is a tissue-specific promoter, optionally wherein the tissuespecific promoter is a muscle-specific promoter or a liver-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7); or(II) the promoter is a constitutive promoter.

271. The method of any one of claims 268-270, wherein the vector is a viral vector, optionally wherein the viral vector is a lentivirus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is a recombinant AAV9 vector or a recombinant AAV8 vector.

272. The method of claim 267, wherein the method comprises administering the human RSPO3, optionally wherein the human RSPO3 is administered to the liver of the nonhuman animal.

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