Rodents expressing humanized polymeric immunoglobulin receptor

Abstract

Provided herein are genetically modified rodents (e.g., mice) comprising a humanized PIGR locus in their genome, for example, positioned at an endogenous mouse PIGR locus. Also provided herein are methods of making and / or using such rodents, cells from such rodents, and rodent ES cells comprising a humanized PIGR locus.

Description

RODENTS EXPRESSING HUMANIZED POLYMERIC IMMUNOGLOBULIN RECEPTORCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Application No.63 / 714,592, filed October 31, 2024, which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been transmitted in electronic form in an XML format and is hereby incorporated by reference in its entirety. Said XML format, created on October 6, 2025, is identified as RPB-03025_SL.xml and is 116,283 bytes in size.BACKGROUND

[0003] Mice are an essential in vivo model for preclinical testing of therapeutic agents due to their small size, well-characterized physiology, and relatively inexpensive maintenance compared to larger mammalian models (e.g., primates). Despite these advantages, there are serious drawbacks to in vivo testing of antibodies and Fc fusion proteins as these therapeutic agents can behave differently in mice than they do in humans. There is an ongoing need for improved humanized mouse models.SUMMARY

[0004] The present disclosure relates to genetically modified rodents (e.g., mice or rats) comprising a humanized polymeric immunoglobulin receptor (PIGR) locus. Provided herein are methods and compositions related to rodents (e.g., mice or rats) that express a human or humanized plgR, as well as cells from such rodents, and methods and uses thereof.

[0005] In some embodiments, provided is a genetically modified rodent (e.g., mouse or rat) whose genome comprises a humanized polymeric immunoglobulin receptor (PIGR) locus encoding a plgR polypeptide comprising human plgR Ig domains 1-5.

[0006] In some embodiments, provided is a cell isolated from a genetically modified rodent as described herein. In some embodiments, provided is an isolated rodent cell that comprises in its genome a humanized PIGR gene encoding a plgR polypeptide comprising human plgR Ig domains 1-5.

[0007] In some embodiments, the humanized PIGR locus encodes a fully human plgR signal peptide. In some embodiments, the humanized PIGR locus encodes a fully rodent plgR signal peptide (e.g., fully mouse or fully rat plgR signal peptide). In some embodiments, the humanized PIGR locus encodes an at least partially rodent plgR signal peptide (e.g., mouse or rat plgR signal peptide). In some embodiments, the humanized PIGR locus encodes a rodent-human chimeric plgR signal peptide. In some embodiments, a rodent-human chimeric plgR signal peptide is about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% human. In some embodiments, a rodent-human chimeric plgR signal peptide is about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% rodent.

[0008] In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising a human plgR cleavage region or a rodent plgR cleavage region (e.g., mouse or rat plgR cleavage region). In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising a human plgR transmembrane domain or rodent plgR transmembrane domain (e.g., mouse or rat plgR transmembrane domain). In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising a human plgR cleavage region and a human plgR transmembrane domain.

[0009] In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising a human plgR cytoplasmic domain, rodent plgR cytoplasmic domain (e.g., mouse or rat plgR cytoplasmic domain), or human-rodent chimeric plgR cytoplasmic domain. In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an at least partially human plgR cytoplasmic domain. In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising a human plgR cytoplasmic domain. In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising a humanrodent chimeric plgR cytoplasmic domain.

[0010] In some embodiments, the humanized PIGR locus comprises rodent plgR exon 1 and rodent plgR exon 2. In some embodiments, the humanized PIGR locus comprises a rodent-human chimeric plgR exon 3. In some embodiments, the humanized PIGR locus comprises a human plgR exon 3. In some embodiments, the humanized PIGR locus comprises human plgR exons 4-6. In some embodiments, the humanized PIGR locus comprises human plgR exons 7-8. In some embodiments, the humanized PIGR locus comprises rodent plgR exons 9-11. In some embodiments, the humanized PIGR locus comprises human plgR exons 9-11.

[0011] In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that is at least 95% identical to any one of the sequences listed in Table 1. In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence of any one of the sequences listed in Table 1.

[0012] In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a signal peptide sequence of SEQ ID NO: 1 or 2. In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a plgR Ig domain sequence of one, two, three, four, or all five of SEQ ID NOs: 3-7. In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a transmembrane domain of SEQ ID NO: 8 or 9. In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a cytoplasmic domain of any one of SEQ ID NOs: 10-12.

[0013] In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13 or 14.

[0014] In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 95% identical to any one of the sequences listed in Table 2. In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence of any one of the sequences listed in Table 2.

[0015] In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an exon 1 sequence of SEQ ID NO: 15. In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an exon 2 sequence of SEQ ID NO: 16. In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an exon 3 sequence of any one of SEQ ID NOs: 17-19. In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an exon 4 sequence of SEQ ID NO: 20. In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an exon 5 sequence of SEQ ID NO:21. In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an exon 6 sequence of SEQ ID NO: 22. In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an exon 7 sequence of SEQ ID NO: 23 or 24. In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an exon 8 sequence of SEQ ID NO: 25 or 26. In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an exon 9 sequence of SEQ ID NO: 27 or 28. In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an exon 10 sequence of SEQ ID NO: 29 or 30. In some embodiments, the humanized PIGR locus comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an exon 11 sequence of SEQ ID NO: 31 or 32.

[0016] In some embodiments, the humanized PIGR locus comprises a genomic sequence found between coordinates 130,826,684 and 130,841,507 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,931,955 and 206,939,452 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,849,332 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly). In some embodiments, the humanized PIGR locus comprises a genomic sequence found between coordinates 130,826,684 and 130,841,507 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,931,955 and 206,939,452 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,849,332 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly).

[0017] In some embodiments, the humanized PIGR locus is at an endogenous PIGR locus. In some embodiments, the humanized PIGR locus is under the control of a PIGR promoter. In some embodiments, the humanized PIGR locus is under the control of an endogenous rodent PIGR promoter or a human PIGR promoter.

[0018] In some embodiments, the genome of the rodent or rodent cell does not encode a functional rodent plgR polypeptide. In some embodiments, at least part of the endogenous rodent PIGR locus has been deleted. In some embodiments, the humanized PIGR locus replaces all or part of the endogenous rodent PIGR locus.

[0019] In some embodiments, a provided genetically modified rodent or rodent cell further comprises in its genome a human or humanized Fc alpha receptor (FcaR) locus. In some embodiments, the FcaR locus is positioned in the rodent leukocyte receptor complex (LRC), wherein the FcaR locus comprises a nucleic acid sequence encoding a FcaR polypeptide comprising a human extracellular domain and a human or rodent cytoplasmic domain. In some embodiments, the FcaR locus is positioned in an intergenic region between the gene loci for the Tthyl protein and the Rdhl3 protein. In some embodiments, the FcaR locus is positioned in an intergenic region between the gene loci for the Lilra5 protein and the Gp6 protein. In some embodiments, the FcaR locus is positioned in an intergenic region between the gene loci for the Pira6 protein and the Ncrl protein, such as between coding nucleic acid sequences for the Pira6 protein and the Ncrl protein. In some embodiments, the FcaR locus comprises a nucleic acid sequence encoding a human FcaR polypeptide. In some embodiments, the FcaR locus comprises human exons 1-5 of the human Fc alpha receptor gene. In some embodiments, the FcaR locus comprises a non-coding portion of non-mouse rodent FcaR exon 1, a coding portion of human FcaR exons 1 and 2, human FcaR exons 3 and 4, and non-mouse rodent FcaR exon 5. In some embodiments, the human or humanized FcaR receptor locus comprises a genomic sequence found between coordinates 54,862,297 and 54,906,185 on human chromosome 19 (+ strand, GRCh38 assembly). In some embodiments, the FcaR locus further comprises a nucleic acid sequence present in a human KIR3DL2 gene, and / or nucleic acid sequence present in the 5’UTR of the human NCR1 gene. In some embodiments, the rodent expresses the FcaR polypeptide on neutrophils, monocytes, macrophages, eosinophils, and dendritic cells (e.g., plasmacytoid dendritic cells).

[0020] In some embodiments, the rodent (e.g., mouse) or rodent cell (e.g., mouse cell) comprises in its genome a human or humanized PIGR locus, a human or humanized FcaR locus, and one or more of: a human or humanized Fc gamma receptor (FcyR) locus, a human or humanized IgH locus, a human or humanized IgK locus, a human or humanized IgA locus, a human or humanized FcRn locus, a human or humanized [32M locus, and / or a human or humanized FcaR la locus.

[0021] In some embodiments, a provided genetically modified rodent (e.g., mouse) is immunodeficient. In some embodiments, the genome of a genetically modified rodent or rodent cell provided herein further comprises (i) a Rag2 gene knock-out, (ii) a IL2rg gene knock-out; and / or (iii) a human or humanized SIRPa knock-in. In some embodiments, the genome of a genetically modified rodent or rodent cell comprises a human or humanizedSIRPa knock-in, and expresses a human or humanized SIRPa protein encoded by a nucleic acid operably linked to a SIRPa promoter. In some embodiments, the genome of a genetically modified rodent or rodent cell comprises a human or humanized SIRPa knock-in, and the rodent or rodent cell expresses a humanized SIRPa protein, wherein the humanized SIRPa protein comprises a functional fragment of a full-length human SIRPa protein human or humanized SIRPa protein (e.g., an extracellular domain of a human SIRPa protein). In some embodiments, the genome of a genetically modified rodent or rodent cell comprises a human or humanized SIRPa knock-in, and the rodent or rodent cell expresses a humanized SIRPa protein that comprises an extracellular portion of a human SIRPa protein and an intracellular portion of an endogenous rodent SIRPa protein.

[0022] In some embodiments, a provided genetically modified rodent or rodent cell expresses a human or humanized thrombopoietin (TPO). In some embodiments, the genome of a genetically modified rodent or rodent cell provided herein comprises a human or humanized TPO gene operably linked to a TPO promoter. In some embodiments, a provided genetically modified rodent or rodent cell expresses a human or humanized GM-CSF. In some embodiments, the genome of a genetically modified rodent or rodent cell provided herein comprises a human or humanized GM-CSF gene operably linked to a GM-CSF promoter. In some embodiments, a provided genetically modified rodent or rodent cell expresses a human or humanized interleukin 3 (IL3). In some embodiments, the genome of a genetically modified rodent or rodent cell provided herein comprises a human or humanized IL3 gene operably linked to an IL3 promoter. In some embodiments, a provided genetically modified rodent or rodent cell expresses a human or humanized interleukin 15 (IL15). In some embodiments, the genome of a genetically modified rodent or rodent cell provided herein comprises a human or humanized IL15 gene operably linked to an IL15 promoter. In some embodiments, a provided genetically modified rodent or rodent cell expresses a human or humanized M-CSF. In some embodiments, the genome of a genetically modified rodent or rodent cell provided herein comprises a human or humanized M-CSF gene operably linked to a M-CSF promoter. In some embodiments, a provided genetically modified rodent or rodent cell expresses a human or humanized CD47. In some embodiments, the genome of a genetically modified rodent or rodent cell provided herein comprises a human or humanized CD47 gene operably linked to a CD47 promoter. In some embodiments, a provided genetically modified rodent or rodent cell expresses a human or humanized Erythropoietin (EPO). In some embodiments, the genome of a genetically modified rodent or rodent cellprovided herein comprises a human or humanized EPO gene operably linked to an EPO promoter. In some embodiments, the genome of a genetically modified rodent or rodent cell comprises a null mutation in at least one corresponding rodent gene at the corresponding rodent gene locus. In some embodiments, the nucleic acids that encode the human or humanized proteins replace the corresponding endogenous rodent gene.

[0023] In some embodiments, a provided genetically modified rodent or rodent cell comprises in its genome an immunoglobulin heavy chain variable region comprising one or more human heavy chain V gene segments, one or more human D gene segments, and one or more human heavy chain J gene segments, wherein the immunoglobulin heavy chain variable region is operably linked to a heavy chain constant region. In some embodiments, the heavy chain constant region is a human heavy chain constant region. In some embodiments, the heavy chain constant region is a rodent heavy chain constant region. In some embodiments, the heavy chain constant region is an endogenous rodent heavy chain constant region.

[0024] In some embodiments, a provided genetically modified rodent or rodent cell comprises in its genome an immunoglobulin light chain variable region comprising one or more unrearranged human VL gene segments and one or more unrearranged human JL gene segments that are operably linked to one or more immunoglobulin constant region genes. In some embodiments, the immunoglobulin light chain variable region comprises one or more human VK gene segments, and one or more human JK gene segments. In some embodiments, the immunoglobulin light chain variable region comprises one or more human VI gene segments, and one or more human Jl gene segments. In some embodiments, the one or more immunoglobulin constant region genes are human immunoglobulin constant region genes. In some embodiments, the one or more immunoglobulin constant region genes are rodent immunoglobulin constant region genes. In some embodiments, the one or more immunoglobulin constant region genes are endogenous rodent immunoglobulin constant region genes.

[0025] In some embodiments, a provided genetically modified rodent is a rat. In some embodiments, a provided genetically modified rodent is a mouse.

[0026] In some embodiments, a provided genetically modified rodent cell is a rat cell. In some embodiments, a provided genetically modified rodent cell is a mouse cell.

[0027] In some embodiments, a provided genetically modified rodent cell is an ES cell (e.g., a rat or mouse ES cell). In some embodiments, a provided genetically modified rodent cell is a mouse ES cell.

[0028] In some embodiments, provided herein is a method of making a genetically modified rodent (e.g., mouse) as described herein. In some embodiments, provided is a method of modifying a rodent genome (e.g. mouse genome), the method comprising inserting a humanized polymeric immunoglobulin receptor (PIGR) locus into an endogenous PIGR locus in the rodent genome, thereby modifying the rodent genome.

[0029] In some embodiments, provided herein is a method of making a rodent (e.g., mouse) comprising a humanized PIGR locus, the method comprising: obtaining or generating a rodent ES cell as described herein, and generating a rodent from said ES cell.

[0030] In some embodiments, provided herein is a method of making a genetically modified rodent ES cell as described herein. In some embodiments, provided is a method of modifying the genome of a rodent ES cell (e.g., mouse ES cell), the method comprising inserting a humanized polymeric immunoglobulin receptor (PIGR) locus into an endogenous PIGR locus in the genome, thereby modifying the rodent ES cell genome. In some embodiments, the rodent ES cell is a mouse ES cell.

[0031] In some embodiments, provided are uses of a genetically modified rodent as described herein for assessing and / or characterizing delivery of a payload. In some embodiments, provided are uses of a genetically modified rodent as described herein for assessing and / or characterizing delivery of a payload to mucosa. In some embodiments, the mucosa is respiratory mucosa, gastrointestinal mucosa, and / or urogenital mucosa. In some embodiments, the payload conjugated to an agent. In some embodiments, the agent is a PIGR-targeting agent. In some embodiments, a PIGR-targeting agent is a polypeptide that specifically binds PIGR (e.g., an anti-PIGR antibody or antigen binding antibody fragment).

[0032] In some embodiments, provided are uses of a genetically modified rodent as described herein for assessing and / or characterizing transcytosis of a molecule. In some embodiments, the molecule conjugated to an agent. In some embodiments, the molecule is a PIGR-targeting agent. In some embodiments, the molecule is an autoantibody. In some embodiments, the molecule is a human IgA autoantibody and / or a human IgM autoantibody.

[0033] In some embodiments, provided are uses of a genetically modified rodent as described herein for removing autoantibodies from circulation. In some embodiments, autoantibodies to be removed from circulation are human IgA autoantibodies and / or human IgM autoantibodies.

[0034] In some embodiments, provided are uses of a genetically modified rodent as described herein for assessing and / or characterizing delivery of a cell therapy to a target celland / or tissue. In some embodiments, provided are uses of a genetically modified rodent as described herein for assessing and / or characterizing delivery of a cell therapy to mucosa. In some embodiments, the mucosa is respiratory mucosa, gastrointestinal mucosa, and / or urogenital mucosa. In some embodiments, the cell therapy is chimeric antigen receptor (CAR)-T cell therapy. In some embodiments, the cell therapy is CAR-T regulatory (Treg) cell therapy.

[0035] In some embodiments, provided are uses of a genetically modified rodent as described herein assessing and / or characterizing targeted immune modulation.

[0036] In some embodiments, provided are uses of a genetically modified rodent as described herein for testing an agent. In some embodiments, the agent is or comprises an IgA antibody or Fea fusion polypeptide. In some embodiments, the agent is or comprises a human IgA antibody or human Fea fusion polypeptide. In some embodiments, the use comprises measuring the immune response generated by the mouse against the IgA antibody or Fea fusion polypeptide. In some embodiments, the use comprises measuring the therapeutic efficacy of the administered human antibody or Fea fusion polypeptide.

[0037] In some embodiments, provided are methods of testing a human IgA antibody or an Fea fusion polypeptide, comprising administering an IgA antibody or Fea fusion polypeptide to a rodent as described herein. In some embodiments, provide methods comprise measuring the immune response generated by the rodent against the human IgA antibody. In some embodiments, provide methods comprise measuring the therapeutic efficacy of the administered human IgA antibody or Fea fusion polypeptide.

[0038] In some embodiments, provided are methods comprising administering a payload to a rodent as described herein, and assessing delivery of the payload to the mucosa of the rodent. In some embodiments, the mucosa is respiratory mucosa, gastrointestinal mucosa, and / or urogenital mucosa. In some embodiments, the payload conjugated to an agent. In some embodiments, the agent is a PIGR-targeting agent. In some embodiments, a PIGR- targeting agent is a polypeptide that specifically binds PIGR (e.g., an anti-PIGR antibody or antigen binding antibody fragment).

[0039] In some embodiments, provided are methods comprising administering a cell therapy to a rodent as described herein; and assessing delivery of the cell therapy to the mucosa of the rodent. In some embodiments, the mucosa is respiratory mucosa, gastrointestinal mucosa, and / or urogenital mucosa. In some embodiments, the cell therapy is CAR-T cell therapy. In some embodiments, the cell therapy is CAR-Treg cell therapy.

[0040] These and other aspects of the invention(s), are described in more detail below and in the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The Drawings included herein is for illustration purposes only and not for limitation.

[0042] FIG. 1A provides schematics of human PIGR gene (top), with representation of the PIGR exons within the gene (middle) and corresponding encoded plgR polypeptide regions (plgR protein, bottom).

[0043] FIG. IB is a schematic of polypeptide domains in a human plgR protein (top) and their concordance with PIGR exons (bottom). From left to right are shown human plgR protein domains: Igl domain (Igl), Ig2 domain (Ig2), Ig3 domain (Ig3), Ig4 domain (Ig4), Ig5 domain (Ig5), transmembrane domain (TM), and cytoplasmic domain, which are encoded by exons 2 to 11 (shown below).

[0044] FIG. 1C provides an annotated alignment of example human and mouse plgR protein sequences. FIG. 1C discloses SEQ. ID. NOs.: 59-60, corresponding to human PIGR protein and mouse PIGR protein, respectively.

[0045] FIG. 2A is a diagram (not to scale) showing construction of an embodiment of a humanized PIGR locus (version 1) in a mouse genome. The resulting version 1 humanized PIGR locus includes mouse exons 1 and 2, a mouse-human chimeric exon 3, human exons 4-8, and mouse exons 9-11. Bacterial homologous recombination (BHR); Ampicillin resistance cassette (Amp); Kanamycin resistance cassette (Kan); Hygromycin resistance cassette (Hyg); Chloramphenicol resistance cassette (CM); Topoisomerase-based cloning (TOPO cloning). Exon regions outlined in bold represent protein encoding regions, with thick bold lines representing protein-encoding regions of exons of human origin.

[0046] FIG. 2B is a diagram (not to scale) depicting a selection cassette swap in an embodiment of a humanized PIGR locus of FIG. 2A. Hygromycin resistance cassette (Hyg); chloramphenicol resistance cassette (CM); Neomycin resistance cassette (Neo). Exon regions outlined in bold represent protein encoding regions, with thick bold lines representing protein-encoding regions of exons of human origin.

[0047] FIG. 2C is a schematic of an example plan for evaluating targeting of additional loci on the same chromosome. Cre may be used to collapse locus and confirm same chromosome targeting, then Rox followed by Cre for cassette removal. Exon regionsoutlined in bold represent protein encoding regions, with thick bold lines representing protein-encoding regions of exons of human origin.

[0048] FIG. 3A is a diagram (not to scale) showing construction of another embodiment of a humanized PIGR locus (version 2) in a mouse genome. The resulting version 2 humanized PIGR locus includes mouse exons 1 and 2, human exons 3-11, and an additional mouse exon 11. Hygromycin resistance cassette (Hyg); Spectinomycin resistance cassette (Spec); Chloramphenicol resistance cassette (CM); Topoisomerase-based cloning (TOPO cloning); Bacterial homologous recombination (BHR). Exon regions outlined in bold represent protein encoding regions, with thick bold lines representing protein-encoding regions of exons of human origin.

[0049] FIG. 3B is a diagram (not to scale) showing an example cloning strategy that includes deletion of a portion of an endogenous mouse PIGR locus. Neomycin resistance cassette (Neo); Chloramphenicol resistance cassette (CM); Bacterial homologous recombination (BHR). Exon regions outlined in bold represent protein encoding regions, with thick bold lines representing protein-encoding regions of exons of human origin.

[0050] FIG. 3C is a diagram (not to scale) depicting an example selection cassette swap in the humanized PIGR locus embodiment depicted in FIG. 3A. Hygromycin resistance cassette (Hyg); Neomycin resistance cassette (Neo); chloramphenicol resistance cassette (CM). Exon regions outlined in bold represent protein encoding regions, with thick bold lines representing protein-encoding regions of exons of human origin.

[0051] FIGs. 4A-4D shows detection of mouse plgR and human plgR in wild type and mice with humanized plgR by Western Blot. FIG. 4A depicts mouse and human plgR protein in WT and example hpIgR+ / _mice was probed with anti-mouse plgR (R& D AF2800) and anti-human plgR antibodies (ThermoScientific: PA- 110769), respectively. FIG. 4B depicts human plgR protein expression in WT and example hpIgR+ / _mice was probed with anti-human plgR antibody (Abeam: ab275020). / / -actin expression (Cell Signaling: 5125S) was used as loading control. FIG. 4C depicts mouse plgR protein in WT and example hpIgR+ / +mice probed with anti-mouse plgR antibody (R& D AF2800). FIG. 4D depicts human plgR protein in WT and example hpIgR+ / +mice probed with two different anti-human plgR antibodies: ThermoScientific: PA-110769 for top panel and Abeam: ab275020 for middle panel. / -actin expression (Cell Signaling: 5125S) was used as loading control, and two biological replicates (#1 and #2) were used in each genotype.DEFINITIONS

[0052] In general, terms used herein are in accordance with their understood meaning in the art, unless clearly indicated otherwise. Explicit definitions of certain terms are provided below; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context. Additional definitions for the following and other terms are set forth throughout the specification. Patent and non-patent literature references cited within this specification, or relevant portions thereof, are incorporated herein by reference in their entireties.

[0053] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0054] The articles “a,” “an,” and “the” as used herein, should be understood to include the plural referents unless clearly indicated to the contrary. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. In some embodiments, exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. In some embodiments, more than one, or all group members are present in, employed in, or otherwise relevant to a given product or process. It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists (e.g., in Markush group or similar format), it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where embodiments or aspects are referred to as “comprising” particular elements, features, etc., provided are embodiments or aspects that “consist,” or “consist essentially of,” such elements, features, etc. For purposes of simplicity, those embodiments have not in every case been specificallyset forth in so many words herein. It should also be understood that any embodiment or aspect can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[0055] Administration: the term “administration” as used herein, includes the administration of a composition (e.g., antigen or antibody) to a subject or system (e.g., to a cell, organ, tissue, organism, or relevant component or set of components thereof). The skilled artisan will appreciate that route of administration may vary depending, for example, on the subject or system to which the composition is being administered, the nature of the composition, the purpose of the administration, etc. For example, in certain embodiments, administration to an animal subject (e.g., to a human or a rodent) may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and / or vitreal. In some embodiments, administration may involve intermittent dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0056] Amino acid: the term “amino acid” is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally- occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing.

[0057] Antibody: the term “antibody” as used herein may refer to both an intact antibody and an antigen binding fragment thereof. Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain includes a heavy chain variable domain and a heavy chain constant domain. Each light chain includes a light chain variable domain and a light chain constant domain. The heavy chain variable domains and light chain variable domains can be further subdivided into three domains of hypervariability, each termed a complementarity determining region (CDR), interspersed with regions that are more conserved, each termed a framework region (FR). Each heavy chain variable domain and light chain variable domain is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminusin the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable domains of the heavy and light chains contain a binding domain that interacts with an antigen.

[0058] Antigen-binding fragment or Antigen-binding portion: the terms “antigen binding fragment” and “antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, single-chain antibodies, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable domain of an intact antibody. These antibody fragments can be obtained using conventional recombinant and / or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.

[0059] Chimeric: as used herein, the term “chimeric” refers to nucleic acids or proteins whose structures (i.e., nucleotide or amino acid sequences) include portions that are from different species. In some embodiments, the “chimeric” nucleic acids or proteins described herein include nucleotide or amino acid sequences that are from both a non-human source and a human. In such embodiments, the “chimeric” nucleic acids or proteins can also be referred to as “humanized” nucleic acids or protein.

[0060] Coding region: as used herein in the context of a gene, includes the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene. A “coding region” of a mRNA molecule also includes the nucleotide residues of the mRNA molecule which are matched with an anti-codon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding region may thus include nucleotide residues comprising codons for amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0061] Correspond to: as used herein, the term “correspond to” refers to exons that encode the same or homologous functional domain or portion of a protein. For example, “exons that correspond to mouse PIGR exons 3-5” refers to the exons from the genetically modified non-human animal that encode the same or homologous functional domain or portion of the protein as encoded by the mouse exons 3-5. These could be exons 3-5 of thegenetically modified non-human animal, or other exons due to the difference in exon configuration among different non-human animal species.

[0062] Derived from: the phrase “derived from” when used concerning a rearranged variable region gene or a variable domain “derived from” an unrearranged variable region and / or unrearranged variable region gene segments refers to the ability to trace the sequence of the rearranged variable region gene or variable domain back to a set of unrearranged variable region gene segments that were rearranged to form the rearranged variable region gene that expresses the variable domain (accounting for, where applicable, splice differences and somatic mutations). For example, a rearranged variable region gene that has undergone somatic mutation does not change the fact that it is derived from the unrearranged variable region gene segments.

[0063] Endogenous gene or Endogenous gene segment: as used herein refer to a gene or gene segment found in a parent or reference organism prior to introduction of a disruption, deletion, replacement, alteration, or modification as described herein. In some embodiments, a reference organism is a wild-type organism. In some embodiments, a reference organism is an engineered organism. In some embodiments, a reference organism is a laboratory-bred organism (whether wild-type or engineered).

[0064] Engineered: as used herein refers, in general, to the aspect of having been manipulated by the hand of man. For example, in some embodiments, a polynucleotide may be considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide. In some embodiments, an engineered polynucleotide may comprise a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence.Alternatively, or additionally, in some embodiments, first and second nucleic acid sequences that each encode polypeptide elements or domains that in nature are not linked to one another may be linked to one another in a single engineered polynucleotide. Comparably, in some embodiments, a cell or organism may be considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, or previously present genetic material has been altered or removed). As is common practice and is understood by persons of skill in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered”even though the actual manipulation was performed on a prior entity. Furthermore, as will be appreciated by persons of skill in the art, a variety of methodologies are available through which “engineering” as described herein may be achieved. For example, in some embodiments, “engineering” may involve selection or design (e.g., of nucleic acid sequences, polypeptide sequences, cells, tissues, and / or organisms) through use of computer systems programmed to perform analysis or comparison, or otherwise to analyze, recommend, and / or select sequences, alterations, etc.). Alternatively, or additionally, in some embodiments, “engineering” may involve use of in vitro chemical synthesis methodologies and / or recombinant nucleic acid technologies such as, for example, nucleic acid amplification (e.g., via the polymerase chain reaction) hybridization, mutation, transformation, transfection, etc., and / or any of a variety of controlled mating methodologies. As will be appreciated by those skilled in the art, a variety of established such techniques (e.g., for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection, etc.)) are well known in the art and described in various general and more specific references that are cited and / or discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989 and Principles of Gene Manipulation: An Introduction to Genetic Manipulation, 5th Ed., ed. By Old, R. W. and S. B. Primrose, Blackwell Science, Inc., 1994, incorporated herein by reference in their entireties.

[0065] Expression: as used herein, the term “expression” of a nucleic acid sequence refers to generation of any gene product from a nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0066] Germline Genome-, as used herein, refers to the genome found in a germ cell (e.g., a gamete, e.g., a sperm or egg) used in the formation of an animal. A germline genome is a source of genomic DNA for cells in an animal. As such, an animal (e.g., a mouse or rat) having a modification in its germline genome is considered to have the modification in the genomic DNA of all of its cells.

[0067] Host cell: as used herein, refers to a cell into which a nucleic acid or protein has been introduced. Persons of skill upon reading this disclosure will understand that such a term refers not only to the particular subject cell, but also is used to refer to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the phrase “host cell.'' In some embodiments, a host cell is or comprises a prokaryotic or eukaryotic cell. In general, a host cell is any cell that is suitable for receiving and / or producing a heterologous nucleic acid or protein, regardless of the Kingdom of life to which the cell is designated. Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of Escherichia coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, a cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, a cell is eukaryotic and is selected from the following cells: Chinese Hamster Ovarian (CHO) (e.g., CHO KI, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2 / 0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell. In some embodiments, a cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER. C6® cell). In some embodiments, a host cell is or comprises an isolated cell. In some embodiments, a host cell is part of a tissue. In some embodiments, a host cell is part of an organism.

[0068] Humanized: is used herein in accordance with its art-understood meaning to refer to nucleic acids or proteins whose structures (i.e., nucleotide or amino acid sequences) include portions that are from a non-human source, which are engineered to have a structure and function more similar to true human nucleic acids or proteins than the original source nucleic acids or proteins. For example, humanizing can involve selecting amino acid substitutions to make a non-human sequence more similar to a human sequence. Humanizing can also involve grafting at least a portion of a non-human protein into a human protein. Togive but one example, in the case of a membrane receptor, a “humanized” gene may encode a polypeptide having an extracellular portion having an amino acid sequence as that of a human extracellular portion and the remaining sequence as that of a non-human (e.g., mouse) polypeptide. In some embodiments, a humanized gene comprises at least a portion of a DNA sequence of a human gene. In some embodiments, a humanized protein comprises a sequence having a portion that appears in a human protein. The term “human” is art recognized, and refers to nucleic acids or proteins whose structures (i.e., nucleotide or amino acid sequences) are entirely from a human source.

[0069] In vitro-. as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0070] In vivo-, as used herein refers to events that occur within a multi-cellular organism, such as a human and / or a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0071] Locus-, as used herein, the term “locus” refers to a location on a chromosome that contains a set of related genetic elements (e.g., genes, gene segments, regulatory elements). A locus can be endogenous or non-endogenous. The term “endogenous locus” refers to a location on a chromosome at which a particular genetic element is naturally found. In some embodiments, an endogenous locus has a sequence found in nature. In some embodiments, an endogenous locus is a wild-type locus. In some embodiments, an endogenous locus is an engineered locus.

[0072] Non-human animal-, as used herein, refers to any vertebrate organism that is not a human. In some embodiments, a non-human animal is a cyclostome, a bony fish, a cartilaginous fish (e.g., a shark or a ray), an amphibian, a reptile, a mammal, and a bird. In some embodiments, a non-human animal is a mammal. In some embodiments, a non-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In some embodiments, a non-human animal is a rodent such as a rat or a mouse.

[0073] Nucleic acid-, as used herein, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a “nucleic acid” is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In someembodiments, “nucleic acid’ refers to an oligonucleotide chain comprising individual nucleic acid residues (alternatively referred to as a polynucleotide). In some embodiments, a “nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid’ is or comprises DNA. In some embodiments, a “nucleic acid’ is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a “nucleic acid’ is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a “nucleic acid’ in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a “nucleic acid’ is, comprises, or consists of one or more “peptide nucleic acids’’, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone. Alternatively, or additionally, in some embodiments, a “nucleic acid’ has one or more phosphorothioate and / or 5’-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a “nucleic acid” is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a “nucleic acid” is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a “nucleic acid” comprises one or more modified sugars (e.g., 2’ -fluororibose, ribose, 2 ’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a “nucleic acid’ has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a “nucleic acid” includes one or more introns. In some embodiments, a “nucleic acid” includes one or more exons. In some embodiments, a “nucleic acid’ is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a “nucleic acid’ is at least, e.g., but not limited to, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a “nucleictzczd” is single stranded; in some embodiments, a “nucleic acid” is double stranded. In some embodiments, a “nucleic acid” has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a “nucleic acid” has enzymatic activity.

[0074] Operably linked', as used herein, refers to a juxtaposition of components, where the components described are in a relationship permitting them to function in their intended manner (e.g., when the components are present in the proper tissue, cell type, cellular activity, etc.). For example, one or more VH gene segments, one or more D gene segments, and one or more JH gene segments are “operably linked” to a heavy chain constant region if the VH, D, and JH gene segments can be spliced to the heavy chain constant region at the proper time in B cell development, regardless of whether such splicing occurs in, e.g., a cell outside the immune system (e.g., a germ cell). A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. “Operably linked” sequences include both expression control sequences that are contiguous with a gene of interest and expression control sequences that act in trans or at a distance to control a gene of interest (or sequence of interest). The term “expression control sequence” includes polynucleotide sequences, which are necessary to affect the expression and processing of coding sequences to which they are ligated. “Expression control sequences” include: appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance polypeptide stability; and when desired, sequences that enhance polypeptide secretion. The nature of such control sequences differs depending upon the host organism. For example, in prokaryotes, such control sequences generally include promoter, ribosomal binding site and transcription termination sequence, while in eukaryotes typically such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0075] Polynucleotide-, as used herein refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and may perform any function.The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0076] Polypeptide or Protein -, as used herein, refers to any polymeric chain of amino acids and encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.). In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that contains portions that occur in nature separately from one another (i.e., from two or more different organisms, for example, human and non-human portions). In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide has an amino acid sequence encoded by a sequence that does not occur in nature (e.g., a sequence that is engineered in that it is designed and / or produced through action of the hand of man to encode said polypeptide).

[0077] Promoter, as used herein includes a DNA sequence operably linked to a nucleic acid sequence to be transcribed such as a nucleic acid sequence encoding a desired molecule. A promoter is generally positioned upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors. In specific embodiments, a promoter is generally positioned upstream of the nucleic acid sequence transcribed to produce the desired molecule, and provides a site for specific binding by RNA polymerase and other transcription factors. The phrase “endogenouspromoter” refers to a promoter that is naturally associated, e.g., in a wild-type organism, with an endogenous gene.

[0078] Replacement', as used herein, refers to a process through which a “replaced” nucleic acid sequence (e.g., a gene) found in a host locus (e.g., in a genome) is removed from that locus, and a different, “replacement” nucleic acid is located in its place. In some embodiments, the replaced nucleic acid sequence and the replacement nucleic acid sequences are comparable to one another in that, for example, they are homologous to one another, contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.), and / or have similar or identical sequences. In some embodiments, a replaced nucleic acid sequence includes one or more of a promoter, an enhancer, a splice donor site, a splice acceptor site, an intron, an exon, an untranslated region (UTR); in some embodiments, a replacement nucleic acid sequence includes one or more coding sequences. In some embodiments, a replacement nucleic acid sequence is a homolog or variant (e.g., mutant) of the replaced nucleic acid sequence. In some embodiments, a replacement nucleic acid sequence is an ortholog or homolog of the replaced sequence. In some embodiments, a replacement nucleic acid sequence is or comprises a human nucleic acid sequence. In some embodiments, including where the replacement nucleic acid sequence is or comprises a human nucleic acid sequence, the replaced nucleic acid sequence is or comprises a rodent sequence (e.g., a mouse or rat sequence). In some embodiments, including where the replacement nucleic acid sequence is or comprises a human nucleic acid sequence, the replaced nucleic acid sequence is or comprises a human sequence. In some embodiments, a replacement nucleic acid sequence is a variant or mutant (i.e., a sequence that contains one or more sequence differences, e.g., substitutions, as compared to the replaced sequence) of the replaced sequence. The nucleic acid sequence so placed may include one or more regulatory sequences that are part of source nucleic acid sequence used to obtain the sequence so placed (e.g., promoters, enhancers, 5'- or 3'-untranslated regions, etc.). For example, in various embodiments, a replacement is a substitution of an endogenous sequence with a heterologous sequence that results in the production of a gene product from the nucleic acid sequence so placed (comprising the heterologous sequence), but not expression of the endogenous sequence; a replacement is of an endogenous genomic sequence with a nucleic acid sequence that encodes a polypeptide that has a similar function as a polypeptide encoded by the endogenous sequence. In various embodiments, an endogenous gene or fragment thereof is replaced with a corresponding human gene or fragment thereof. A corresponding human geneor fragment thereof is a human gene or fragment that is an ortholog of, or is substantially similar or the same in structure and / or function, as the endogenous gene or fragment thereof that is replaced.

[0079] Targeting construct or targeting vector, as used herein, refers to a polynucleotide molecule that comprises a targeting region. A targeting region comprises a sequence that is identical or substantially identical to a sequence in a target cell, tissue or animal and provides for integration of the targeting construct into a position within the genome of the cell, tissue or animal via homologous recombination. Targeting regions that target using site-specific recombinase recognition sites (e.g., loxP or Frt sites) are also included and described herein. In some embodiments, a targeting construct as described herein further comprises a nucleic acid sequence or gene of particular interest, a selectable marker, control and / or regulatory sequences, and other nucleic acid sequences that allow for recombination mediated through exogenous addition of proteins that aid in or facilitate recombination involving such sequences. In some embodiments, a targeting construct as described herein further comprises a gene of interest in whole or in part, wherein the gene of interest is a heterologous gene that encodes a polypeptide, in whole or in part, that has a similar function as a protein encoded by an endogenous sequence. In some embodiments, a targeting construct as described herein further comprises a humanized gene of interest, in whole or in part, wherein the humanized gene of interest encodes a polypeptide, in whole or in part, that has a similar function as a polypeptide encoded by an endogenous sequence. In some embodiments, a targeting construct (or targeting vector) may comprise a nucleic acid sequence manipulated by the hand of man. For example, in some embodiments, a targeting construct (or targeting vector) may be constructed to contain an engineered or recombinant polynucleotide that contains two or more sequences that are not linked together in that order in nature yet manipulated by the hand of man to be directly linked to one another in the engineered or recombinant polynucleotide.

[0080] Transgenic animal, transgenic non-human animal or Tg+: are used interchangeably herein and refer to any non-naturally occurring non-human animal in which one or more of the cells of the non-human animal contain heterologous nucleic acid and / or gene encoding a polypeptide of interest, in whole or in part. In some embodiments, a heterologous nucleic acid sequence and / or gene is introduced into the cell, directly or indirectly by introduction into a precursor cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term geneticmanipulation does not include classic breeding techniques, but rather is directed to introduction of recombinant DNA molecule(s). This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. The term “7 <+” includes animals that are heterozygous or homozygous for a heterologous nucleic acid and / or gene, and / or animals that have single or multi-copies of a heterologous nucleic acid and / or gene.

[0081] Variant', as used herein, includes a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, e.g., due to the degeneracy of the genetic code, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

[0082] Vector, as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. In some embodiments, vectors are capable of extra-chromosomal replication and / or expression of nucleic acids to which they are linked in a host cell such as a eukaryotic and / or prokaryotic cell. Vectors capable of directing the expression of operably linked genes are referred to herein as “expression vectors.

[0083] Wild-type-. as used herein, refers to an entity having a structure and / or activity as found in nature in a “normar (as contrasted with mutant, diseased, altered, engineered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).DETAILED DESCRIPTIONPolymeric Immunoglobulin Receptor

[0084] Human polymeric immunoglobulin receptor (plgR) is a -120 kD type 1 transmembrane protein. Human plgR has been shown to have a role in protein trafficking andtranscytosis. Kaetzel, C. S., (2001) Current Biology, 11(1): R35 - R38, which is incorporated herein by reference in its entirety. For example, plgR is sorted to the basolateral membrane of polarized epithelial cells, undergoes endocytosis through clathrin-coated pits to basolateral early endosomes, and microtubule-dependent translocation to apical recycling endosomes. Human plgR is also involved in delivery from apical endosomes to the plasma membrane.

[0085] During human fetal development and early infancy production of plgR precedes IgA by several months. In humans, plgR is expressed by various secretory epithelial cells, including those lining the gastrointestinal tract and those of mammary glands (e.g., to transport IgA into milk).

[0086] Human and mouse plgR have an extracellular region comprising five Ig homology domains with conserved disulfide bonds, a cleavage region, a transmembrane domain, and a cytoplasmic domain. plgR Ig domains 1, 4 and 5 are highly conserved among species, and contain signals for non-covalent and disulfide bonding to polymeric IgA, while Ig domains 2 and 3 are less conserved, and are absent in some splice variants of rabbit plgR. Kaetzel, C. S., (2001) supra. In humans, the extracellular ligand-binding region of plgR, known as secretory component (SC), is cleaved and released in free form or as a component of secretory IgA (SIgA). Kaetzel, C. S., (2005) Immuno. Rev., 206: 83-99, which is incorporated herein by reference in its entirety.

[0087] In some embodiments, a plgR protein comprises an Igl domain, an Ig2 domain, an Ig3 domain, an Ig4 domain, and an Ig5 domain. In some embodiments, plgR binds to IgA, e.g., dimeric and / or polymeric IgA. In some embodiments, a plgR protein binds to a first IgA through its Igl domain and a second IgA through its Ig5 domain. In some embodiments, binding of IgA to the plgR Igl domain induces a conformational change that then allows binding of a second IgA to a plgR Ig5 domain. plgR binding to IgA may be covalent via disulfide exchange (e.g., Ig5-C468-Fc-C311 a disulfide bond), for example, in transcytosis. In some embodiments, binding may be non-covalent (e.g., to Igl domain).

[0088] Exemplary plgR nucleic acid and amino acid sequences are available to the public at the GenBank database. Exemplary human plgR nucleic acid and amino acid sequences are available to the public at the GenBank database under NM_002644.4 and NP_002635.2. Exemplary murine plgR nucleic acid and amino acid sequences are available to the public at the GenBank database, for example, under NM_011082.3 and NP_035212.2. Exemplary rat plgR nucleic acid and amino acid sequences are available to the public at the GenBank database, for example, under NM_012723.4 and NP_036855.3. In addition, nucleicacid and polypeptide sequences of plgR orthologs in other organisms are known in the art and any of these are also included within the scope of the disclosure.

[0089] In some embodiments, plgR is glycosylated. Without being bound by theory, it is envisioned that plgR glycosylation may prevent proteolysis.Rodents with Humanized plgR

[0090] In some embodiments, provided are genetically modified non-human animals (e.g., rodents) and cells (e.g., ES cells) comprise a humanized PIGR locus. In some embodiments, provided are genetically modified rodents (e.g., mice or rats) and cells (e.g., ES cells) that comprise a humanized PIGR locus.

[0091] In some embodiments provided genetically modified rodents (e.g., mice) comprise a humanized PIGR locus, wherein the encoded polypeptide comprises a fully human plgR signal peptide. In some embodiments, provided genetically modified rodents (e.g., mice) comprise a humanized PIGR locus, wherein the encoded polypeptide comprises an at least partially rodent plgR signal peptide (e.g., mouse plgR signal peptide). In some embodiments, an encoded plgR polypeptide comprises a rodent plgR signal peptide (e.g., mouse plgR signal peptide). In some embodiments, an encoded plgR polypeptide comprises rodent-human chimeric plgR signal peptide. In some embodiments, a rodent-human chimeric plgR signal peptide is about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% human. In some embodiments, a rodent-human chimeric plgR signal peptide is about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% rodent.

[0092] In some embodiments, an encoded plgR polypeptide comprises a human, rodent, or human-rodent chimeric plgR cleavage region. In some embodiments, an encoded plgR polypeptide comprises a human plgR cleavage region. In some embodiments, an encoded plgR polypeptide comprises a rodent plgR cleavage region. In some embodiments, an encoded plgR polypeptide comprises a mouse plgR cleavage region. In some embodiments, an encoded plgR polypeptide comprises a human-mouse chimeric plgR cleavage region. In some embodiments, an encoded plgR polypeptide comprises a rat plgR cleavage region. In some embodiments, an encoded plgR polypeptide comprises a human-rat chimeric plgR cleavage region.

[0093] In some embodiments, an encoded plgR polypeptide comprises a human, rodent, or human-rodent chimeric plgR transmembrane domain. In some embodiments, anencoded plgR polypeptide comprises a human plgR transmembrane domain. In some embodiments, an encoded plgR polypeptide comprises a mouse plgR transmembrane domain. In some embodiments, an encoded plgR polypeptide comprises a human-mouse chimeric plgR transmembrane domain. In some embodiments, an encoded plgR polypeptide comprises a rat plgR transmembrane domain. In some embodiments, an encoded plgR polypeptide comprises a human-rat chimeric plgR transmembrane domain.

[0094] In some embodiments, a rodent-human chimeric plgR transmembrane domain is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% human. In some embodiments, a rodenthuman chimeric plgR transmembrane domain is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% rodent.

[0095] In some embodiments, a rodent-human chimeric plgR transmembrane domain is about 85%, about 90%, or about 95% human. In some embodiments, a rodent- human chimeric plgR transmembrane domain is about 85%, about 90%, or about 95% rodent.

[0096] In some embodiments, an encoded plgR polypeptide comprises a human, rodent, or human-rodent chimeric plgR cytoplasmic domain. In some embodiments, an encoded plgR polypeptide comprises an at least partially human plgR cytoplasmic domain. In some embodiments, an encoded plgR polypeptide comprises a human plgR cytoplasmic domain. In some embodiments, an encoded plgR polypeptide comprises a mouse plgR cytoplasmic domain. In some embodiments, an encoded plgR polypeptide comprises a human-mouse chimeric plgR cytoplasmic domain. In some embodiments, an encoded plgR polypeptide comprises a rat plgR cytoplasmic domain. In some embodiments, an encoded plgR polypeptide comprises a human-rat chimeric plgR cytoplasmic domain.

[0097] In some embodiments, a rodent-human chimeric plgR cytoplasmic domain is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, or about 98% human. In some embodiments, a rodent-human chimeric plgR cytoplasmic domain is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, or about 98% rodent.

[0098] In some embodiments, a rodent-human chimeric plgR cytoplasmic domain is about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, or about 98% human. In some embodiments, a rodent-human chimeric plgR cytoplasmic domain is about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 92%, about 94%, about 96%, or about 98% rodent.

[0099] In some embodiments, humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences listed in Table 1 below.

[0100] In some embodiments, humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that is at least 98% identical to any one of the sequences listed in Table 1 below. In some embodiments, humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that is at least 99% identical to any one of the sequences listed in Table 1 below.

[0101] In some embodiments, a humanized PIGR locus encodes a plgR polypeptide comprising one or more of the sequences listed in Table 1.

[0102] Table 1 - plgR polypeptide sequences

[0103] In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that differs by no more than 3 amino acids, differs by no more than 2 amino acids, or differs by no more than 1 amino acid from a sequence of SEQ ID NO: 1 or 2. In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an Igl domain, Ig2 domain, Ig3 domain, Ig4 domain, and Ig5 domain, wherein the Igl domain comprises an amino acid sequence that differs by no more than 3 amino acids, differs by no more than 2 amino acids, or differs by no more than 1 amino acid from SEQ ID NO: 3; wherein the Ig2 domain comprises an amino acid sequence that differs by no more than 3 amino acids, differs by no more than 2 amino acids, or differs by no more than 1 amino acid from SEQ ID NO: 4; wherein the Ig3 domain comprises an amino acid sequence that differs by no more than 3 amino acids, differs by no more than 2 amino acids, or differs by no more than 1 amino acid from SEQ ID NO: 5; wherein the Ig4 domain comprises an amino acid sequence that differs by no more than 3 amino acids, differs by no more than 2 amino acids, or differs by no more than 1 amino acid from SEQ ID NO: 6; and wherein the Ig5 domain comprises an amino acid sequence that differs by no more than 3 amino acids, differs by no more than 2 amino acids, or differs by no more than 1 amino acid from SEQ ID NO: 7.

[0104] In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence a transmembrane domain that differs by no more than 3 amino acids, differs by no more than 2 amino acids, or differs by no more than 1 amino acid from SEQ ID NO: 8 or 9. In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising a cytoplasmic domain that differs by no more than 3 amino acids, differs by no more than 2 amino acids, or differs by no more than 1 amino acid from any one of SEQ ID NOs: 10-12.

[0105] In some embodiments, the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that that differs by no more than 5 amino acids, differs by no more than 4 amino acids, differs by no more than 3 amino acids, differs by no more than 2 amino acids, or differs by no more than 1 amino acid from a sequence of SEQ ID NO: 13 or 14.

[0106] In some embodiments, provided genetically modified rodents (e.g., mice) comprise a humanized PIGR locus, wherein the humanized PIGR locus comprises both rodent plgR exons and human plgR exons. In some embodiments, a PIGR locus comprises one or more of (i) a rodent plgR exon 1, (ii) a rodent plgR exon 2, (iii) a human, rodent, orrodent-human chimeric plgR exon 3, (iv) a human plgR exon 4, (v) a human plgR exon 5, (vi) a human plgR exon 6, (vii) a human, rodent, or human-rodent chimeric plgR exon 7, (viii) a human, rodent, or human-rodent chimeric plgR exon 8, (ix) a human, rodent, or human-rodent chimeric plgR exon 9, (x) a human, rodent, or human-rodent chimeric plgR exon 10, and (xi) a human, rodent, or human-rodent chimeric plgR exon 11.

[0107] In some embodiments, a PIGR locus comprises: (i) a rodent plgR exon 1, (ii) a rodent plgR exon 2, (iii) a rodent-human chimeric plgR exon 3, (iv) a human plgR exon 4, (v) a human plgR exon 5, (vi) a human plgR exon 6, (vii) a human plgR exon 7, (viii) a human plgR exon 8, (ix) a human plgR exon 9, (x) a human plgR exon 10, and (xi) a human plgR exon 11. In some embodiments, a PIGR locus further comprises a rodent plgR exon 11.

[0108] In some embodiments, a PIGR locus comprises: (i) a rodent plgR exon 1, (ii) a rodent plgR exon 2, (iii) a human plgR exon 3, (iv) a human plgR exon 4, (v) a human plgR exon 5, (vi) a human plgR exon 6, (vii) a human plgR exon 7, (viii) a human plgR exon 8, (ix) a human plgR exon 9, (x) a human plgR exon 10, and (xi) a human plgR exon 11. In some embodiments, a PIGR locus further comprises a rodent plgR exon 11.

[0109] In some embodiments, a PIGR locus comprises: (i) a rodent plgR exon 1, (ii) a rodent plgR exon 2, (iii) a rodent-human chimeric plgR exon 3, (iv) a human plgR exon 4, (v) a human plgR exon 5, (vi) a human plgR exon 6, (vii) a human plgR exon 7, (viii) a human plgR exon 8, (ix) a rodent plgR exon 9, (x) a rodent plgR exon 10, and (xi) a rodent plgR exon 11.

[0110] In some embodiments, a PIGR locus comprises: (i) a rodent plgR exon 1, (ii) a rodent plgR exon 2, (iii) a human plgR exon 3, (iv) a human plgR exon 4, (v) a human plgR exon 5, (vi) a human plgR exon 6, (vii) a human plgR exon 7, (viii) a human plgR exon 8, (ix) a rodent plgR exon 9, (x) a rodent plgR exon 10, and (xi) a rodent plgR exon 11.

[0111] In some embodiments, a PIGR locus comprises: (i) a rodent plgR exon 1, (ii) a rodent plgR exon 2, (iii) a human plgR exon 3, (iv) a human plgR exon 4, (v) a human plgR exon 5, (vi) a human plgR exon 6, (vii) a human plgR exon 7, (viii) a human plgR exon 8, (ix) a rodent plgR exon 9, (x) a rodent plgR exon 10, and (xi) a rodent plgR exon 11.

[0112] In some embodiments, humanized PIGR locus comprises a nucleic acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences listed in Table 2 below. In some embodiments, humanized PIGR locus comprises a nucleic acid sequence that is at least 98% identical to any one of the sequenceslisted in Table 2 below. In some embodiments, humanized PIGR locus comprises a nucleic acid sequence that is at least 99% identical to any one of the sequences listed in Table 2 below.

[0113] In some embodiments, humanized PIGR locus comprises one or more of the nucleic acid sequences listed in Table 2.

[0114] Table 2 - Nucleic acid sequences encoding PIGR

[0115] In some embodiments, a humanized PIGR locus comprises a nucleic acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to: a genomic sequence found between coordinates 130,826,684 and 130,841,507 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,931,955 and 206,939,452 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,849,332 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly). In some embodiments, a humanized PIGR locus comprises a nucleic acid sequence that is at least 98% identical to: a genomic sequence found between coordinates 130,826,684 and 130,841,507 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,931,955 and 206,939,452 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,849,332 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly). In some embodiments,a humanized PIGR locus comprises a nucleic acid sequence that is at least 99% identical to: a genomic sequence found between coordinates 130,826,684 and 130,841,507 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,931,955 and 206,939,452 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,849,332 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly). In some embodiments, a humanized PIGR locus comprises a genomic sequence found between coordinates 130,826,684 and 130,841,507 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,931,955 and 206,939,452 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,849,332 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly).

[0116] In some embodiments, a humanized PIGR locus comprises a nucleic acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to: a genomic sequence found between coordinates 130,826,684 and 130,838,394 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,927,828 and 206,940,203 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,850,896 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly). In some embodiments, a humanized PIGR locus comprises a nucleic acid sequence that is at least 98% identical to: a genomic sequence found between coordinates 130,826,684 and 130,838,394 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,927,828 and 206,940,203 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,850,896 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly). In some embodiments, a humanized PIGR locus comprises a nucleic acid sequence that is at least 99% identical to: a genomic sequence found between coordinates 130,826,684 and 130,838,394 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,927,828 and 206,940,203 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,850,896 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly). In some embodiments, a humanized PIGR locus comprises a genomic sequence found between coordinates130,826,684 and 130,838,394 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,927,828 and 206,940,203 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,850,896 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly).

[0117] In some embodiments, a humanized PIGR locus is at an endogenous PIGR locus. In some embodiments, a humanized PIGR locus is under the control of a PIGR promoter. In some embodiments, a humanized PIGR locus is under the control of an endogenous rodent PIGR promoter. In some embodiments, humanized PIGR locus is under the control of a human PIGR promoter.

[0118] In some embodiments, provided genetically modified rodents (e.g., mice) do not express a functional rodent plgR polypeptide. In some embodiments, a rodent genome does not encode a functional rodent plgR polypeptide. In some embodiments, at least part of the endogenous rodent PIGR locus has been deleted from the rodent genome. In some embodiments, a humanized PIGR locus replaces all or part of the endogenous rodent PIGR locus.Rodents with Humanized Fc Receptors and Humanized plgRHumanized FcaR Loci

[0119] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein further comprise a human or humanized FcaR locus.

[0120] In some embodiments, a rodent or rodent ES cell genome comprises a human or humanized FcaR locus at an endogenous FcaR locus or at a position corresponding to an FcaR locus in a human genome. Mice do not have an endogenous FcaR homolog. In some embodiments, a nucleic acid sequence encoding a human or humanized FcaR is inserted into a mouse genome at a position that corresponds to the position of human endogenous FcaR locus or a position in the mouse genome that corresponds to loci located near the position of human endogenous FcaR locus in the human genome.

[0121] In some embodiments, the FcaR locus comprises a nucleic acid sequence encoding an FcaR polypeptide comprising a human extracellular domain, a rodent (that is not a mouse, e.g., rat) transmembrane domain and a rodent (that is not a mouse, e.g., rat) cytoplasmic domain. In some embodiments, the FcaR locus comprises a nucleic acid sequence encoding an FcaR polypeptide comprising a human extracellular domain, a humantransmembrane domain and a rodent (that is not a mouse, e.g., rat) cytoplasmic domain. In some embodiments, the FcaR locus comprises a nucleic acid sequence encoding an FcaR polypeptide comprising a human extracellular domain, a human transmembrane domain and a human cytoplasmic domain.

[0122] In some embodiments, the FcaR gene locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers). In other embodiments, the FcaR gene locus comprises human regulatory elements (e.g., human promoters and / or enhancers).

[0123] As is described in the art (see, e.g., Monteiro and van de Winkel, (2003) Annu. Rev. Immunol. 21:177-204, which is incorporated herein by reference), the human FcaR gene consists of 5 exons: exon 1 including the 5’ UTR, ATG translation initiation codon and part of the leader peptide encoding sequence; exon 2 encoding the remainder of the leader peptide; exons 3 and 4 encoding two extracellular domains, ECI and EC2, of FcaR, with ECI being the domain that binds to an IgA Fc; and exon 5 encoding the cytoplasmic and transmembrane domains of the protein.

[0124] In some embodiments, a nucleic acid encoding a human or humanized FcaR polypeptide provided herein encodes a fully human FcaR polypeptide. In certain embodiments, such a nucleic acid comprises a humanized exon 1 (e.g., comprising a rodent (non-mouse, e.g., rat) 5’ UTR and a human coding sequence), human coding exons 2-5 through the stop codon, and a human or rodent (non-mouse, e.g., rat) 3’UTR and polyA. In some embodiments, the nucleic acid encoding human or humanized FcaR polypeptide comprises exons 1-5 of the human FcaR gene.

[0125] In some embodiments, a nucleic acid encoding a FcaR polypeptide is a chimeric rodent (non-mouse, e.g., rat) / human sequence. In some embodiments, the chimeric rodent (non-mouse, e.g., rat) / human sequence comprises a human, rat or chimeric rat / human exon 1 (such that the nucleic acid sequence encoding the leader sequence is either human or rat); a human or rat exon 2, human exons 3-4; and rat exon 5. In these embodiments, the nucleic acid encodes a FcaR polypeptide comprising human FcaR extracellular domain and rat transmembrane and cytoplasmic domains. In some embodiments, the regulatory regions (e.g., promoters and UTRs) are rodent (e.g., rat) regulatory regions.

[0126] NCBI Reference Sequence Number NW_016107304.1 is a representative source sequence of a human FcaR gene. NCBI Reference Sequence Numbers NM_002000.4and NP_001991.1, NM.133269,4 and NP_579803.1, NM_133271.4 and NP_579805.1, NM_133272.4 and NP_579806.1, NM_133273.4 and NP_579807.1, NM_133274.4 and NP_579808.1, NM_133277.4 and NP_579811.1, NM_133278.4 and NP_579812.1, XM_011526625.3 and XP_011524927.1, XM_017026473.1 and XP_016881962.1, XM_017026474.2 and XP_016881963.1, provide representative source sequences of human FcaR cDNA and polypeptides from which a desired human portion may be obtained.

[0127] NCBI Reference Sequence Number NC_005100.4 provides a representative source sequence of a Rattus norvegicus FcaR gene and NCBI Reference Sequence Numbers NM_201992.1 — NP_973721.1 provide representative source sequences of Rattus norvegicus FcaR cDNA and polypeptides from which a desired Rattus norvegicus portion may be obtained and / or which can be used in the design of targeting vector homology arms.

[0128] In some embodiments, a rodent (e.g., mouse) is heterozygous for the genetically modified FcaR locus. In some embodiments, a rodent (e.g., mouse) is homozygous for the genetically modified FcaR locus.

[0129] In some embodiments, a human or humanized FcaR locus is present is positioned in a mouse genome in the leukocyte receptor complex (LRC) on mouse chromosome 7. In some embodiments, a sequence encoding a human or humanized Fc alpha receptor (FcaR) protein is positioned in an intergenic region between the gene loci for the Tthyl protein and the Rdhl3 protein, the Lilra5 polypeptide and the Gp6 polypeptide, the Pira6 protein and the Gp6 protein, and / or the Pira6 protein and the Ncrl protein. In some embodiments, a sequence encoding a human or humanized Fc alpha receptor (FcaR) protein is positioned between coordinates chr7:4,303,905 - 4,312,280 in the mouse genome (+ strand, GRCm38 assembly). In some embodiments, the intergenic region is the 54kb region between the Pira6 and Ncrl loci. In some embodiments, the nucleic acid sequence that encodes the FcaR further comprises a nucleic acid sequence that encodes all or part of the human KIR3DL2 gene, and / or further comprises a nucleic acid sequence of all or part of the human NCR1 gene, e.g., a nucleic acid sequence present in the 5’UTR of human NCR1 gene.

[0130] In some embodiments, provided genetically modified rodents (e.g., mice) comprise genetically modified loci that encode antibody heavy chains comprising a human Fc (e.g., a human IgAl Fc, a human IgA2 Fc). Such loci are disclosed in W02019 / 190990, the contents of which are incorporated by reference in their entirety.

[0131] In some embodiments, rodents (e.g., mice) comprising a humanized plgR locus described herein can be combined with a genetically modified FcaR locus by breedingor retargeting of ES cells in accordance with the techniques described herein and known in the art. In some embodiments, a mouse comprising a humanized plgR locus (mouse chr. 1) is bred with a mouse comprising a genetically modified FcaR locus (mouse chr. 7) using standard techniques. The resultant mice may be heterozygous or homozygous for either of these humanized genes.Humanized Low Affinity Fc Gamma Receptors

[0132] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein further comprise a locus encoding human low affinity Fc gamma receptor (FcyR) polypeptide (e.g., a human FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa or FcyRIIIb polypeptide; see, for example, U. S Patent No. 8,658,154, the contents of which are incorporated herein by reference in their entirety).

[0133] In some embodiments, the low affinity FcyR locus comprises a nucleic acid sequence encoding a human FcyRIIa polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcyRIIa polypeptide is positioned at an endogenous mouse low affinity FcyR locus. In some embodiments, the nucleic acid sequence encoding the FcyRIIa polypeptide replaces all or part of an endogenous mouse low affinity FcyR locus. In some embodiments, the human FcyRIIa gene comprises a polymorphism, wherein the polymorphism is selected from a 131His low responder polymorphism and a 131 Arg high responder polymorphism. In some embodiments, the FcyRIIa polymorphism is the 131His low responder polymorphism. In some embodiments, the mouse does not express a mouse low affinity FcyR polypeptide (e.g., does not express a mouse FcyRIIb, FcyRIV and / or FcyRIII polypeptide, or does not express functional mouse FcyRIIb, FcyRIV and / or FcyRIII polypeptide). In some embodiments, the FcyRIIa gene locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0134] In some embodiments, the low affinity FcyR locus comprises a nucleic acid sequence encoding a human FcyRIIb polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcyRIIb polypeptide is positioned at an endogenous mouse low affinity FcyR locus. In some embodiments, the nucleic acid sequence encoding the FcyRIIb polypeptide replaces all or part of an endogenous mouse low affinity FcyR locus. In some embodiments, the human FcyRIIb gene comprises an amino acid substitution, whereinthe substitution is selected from an 187Ile or a 187Thr substitution. In some embodiments, the mouse does not express a mouse low affinity FcyR polypeptide (e.g., does not express a mouse FcyRIIb, FcyRIV and / or FcyRIII polypeptide, or does not express functional mouse FcyRIIb, FcyRIV and / or FcyRIII polypeptide). In some embodiments, the FcyRIIb gene locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0135] In some embodiments, the low affinity FcyR locus comprises a nucleic acid sequence encoding a human FcyRIIc polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcyRIIc polypeptide is positioned at an endogenous mouse low affinity FcyR locus. In some embodiments, the nucleic acid sequence encoding the FcyRIIc polypeptide replaces all or part of an endogenous mouse low affinity FcyR locus. In one embodiment, the FcyRIIc gene is a specific allelic variant, wherein the allelic variant is selected from a 57Stop variant and a 57Q variant. In some embodiments, the mouse does not express a mouse low affinity FcyR polypeptide (e.g., does not express a mouse FcyRIIB, FcyRIV and / or FcyRIII polypeptide). In some embodiments, the FcyRIIc gene locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0136] In some embodiments, the low affinity FcyR locus comprises a nucleic acid sequence encoding a human FcyRIIIa polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcyRIIIa polypeptide is positioned at an endogenous mouse low affinity FcyR locus. In some embodiments, the nucleic acid sequence encoding the FcyRIIIa polypeptide replaces all or part of an endogenous mouse low affinity FcyR locus. In some embodiments, the mouse does not express a mouse low affinity FcyR polypeptide (e.g., does not express a mouse FcyRIIb, FcyRIV and / or FcyRIII polypeptide, or does not express functional mouse FcyRIIb, FcyRIV and / or FcyRIII polypeptide). In one embodiment, the FcyRIIIa gene is a specific allelic variant, wherein the allelic variant is selected from a 176Val variant and a 176Phe variant. In some embodiments, the FcyRIIIa allelic variant is the 176Val variant. In some embodiments, the FcyRIIIa gene locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0137] In some embodiments, the low affinity FcyR locus comprises a nucleic acid sequence encoding a human FcyRIIIb polypeptide. In some embodiments, the nucleic acid sequence encoding the human FcyRIIIb polypeptide is positioned at an endogenous mouse low affinity FcyR locus. In some embodiments, the nucleic acid sequence encoding the FcyRIIIb polypeptide replaces all or part of an endogenous mouse low affinity FcyR locus. In some embodiments, the FcyRIIIb gene is a specific allelic variant, wherein the allelic variant is selected from a NA1 variant and a NA2 variant. In another specific embodiment, the FcyRIIIb allelic variant is a NA2 variant. In some embodiments, the mouse does not express a mouse low affinity FcyR polypeptide (e.g., does not express a mouse FcyRIIb, FcyRIV and / or FcyRIII polypeptide, or does not express functional mouse FcyRIIb, FcyRIV and / or FcyRIII polypeptide). In some embodiments, the FcyRIIIb gene locus comprises non-human regulatory elements e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are mouse regulatory elements (e.g., rat or mouse promoters or enhancers).

[0138] In some embodiments, a rodent (e.g., mouse) provided herein comprises one or more human low affinity FcyR genes as described in U. S. Patent Nos. 9,221,894, 9,056,130, 9,089,599, 8,658,154, 8,883,496 or 8,658,853, each of which is incorporated by reference herein. In some embodiments, a rodent (e.g., mouse) provided herein comprises at least two low affinity human FcyR genes and an endogenous mouse Fc y-chain gene, wherein the low affinity human FcyR genes are selected from the group consisting of human FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa and FcyRIIIb. In some certain embodiments, a rodent (e.g., mouse) provided herein comprises a human FcyRIIa and FcyRIIIb, and an endogenous rodent Fc y-chain gene. In some embodiments, a rodent (e.g., mouse) provided herein comprises FcyRIIa, FcyRIIIa, FcyRIIb, FcyRIIc and FcyRIIId genes, and an endogenous rodent Fc y-chain gene. In some embodiments, a rodent (e.g., mouse) comprising one or more human FcyRs further comprises a homozygous disruption in endogenous rodent Fey RUB, FcyRIV and FcyRIII genes (e.g., endogenous mouse FcyRIIb, FcyRIV and FcyRIII a-chain encoding sequences). In various embodiments, a rodent (e.g., mouse) comprising one or more human low affinity FcyRs as described herein does not detectably express an endogenous rodent low affinity FcyR polypeptide (e.g., an endogenous low affinity FcyR a-chain polypeptide).

[0139] In some embodiments, a rodent (e.g., mouse) is heterozygous for the genetically modified low affinity FcyR locus. In some embodiments, a rodent (e.g., mouse) is homozygous for the genetically modified low affinity FcyR locus.

[0140] In some embodiments, rodents (e.g., mice) comprising a humanized plgR locus described herein can be combined with a genetically modified low affinity FcyR locus by breeding or retargeting of ES cells in accordance with the techniques described herein and known in the art. The resultant mice may be heterozygous or homozygous for either of these humanized genes.Humanized Neonatal Fc Receptor Loci

[0141] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein further comprise a humanized or human neonatal Fc receptor (FcRn) locus. FcRn, also known as the Brambell receptor, is a protein that is expressed by endothelial cells and associates with beta-2-microglobulin (P2M) and binds to both the Fc domains of IgG antibodies and serum albumin. FcRn extends the half-life of IgG and serum albumin. Specifically, by binding IgG and serum albumin in a pH dependent manner, FcRn is able to rescue these serum proteins from lysosomal degradation by endothelial cells, thereby increasing the serum half-life of such proteins.

[0142] In some embodiments, the FcRn locus comprises a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain, a rodent (e.g., mouse or rat) transmembrane domain and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcRn locus comprises a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcRn locus comprises a nucleic acid sequence encoding an FcRn polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain and a human (e.g., mouse or rat) cytoplasmic domain.

[0143] In some embodiments, the nucleic acid sequence encoding the FcRn polypeptide is positioned at an endogenous rodent (e.g., mouse) FcRn locus. In certain embodiments, the nucleic acid sequence encoding the FcRn polypeptide replaces all or part of an endogenous rodent (e.g., mouse) FcRn gene. For example, in some embodiments, the nucleic acid sequence encoding the extracellular domain of an endogenous FcRn at an endogenous FcRn locus is replaced with a nucleic acid sequence encoding the extracellular domain of a human FcRn such that a rodent (e.g., mouse) comprising such a locus expresses an FcRn with a human extracellular domain and a rodent (e.g., rat or mouse) transmembrane and cytoplasmic domain. In some embodiments, the rodent (e.g., mouse) does not express amouse FcRn, or does not express a functional rodent FcRn. In some embodiments, the FcRn gene locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0144] In certain embodiments, the rodent (e.g., mouse) exons encoding alpha 1, alpha 2, and alpha 3 domains (exons 3, 4, and 5, which are the first three coding exons) of the rodent FcRn gene are replaced with human exons encoding alpha 1, alpha 2, and alpha 3 domains (exons 3, 4, and 5) of the human FcRn gene. In some embodiments, the FcRn gene comprises mouse exon 1 (non-coding exon), mouse exon 2 (comprising nucleic acid sequence encoding the signal peptide), and human exons 3-6, mouse exons 6 and 7 (encoding transmembrane and cytoplasmic domains). Exemplary humanized FcRn locus is described in W02019 / 190990 (e.g., in Figure 4), incorporated herein by reference.

[0145] GenBank accession nos. NC_000019.10 (49512279-49526428), NM_001136019.1, and NP_001129491.1 provide representative source sequences of a human FcRn gene, cDNA and polypeptide from which a desired human portion may be obtained. GenBank accession nos. NC_000073.6 (45092992-45103846), NM_010189.1, and NP_034319.1 provide representative source sequences of a mouse FcRn gene, cDNA and polypeptide from which a desired mouse portion may be obtained and / or which can be used in the design of targeting vector homology arms.

[0146] In some embodiments, the rodent (e.g., mouse) is heterozygous for the genetically modified FcRn locus. In some embodiments, the rodent (e.g., mouse) is homozygous for the genetically modified FcRn locus.

[0147] In some embodiments, rodents (e.g., mice) comprising a humanized plgR locus described herein can be combined with a genetically modified FcRn locus by breeding or retargeting of ES cells in accordance with the techniques described herein and known in the art. In some embodiments, a mouse comprising a humanized plgR locus (mouse chr. 1) is bred with a mouse comprising a genetically modified FcRn locus (mouse chr. 7) using standard techniques. The resultant mice may be heterozygous or homozygous for either of these humanized genes.Humanized B-2-microglobulin

[0148] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein further comprise a locus encoding humanized P-2-microglobulin (P2M) polypeptide. 2M is a polypeptide that lacks a transmembrane domain and that associates with the FcRn and MHC class I molecules.

[0149] In some embodiments, the 2M locus comprises a nucleic acid sequence encoding a human P2M polypeptide. In some embodiments, the nucleic acid sequence encoding the human P2M polypeptide is positioned at an endogenous mouse 2M locus. In certain embodiments, the nucleic acid sequence encoding the 2M polypeptide replaces all or part of an endogenous mouse P2M gene. In some embodiments, the rodent (e.g., mouse) does not express a mouse P2M, or does not express a functional mouse P2M polypeptide. In some embodiments, the P2M gene locus comprises non-human regulatory elements (e.g., nonhuman promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0150] Humanized P2M polypeptides, loci encoding humanized 2M polypeptides and mice expressing humanized 2M polypeptides are described in U. S. Pat. Pub. Nos.2013 / 0111617 and 2013 / 0185819, each of which is incorporated by reference herein. Thus, as described in U. S. Pat. Pub. Nos. 2013 / 0111617 and 2013 / 0185819, in some embodiments, rodents (e.g., mice) comprise a humanized 2M gene, wherein the gene comprises exons 2, 3, and 4 of the human 2M gene, and in some embodiments, the humanized 2M gene comprises exon 1 of the rodent (e.g., mouse) 2M gene. In some embodiments, the rodent (e.g., mouse) is heterozygous for the genetically modified 2M locus. In some embodiments, the rodent (e.g., mouse) is homozygous for the genetically modified 2M locus.

[0151] In some embodiments, rodents (e.g., mice) comprising a humanized plgR locus described herein can be combined with a human or humanized 2M locus by breeding or retargeting of ES cells in accordance with the techniques described herein and known in the art. In some embodiments, a mouse comprising a humanized plgR locus (mouse chr. 1) is bred with a mouse comprising a genetically modified FcRn locus (mouse chr. 7) and / or a human or humanized 2M locus (mouse chr. 2) using standard techniques to generate a mouse comprising all three genetic modifications. The resultant mice may be heterozygous or homozygous for any of these humanized genes.Humanized Fc epsilon receptor 1 alpha

[0152] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein further comprise a humanized or human Fc epsilonreceptor 1 alpha (FcsRla) locus. FcsRla associates with FcsRip and FcsRly to form FcsRl, a high-affinity receptor for IgE that is expressed on epidermal Langerhans cells, eosinophils, mast cells and basophils. The IgE binding site of FcsRl is found in the FcsRla subunit.

[0153] In some embodiments, the FcsRla locus comprises a nucleic acid sequence encoding an FcsRla polypeptide comprising a human extracellular domain, a rodent (e.g., mouse or rat) transmembrane domain and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcsRla locus comprises a nucleic acid sequence encoding an FcsRla polypeptide comprising a human extracellular domain, a human transmembrane domain and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcsRla locus comprises a nucleic acid sequence encoding an FcsRla polypeptide comprising a human extracellular domain, a human transmembrane domain and a human cytoplasmic domain. An exemplary embodiment of an engineered FcsRla locus is described in W02019 / 190990, which is incorporated herein by reference.

[0154] In some embodiments, the nucleic acid sequence encoding the FcsRla polypeptide is positioned at an endogenous rodent (e.g., mouse) FcsRla locus. In certain embodiments, the nucleic acid sequence encoding the FcsRla polypeptide replaces all or part of an endogenous rodent (e.g., mouse) FcsRla gene. For example, in some embodiments, the nucleic acid sequence encoding the extracellular domain of an endogenous FcsRla at an endogenous FcsRla locus is replaced with a nucleic acid sequence encoding the extracellular domain of a human FcsRla such that a rodent (e.g., mouse) comprising such a locus expresses a FcsRla with a human extracellular domain and a rodent (e.g., rat or mouse) transmembrane and cytoplasmic domain. In some embodiments, the nucleic acid sequence encoding an FcsRla polypeptide comprising a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain is positioned at an endogenous rodent (e.g., mouse) FcsRla locus. In some embodiments, the nucleic acid sequence encoding a FcsRla polypeptide comprising a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain replaces all or part of an endogenous rodent (e.g., mouse) FcsRla gene. In some embodiments, the mouse does not express a rodent (e.g., mouse) FcsRla, or does not express a functional rodent (e.g., mouse) FcsRla. In some embodiments, the FcsRla gene locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0155] In certain embodiments, part of rodent (e.g., mouse) coding exon 1, coding exon 2, coding exon 3, coding exon 4, and coding exon 5 of the rodent (e.g., mouse) FCERIOC are replaced by part of human coding exon 1, coding exon 2, coding exon 3, coding exon 4, and coding exon 5 of the human FCERIOC gene. In some embodiments, the FCERIOC gene comprises chimeric mouse / human exon 1 (comprising mouse promoter and 5’ UTR), human coding exons 2-5 through the stop codon, human 3 ’UTR and polyA, followed by the mouse 3 ’UTR and polyA. In some embodiments, chimeric gene exons 1 (partial) and 2 encode the signal peptide, exon 3 and 4 encode the two Ig-like domains of FCERIOC that are believed to interact with IgE, and exon 5 encodes the cytoplasmic and transmembrane domains of the protein (see Figure 9 of W02019 / 190990).

[0156] GenBank accession nos. NC_000001.ll (159283888-159308224), NM_002001.3, and NP_001992.1 provide representative source sequences of a human FcsRla gene, cDNA and polypeptide from which a desired human portion may be obtained. GenBank accession nos. NC_000067.6 (173221269-173227232), NM_010184.1, and NP_034314.1 provide representative source sequences of a mouse FcsRla gene, cDNA and polypeptide from which a desired mouse portion may be obtained and / or which can be used in the design of targeting vector homology arms.

[0157] In some embodiments, the rodent (e.g., mouse) is heterozygous for the genetically modified FcsRla locus. In some embodiments, the rodent (e.g., mouse) is homozygous for the genetically modified FcsRla locus.

[0158] In some embodiments, rodents (e.g., mice) comprising a humanized plgR locus described herein can be combined with a genetically modified FcsRla locus by breeding or retargeting of ES cells in accordance with the techniques described herein and known in the art. The resultant mice may be heterozygous or homozygous for either of these humanized genes.Humanized Fc gamma receptor la

[0159] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein further comprise a humanized or human Fc gamma receptor la (FcyRla) locus. FcyRla is a high affinity FcyR protein expressed on monocytes that binds to the Fc portion of IgG and causes activation of the host cell.

[0160] In some embodiments, the FcyRla locus comprises a nucleic acid sequence encoding a FcyRla polypeptide comprising a human extracellular domain, a rodent (e.g., mouse or rat) transmembrane domain and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcyRla locus comprises a nucleic acid sequence encoding a FcyRla polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FcyRla locus comprises a nucleic acid sequence encoding a FcyRla polypeptide comprising a human extracellular domain, a human (e.g., mouse or rat) transmembrane domain and a human (e.g., mouse or rat) cytoplasmic domain.

[0161] In some embodiments, the nucleic acid sequence encoding the FcyRla polypeptide is positioned at an endogenous rodent (e.g., mouse) FcyRla locus. In certain embodiments, the nucleic acid sequence encoding the FcyRla polypeptide replaces all or part of an endogenous rodent (e.g., mouse) FcyRla gene. For example, in some embodiments, the nucleic acid sequence encoding the extracellular domain of an endogenous FcyRla at an endogenous FcyRla locus is replaced with a nucleic acid sequence encoding the extracellular domain of a human FcyRla such that a rodent (e.g., mouse) comprising such a locus expresses a FcyRla with a human extracellular domain and a rodent (e.g., rat or mouse) transmembrane and cytoplasmic domain. In some embodiments, the rodent (e.g., mouse) does not express a rodent (e.g., mouse) FcyRla, or does not express a functional rodent (e.g., mouse) FcyRla. In some embodiments, the FcyRla gene locus comprises non-human regulatory elements (e.g., non-human promoters and / or enhancers). In some embodiments, the non-human regulatory elements are rodent regulatory elements (e.g., rat or mouse promoters or enhancers).

[0162] Humanized FcyRla polypeptides, loci encoding humanized FcyRla polypeptides and mice expressing humanized FcyRla polypeptides are described in U. S. Pat. No. 9,474,255 and U. S. Pat. Pub. No. 2017 / 0086432, each of which is incorporated by reference herein.

[0163] In some embodiments, the rodent (e.g., mouse) is heterozygous for the genetically modified FcyRla locus. In some embodiments, the rodent (e.g., mouse) is homozygous for the genetically modified FcyRla locus.

[0164] In some embodiments, rodents (e.g., mice) comprising a humanized plgR locus described herein can be combined with a genetically modified FcyRla locus by breeding or retargeting of ES cells in accordance with the techniques described herein andknown in the art. In some embodiments, a mouse comprising a humanized plgR locus (mouse chr. 1) is bred with a mouse comprising a genetically modified FcyRla locus (mouse chr. 3) using standard techniques. The resultant mice may be heterozygous or homozygous for either of these humanized genes.Rodents with Humanized Immunoglobulins and Humanized plgR

[0165] In some embodiments, provided genetically modified rodents (e.g., mice or rats) and cells (e.g., ES cells) described herein further comprise a humanized or human immunoglobulin locus.

[0166] The ability to generate antibodies has been harnessed in genetically modified animals, which are able to generate therapeutic antibodies or antibody fragments against human targets. Exemplary genetically modified mice, comprising human V(D)J gene segments, for generation of therapeutic antibodies are those described in U. S. Pat. Nos.5,633,425, 5,770,429, 5,814,318, 6,075,181, 6,114,598, 6,150,584, 6,998,514, 7,795,494, 7,910,798, 8,232,449, 8,703,485, 8,907,157, and 9,145,588, each of which is hereby incorporated by reference in its entirety, as well as in U. S. Pat. Pub. Nos. 2008 / 0098490, 2010 / 0146647, 2013 / 0145484, 2012 / 0167237, 2013 / 0167256, 2013 / 0219535, 2012 / 0207278, 2015 / 0113668, 2019 / 0127757, and 2021 / 0059229 each of which is hereby incorporated by reference in its entirety, and in PCT Pub. Nos. W02007117410, W02008151081, WO2009157771, W02010039900, WO2011004192, WO2011123708, WO2013045916, WO2015049517, W02014093908, W02014093908, W02006008548, W02010109165, W02016062990, W02018039180, WO2011158009, WO2013041844, WO2013041846, WO2013079953, W02013061098, WO2013144567, WO2013144566, W02013171505, W02012018610, WO2022126113, WO2020132557, WO2017035274, WO2019236670, WO2019236671, W02019008123, WO2021123090, W02020169022, and WO2021244522, each of which are hereby incorporated by reference in its entirety. Other exemplary genetically modified mice, comprising human V(D)J gene segments, for generation of therapeutic antibodies are those described in U. S. Pat. Nos. 6,596,541, 6,586,251, 8,642,835, 9,706,759, 10,238,093, 8,754,287, 10,143,186, 9,796,788, 10,130,081, 9,226,484, 9,012,717, 10,246,509, 9,204,624, and 9,686,970, each of which is hereby incorporated by reference in its entirety, as well as in U. S. Pat. Pub. Nos. 2013 / 0212719, 2015 / 0289489, 2017 / 0347633, 2019 / 0223418, 2018 / 0125043, 2019 / 0261612, and 2019 / 0380316, each of which is hereby incorporated by reference in its entirety, in PCT Pub. Nos. WO2013138680,WO2013138712, WO2013138681, W02015042250, WO2012148873, WO2013134263, WO2013184761, W02014160179, WO2017214089, WO2016149678, and WO2017123808, WO 2012018764, WO2019241692, WO2017123804, WO2022140219 and Murphy, A., “Veloclmmune: Immunoglobulin Variable Region Humanized Mouse,” in Recombinant Antibodies for Immunotherapy, New York, NY, Cambridge University Press, 101-107 (2009), each of which are hereby incorporated by reference in its entirety. Additional detailed embodiments of certain exemplary genetically engineered non-human animals, e.g., rodents, e.g., rats or mice, are described below.

[0167] Various embodiments of the genetically modified non-human animals, e.g., rodents, e.g., rats or mice, are described in more detail herein below. In some embodiments, the immunized non-human animal host is a rodent such as a rat or mouse.

[0168] In some embodiments, the host is a genetically modified rodent that comprises in its genome an immunoglobulin heavy chain variable region comprising one or more human heavy chain V gene segments, one or more human D gene segments, and one or more human heavy chain J gene segments, wherein the immunoglobulin heavy chain variable region is operably linked to a constant region, and an immunoglobulin light chain variable region comprising one or more human light chain V gene segments and one or more human light chain J gene segments, wherein the light chain is operably linked to a constant region.

[0169] The constant region locus includes one or more Ig heavy chain constant region gene segments (CH). In some embodiments, the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region such that the rodent produces antibodies comprising variable domains derived from the VH gene segment, the DH gene segment and the JH gene segment and heavy chain constant domains derived from the CH gene segment.

[0170] In some embodiments, provided herein are rodents (e.g., rats or mice) and rodent ES cells comprising in their genome a genetically modified immunoglobulin heavy chain constant region. In some embodiments, a genetically modified immunoglobulin heavy chain constant region comprises human or humanized CH gene segments. Exemplary genetically modified mice, comprising human or humanized CH gene segments, are described in U. S. Pat. No. 11,576,984 and in PCT Pub. No. W02019190990, each of which are hereby incorporated by reference in its entirety.

[0171] In some embodiments the IgH locus comprises: (i) an immunoglobulin heavy chain variable region comprising one or more VH gene segments, one or more DH genesegments and one or more JH gene segments (e.g., rat or mouse VH gene segments, DH gene segments and JH gene segments); and (ii) an immunoglobulin heavy chain constant region comprising one or more CH gene segments encoding an IgG constant domain comprising a human CHI domain, a human hinge region, a human CH2 domain, a human CH3 domain, a human or rodent IgG transmembrane domain and a human or rodent IgG cytoplasmic domain. In some embodiments all the CH gene segments in the immunoglobulin heavy chain constant region are human. In some embodiments the immunoglobulin heavy chain variable region is operably linked to the immunoglobulin heavy chain constant region such that the rodent produces IgG antibodies comprising variable domains derived from the VH gene segment, the DH gene segment and the JH gene segment and heavy chain constant domains derived from the CH gene segment. In some embodiments, the locus is positioned at the endogenous rodent immunoglobulin heavy chain locus. In some embodiments, a rodent provided herein expresses antibodies having human variable domains and human IgGl constant domains. In some embodiments, a rodent provided herein expresses antibodies having human variable domains and human IgG2 constant domains. In some embodiments, a rodent provided herein expresses antibodies having human variable domains and human IgG3 constant domains. In some embodiments, a rodent provided herein expresses antibodies having human variable domains and human IgG4 constant domains. In some embodiments, a rodent provided herein expresses antibodies having a human K light chain, e.g., expresses antibodies having a human K constant domain, or expresses antibodies having human K variable and constant domains. In some embodiments, a rodent provided herein expresses antibodies having a human A light chain, e.g., expresses antibodies having a human A constant domain, or expresses antibodies having human 1 variable and constant domains.

[0172] In some embodiments, provided herein are rodents (e.g., rats or mice) comprising in their genome a genetically modified immunoglobulin kappa (IgK) chain locus. In some embodiments the IgK locus comprises: (1) an immunoglobulin K chain variable region comprising one or more human VKgene segments and one or more human JKgene segments; and (2) an immunoglobulin K chain constant region comprising a human CKgene segment. In some embodiments, the immunoglobulin K chain variable region is operably linked to the immunoglobulin K chain constant region such that the rodent produces antibodies comprising light chain variable domains derived from the human VKgene segment and the human JKgene segment and light chain constant domains derived from the CKgenesegment. In certain embodiments the locus is positioned at the endogenous rodent immunoglobulin K chain locus.

[0173] In some embodiments, provided herein are rodents (e.g., rats or mice) comprising in their genome a genetically modified immunoglobulin lambda (IgA.) chain locus. In certain embodiments the IgA locus comprises one or more human Vz gene segments, one or more human Jz gene segments and one or more Cz gene segments. In some embodiments, the human Jz gene segments and the Cz gene segments are arranged as one or more Jz-Cz clusters. In some embodiments, the human Jz gene segments and the Cz gene segments are arranged such that the human Jz gene segments are collectively positioned upstream of one or more Cz gene segments. In some embodiments, the human Jz gene segments and the Cz gene segments are arranged such that some of the human Jz gene segments and the Cz gene segments are arranged as one or more Jz-Cz clusters, while other Jz gene segments are collectively positioned upstream of one or more Cz gene segments. In some embodiments, the human Vz gene segment and the human Jz gene segment are operably linked to the human Cz gene segment such that the rodent produces antibodies comprising light chain variable domains derived from the human Vz gene segment and the human Jz gene segment and light chain constant domains derived from the Cz gene segment. In certain embodiments the locus is positioned at the endogenous rodent immunoglobulin 1 chain locus.

[0174] In some embodiments, the host is a genetically modified mouse that comprises in its genome an immunoglobulin heavy chain variable region comprising one or more human heavy chain V gene segments, one or more human D gene segments, and one or more human heavy chain J gene segments, wherein the heavy chain variable region is operably linked to a murine (e.g., a rat or mouse) constant region, and an immunoglobulin light chain variable region comprising one or more human light chain V gene segments and one or more human light chain J gene segments, wherein the light chain is operably linked to a murine constant region.

[0175] In one aspect, the immunoglobulin heavy chain variable region is operably linked to a mouse heavy chain constant region, and the immunoglobulin light chain variable region is operably linked to a mouse light chain constant region. In a further aspect, the immunoglobulin heavy chain variable region operably linked to a mouse heavy chain constant region resides at the endogenous mouse heavy chain locus, and the immunoglobulin light chain variable region operably linked to a mouse light chain constant region resides at the endogenous mouse light chain locus. One exemplary embodiment is described inMacdonald et al, Proc. Natl. Acad. Sci. USA 111:5147-52 and supporting information (www.pnas.org / cgi / content / short / 1323896111), which is hereby incorporated by reference in its entirety. Various embodiments of the genetically modified non-human animals, e.g., rodents, e.g., rats or mice, are described in more detail herein below.

[0176] In some embodiments, a genetically modified rodent comprises in its genome (e.g., its germline genome) an engineered immunoglobulin heavy chain locus (e.g., an engineered endogenous rodent immunoglobulin heavy chain locus) comprising one or more unrearranged human VH gene segments, one or more unrearranged human DH gene segments, and one or more unrearranged human JH gene segments that are upstream of (e.g., operably linked to) one or more rodent (e.g., rat or mouse) immunoglobulin heavy chain constant region genes (e.g., one or more endogenous rodent (e.g., rat or mouse) immunoglobulin heavy chain constant region genes). Such an engineered immunoglobulin heavy chain locus is referred to herein as an “HoH locus.” Rodents including an HoH locus are exemplified in, e.g., U. S. Patent Nos. 6,596,541; 8,642,835; and 8,697,940, and Murphy, A., “Veloclmmune: Immunoglobulin Variable Region Humanized Mouse,” in Recombinant Antibodies for Immunotherapy, New York, NY, Cambridge University Press, 101-107 (2009), each of which is incorporated by reference in its entirety. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is homozygous at an HoH locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is heterozygous at an HoH locus.

[0177] In some embodiments, one or more unrearranged human VH gene segments includes at least six human VH gene segments. In some embodiments, one or more unrearranged human VH gene segments includes at least 18 human VH gene segments. In some embodiments, one or more unrearranged human VH gene segments includes at least 39 human VH gene segments. In some embodiments, one or more unrearranged human VH gene segments includes at least 80 human VH gene segments. In some embodiments, one or more unrearranged human DH gene segments includes at least 27 human DH gene segments. In some embodiments, one or more unrearranged human JH gene segments includes at least six human JH gene segments.

[0178] In some embodiments, one or more unrearranged human VH gene segments includes all functional human VH gene segments. In some embodiments, one or more unrearranged human VH gene segments includes less than 80 human VH gene segments. In some embodiments, one or more unrearranged human VH gene segments includes less than 39 human VH gene segments. In some embodiments, one or more unrearranged human VHgene segments includes less than 18 human VH gene segments. In some embodiments, one or more unrearranged human VH gene segments includes less than 10 human VH gene segments. In some embodiments, one or more unrearranged human VH gene segments includes all human VH gene segments. In some embodiments, one or more unrearranged human VH gene segments includes a contiguous (e.g., unmodified) human sequence including human IGHV(III)-82 to human IGHV6-1 of a human heavy chain immunoglobulin gene locus.

[0179] In some embodiments, one or more unrearranged human DH gene segments and one or more unrearranged human JH include all human DH and JH gene segments. In some embodiments, one or more unrearranged human DH gene segments includes a contiguous (e.g., unmodified) human sequence including human IGHD1-1 to human IGHJ-6 of a human heavy chain immunoglobulin gene locus.

[0180] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin heavy chain locus (e.g., an engineered endogenous rodent immunoglobulin light chain locus) that comprises a deletion of all endogenous mouse immunoglobulin heavy chain variable region gene segments (e.g., deletion of a contiguous mouse immunoglobulin heavy chain gene sequence from mouse IGHV1-86 to IGHJ4 on mouse chromosome 12). In some embodiments, an endogenous mouse immunoglobulin heavy chain variable region sequence is replaced (e.g., by homologous recombination) with a contiguous sequence of human immunoglobulin heavy chain variable region sequence. In some embodiments, the human immunoglobulin heavy chain variable region sequence comprises all human immunoglobulin heavy chain variable gene segments (i.e., a contiguous sequence from human IGHV(III)-82 to human IGHV6-1 on chromosome 14). In some embodiments, the human immunoglobulin K sequence is at least 500 kb in size (i.e., at least 500kb, at least lOOkb, at least l,000kb). In some embodiments, the replacement of the endogenous mouse immunoglobulin heavy chain locus is performed with a single recombination step. Exemplary embodiments can be found, e.g., in PCT Publication No. W02020169022, which is hereby incorporated by reference in its entirety).

[0181] In some embodiments, one or more unrearranged human VH gene segments includes at least 18 human VH gene segments, one or more unrearranged human DH gene segments includes 27 human DH gene segments, and one or more unrearranged human JH gene segments includes six human JH gene segments. Such an engineered immunoglobulin heavy chain locus is referred to herein as a “Veloclmmune® 1 HoH locus.” In someembodiments, one or more unrearranged human VH gene segments includes at least 39 human VH gene segments, one or more unrearranged human DH gene segments includes 27 human DH gene segments, and one or more unrearranged human JH gene segments includes six human JH gene segments. Such an engineered immunoglobulin heavy chain locus is referred to herein as a “Veloclmmune® 2 HoH locus.” In some embodiments, one or more unrearranged human VH gene segments includes at least 80 human VH gene segments, one or more unrearranged human DH gene segments includes 27 human DH gene segments, and one or more unrearranged human JH gene segments includes six human JH gene segments. Such an engineered immunoglobulin heavy chain locus is referred to herein as a “Veloclmmune® 3 HoH locus.”

[0182] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises an HoH locus, produces an antibody comprising, inter alia, heavy chains, wherein each heavy chain comprises a human heavy chain variable domain operably linked to a rodent (e.g., rat or mouse) heavy chain constant domain, e.g., in response to antigenic stimulation.

[0183] In some embodiments, a genetically modified rodent comprises in its genome (e.g., its germline genome) an engineered immunoglobulin heavy chain locus (e.g., an engineered endogenous rodent immunoglobulin heavy chain locus) comprising one or more unrearranged human VH gene segments, one or more unrearranged human DH gene segments, and one or more unrearranged human JH gene segments, which further comprises substitution or insertion of at least one histidine for a non-histidine residue, such that the unrearranged immunoglobulin heavy chain variable gene sequence comprises in a complementarity determining region 3 (CDR3) encoding sequence a substitution of at least one non histidine codon with a histidine codon or an insertion of at least one histidine codon (see, e.g., PCT Pub. Nos. WO2013 / 138712 and WO2013 / 138681, incorporated herein by reference in their entireties).

[0184] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin heavy chain locus, such as comprising a restricted heavy chain variable region sequence, comprising a limited human heavy chain variable region repertoire.

[0185] In some embodiments, a genetically modified rodent comprises in its genome (e.g., its germline genome) an engineered immunoglobulin heavy chain locus (e.g., an engineered endogenous rodent immunoglobulin heavy chain locus) comprising a singlehuman VH gene segment, one or more unrearranged human DH gene segments, and one or more unrearranged human JH gene segments that are upstream of (e.g., operably linked to) one or more rodent (e.g., rat or mouse) immunoglobulin heavy chain constant region genes (e.g., one or more endogenous rodent (e.g., rat or mouse) immunoglobulin heavy chain constant region genes). A genetically modified rodent having such an engineered immunoglobulin heavy chain locus (e.g., an engineered endogenous rodent immunoglobulin heavy chain locus) is exemplified in, e.g., U. S. Patent Publication No. 2019 / 0261612 and U. S. Patent No. 10,238,093, each of which is incorporated by reference in its entirety.

[0186] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin heavy chain locus (e.g., an engineered endogenous rodent immunoglobulin heavy chain locus) comprising a single rearranged human heavy chain variable region upstream of (e.g., operably linked to) one or more rodent (e.g., rat or mouse) constant region genes. Such an engineered immunoglobulin heavy chain locus is referred to herein as a “UHC locus” or a “universal heavy chain locus” or a “common heavy chain locus.” Rodents including a UHC locus are exemplified in, e.g., U. S. Patent No. 9,204,624, which is incorporated by reference in its entirety.

[0187] In some embodiments, a single rearranged human heavy chain variable region comprises a single human VH gene segment, a single human DH gene segment, and a single human JH gene segment. In some embodiments, a single human VH gene segment is a human VH3-23, a single human DH gene segment is a human DH4-4, and a single human JH gene segment is a human JH4.

[0188] In some embodiments, a single rearranged human heavy chain variable region comprises a single human VH gene segment and a single human JH gene segment, which are separated by two amino acids. In some embodiments, a single human VH gene segment is a human VH3-23, a single human JH gene segment is a human JH4, and two amino acids are glycine and tyrosine.

[0189] In some embodiments, one or more rodent (e.g., mouse or rat) heavy chain constant region genes are one or more endogenous rodent (e.g., mouse or rat) heavy chain constant region genes.

[0190] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a UHC locus, produces an antibody comprising, inter alia, immunoglobulinchains, where each immunoglobulin chain comprises a human heavy chain variable domain operably linked to a constant domain, e.g., in response to antigenic stimulation.

[0191] In certain embodiments, the immunoglobulin constant region comprises a CH gene segment encoding an IgG constant domain comprising a human CHI domain, a human hinge region, a human CH2 domain, a human CH3 domain, an IgG transmembrane domain and an IgG cytoplasmic domain. In some embodiments, the IgG transmembrane domain is a rodent IgG transmembrane domain (e.g., a mouse or rat transmembrane domain). In certain embodiments, transmembrane domain is a human IgG transmembrane domain. In some embodiments, the IgG cytoplasmic domain is a rodent IgG cytoplasmic domain (e.g., a mouse or rat cytoplasmic domain). In some embodiments, the IgG cytoplasmic domain is a human IgG cytoplasmic domain. In some embodiments, the IgG connecting region is a rodent IgG connecting region (e.g., a mouse or rat connecting domain). In certain embodiments, IgG connecting region is a human IgG connecting region.

[0192] In certain embodiments, the human CHI domain, the human hinge region, the human CH2 domain and the human CH3 domain are IgGl domains. In some embodiments, such an IgGl domain is encoded by an allele selected from IGHGl*01, IGHG1*O2, IGHG1*O3, IGHGl*04 and IGHG1*O5.

[0193] In certain embodiments, the human CHI domain, the human hinge region, the human CH2 domain and the human CH3 domain are IgG2 domains. In some embodiments, such an IgG2 domain is encoded by an allele selected from IGHG2*01, IGHG2*02, IGHG2*03, IGHG2*04, IGHG2*05 and IGHG2*06.

[0194] In certain embodiments, the human CHI domain, the human hinge region, the human CH2 domain and the human CH3 domain are IgG3 domains. In some embodiments, such an IgG3 domain is encoded by an allele selected from IGHG3*01, IGHG3*02, IGHG3*03, IGHG3*04, IGHG3*05, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12, IGHG3*13, IGHG3*14, IGHG3*15, IGHG3*16, IGHG3*17, IGHG3*18 and IGHG3*19.

[0195] In certain embodiments, the human CHI domain, the human hinge region, the human CH2 domain and the human CH3 domain are IgG4 domains. In some embodiments, such an IgG4 domain is encoded by an allele selected from IGHG4*01, IGHG4*02, IGHG4*03 and IGHG4*04.

[0196] In some embodiments, the CH gene segment encodes variant human immunoglobulin heavy chain constant region sequence (i.e., a human immunoglobulin heavychain constant region sequence that includes one or more additions, deletions, and / or substitutions relative to an appropriate reference human immunoglobulin heavy chain constant region sequence) that is characterized in that effector function and / or affinity for an FcR is enhanced or diminished relative to a reference human immunoglobulin heavy chain constant region.

[0197] In some embodiments, the CH gene segment encodes a human immunoglobulin heavy chain constant region characterized by an altered affinity for activating and / or inhibitory receptors. In some embodiments, the CH gene segment encodes a human immunoglobulin heavy chain constant region characterized by enhanced or diminished binding to an FcRn receptor, e.g., at acidic pH as compared to neutral pH. In some embodiments, the CH gene segment encodes a human immunoglobulin heavy chain constant region, in whole or in part, encodes a human immunoglobulin heavy chain constant region having one or more amino acid modifications. Exemplary amino acid modifications include, but are not limited to, a substitution at position 297 (e.g., N297A), position 250 (e.g., 250E or 250Q), position 252 (e.g., 252L, 252Y, 252F, 252W or 252T), position 254 (e.g., 254S or 254T), position 256 (e.g., 256S, 256R, 256Q, 256E, 256D, or 256T), position 307 (e.g., 307P or 307 A), position 308 (e.g., 308F or 308V), position 428 (e.g., 428L or 428F), position 433 (e.g., 433H, 433Lm, 433R, 433S, 433P, 433Q or 433K), position 434 (e.g., 434A, 434W, 434H, 434F or 434Y), and combinations thereof. In some embodiments, the CH gene segment encodes a human immunoglobulin heavy chain constant region having one or more pairs or groups of amino acid modifications selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 2571 and 3111 (e.g., P257I and Q311I); 2571 and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F).

[0198] In some embodiments, the CH gene segment encodes a chimeric immunoglobulin heavy chain constant domain that includes segments or portions derived from (or that appear in) more than one human immunoglobulin isotypes. For example, such a chimeric CH region may comprise a CH2 domain derived from a human IgGl, human IgG2 or human IgG4 molecule, combined with a CH3 domain derived from a human IgGl, human IgG2 or human IgG4 molecule. In some certain embodiments, the chimeric CH region further comprises a chimeric hinge region. For example, a chimeric hinge may comprise an "upperhinge" amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from a human IgGl, a human IgG2 or a human IgG4 hinge region, combined with a "lower hinge" sequence (amino acid residues from positions 228 to 236 according to EU numbering) derived from a human IgGl, a human IgG2 or a human IgG4 hinge region. In some certain embodiments, a chimeric hinge region comprises amino acid residues derived from a human IgGl or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge.

[0199] In certain embodiments, the modified CH gene segment is located at an endogenous CH gene segment locus. In certain embodiments, the modified CH gene segment is located at an endogenous CYigene segment locus, an endogenous CY2a gene segment locus, an endogenous CY2b gene segment locus, an endogenous CY2c gene segment locus or an endogenous CY3 gene segment locus.

[0200] The endogenous immunoglobulin heavy chain constant region gene structure can vary between rodents. For example, the Norwegian rat does not have a CY3 gene segment at the endogenous immunoglobulin heavy chain locus, whereas mouse strains generally do. Even for the same species, the immunoglobulin heavy chain constant region gene structure can vary from strain to strain. For example, while some mouse stains have a CY2a gene segment at the endogenous immunoglobulin heavy chain locus, other mouse stains (e.g., mouse strains with the Ighl-b allele) have a CY2c gene segment instead, and some mouse strains may have both CY2a and CY2c gene segments. The rodent CYi, CY2a, CY2b and CY3 gene segments disclosed in the figures, examples, and / or descriptions herein are therefore exemplary rodent CH gene segments and one of skill in the art would appreciate that the specific constant region gene structure will vary from one rodent strain to another. Thus, for example, one of skill in the art would appreciate that the disclosure contemplates rodents comprising a rodent CY2c gene segment in place of or in addition to any disclosed rodent CY2a gene segment.

[0201] In some embodiments, the modified CH gene segment is a human CYigene segment (or at least the portion of the human CYigene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgGl constant domain) and it is positioned at an endogenous CY2a gene segment locus. In some embodiments, the portion of the human CYigene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgGl constant domain is operably linked to the portion of theendogenous rodent CY2a gene segment encoding the IgG2a transmembrane and / or cytoplasmic domain.

[0202] In some embodiments, the modified CH gene segment is a human CYigene segment (or at least the portion of the human CYigene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgGl constant domain) and it is positioned at an endogenous CY2c gene segment locus. In some embodiments, the portion of the human CYigene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgGl constant domain is operably linked to the portion of the endogenous rodent CY2c gene segment encoding the IgG2c transmembrane and / or cytoplasmic domain.

[0203] In some embodiments, the modified CH gene segment is a human CY4 gene segment (or at least the portion of the human CY4 gene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgG4 constant domain) and it is positioned at an endogenous CYigene segment locus. In some embodiments, the portion of the human CY4 gene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgG4 constant domain is operably linked to the portion of the endogenous rodent CYigene segment encoding the IgGl transmembrane and / or cytoplasmic domain.

[0204] In certain embodiments, the modified CH gene segment replaces all or part of an endogenous CH gene segment. In certain embodiments, the modified CH gene segment replaces all or part of an endogenous CYigene segment, an endogenous CY2a gene segment, an endogenous CY2b gene segment, an endogenous CY2c gene segment, or an endogenous CY3 gene segment.

[0205] In some embodiments, the modified CH gene segment is a human CYigene segment (or at least the portion of the human CYigene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgGl constant domain) and it replaces all or part of an endogenous CY2agene segment locus. In some embodiments, the portion of the human CYigene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgGl constant domain replaces the portion of the endogenous rodent CY2agene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgG2a constant domain such that the portion of the human CYigene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3domain of the IgGl constant domain is operably linked to the portion of the endogenous rodent CY2a gene segment encoding the IgG2a transmembrane and / or cytoplasmic domain.

[0206] In some embodiments, the modified CH gene segment is a human CY4 gene segment (or at least the portion of the human CY4 gene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgG4 constant domain) and it replaces all or part of an endogenous CYigene segment locus. In some embodiments, the portion of the human CY4 gene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgG4 constant domain replaces the portion of the endogenous rodent CYigene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgGl constant domain such that the portion of the human CY4 gene segment encoding the CHI domain, the hinge region, the CH2 domain and the CH3 domain of the IgG4 constant domain is operably linked to the portion of the endogenous rodent CYigene segment encoding the IgGl transmembrane and / or cytoplasmic domain.

[0207] In certain embodiments the Ig heavy chain constant region includes one or more rodent (e.g., rat or mouse) CH gene segments. In some embodiments, the Ig constant region includes a rodent (e.g., rat or mouse) Cugene segment. In some embodiments, the Ig constant region includes a rodent (e.g., rat or mouse) Cg gene segment. In some embodiments, the Ig constant region includes a rodent (e.g., rat or mouse) CYigene segment. In some embodiments, the Ig constant region includes a rodent (e.g., rat or mouse) CY2a gene segment. In some embodiments, the Ig constant region includes a rodent (e.g., rat or mouse) CY2b gene segment. In some embodiments, the Ig constant region includes a rodent (e.g., rat or mouse) CY2C gene segment. In some embodiments, the Ig constant region includes a rodent (e.g., mouse) CY3 gene segment. In some embodiments, the Ig constant region includes a rodent (e.g., rat or mouse) Cegene segment. In some embodiments, the Ig constant region includes a rodent (e.g., rat or mouse) Cegene segment. In some embodiments, the one or more rodent constant region gene segments are endogenous constant region gene segments. In some embodiments, the modified CH gene segment described above is the only modified CH gene segment in the Ig heavy chain constant region.

[0208] In some embodiments, the modified CH gene segment described above is one of a plurality of modified CH gene segments in the Ig heavy chain constant region (e.g., one of 2, 3, 4, 5, 6, 7 or 8 modified gene segments that are partially or fully humanized in the Ig heavy chain constant region). In some embodiments, the Ig heavy chain constant region includes a human or partially human Cugene segment. In some embodiments, the Ig heavychain constant region includes a human or partially human Cg gene segment. In some embodiments, the Ig heavy chain constant region includes a human or partially human CYigene segment. In some embodiments, the Ig heavy chain constant region includes a human or partially human CY2 gene segment. In some embodiments, the Ig heavy chain constant region includes a human or partially human CY3 gene segment. In some embodiments, the Ig heavy chain constant region includes a human or partially human CY4 gene segment. In some embodiments, the Ig heavy chain constant region includes a human or partially human Cegene segment. In some embodiments, the Ig heavy chain constant region includes a human or partially human Cagene segment. In some embodiments, the Ig heavy chain constant region comprises a human Cggene segment, a human Cg gene segment, a human Cyigene segment and a human Cy3 gene segment. In some embodiments, the Ig heavy chain constant region further comprises a human Cy2 gene segment and a human Cy4 gene segment. In some embodiments, the Ig heavy chain constant region further comprises a human Cagene segment. In some embodiments, the Ig heavy chain constant region further comprises a human Cegene segment.

[0209] In some embodiments, the IgH locus comprises human or rodent (e.g., rat or mouse) regulatory elements. In some embodiments, the regulatory element is an endogenous regulatory element. In certain embodiments, the IgH locus comprises a rodent (e.g., rat or mouse) or human intronic enhancer (Ei). In some embodiments, the IgH locus comprises a rodent (e.g., rat or mouse) or human 3' regulatory region (3' RR).

[0210] In some embodiments, the modified immunoglobulin heavy chain locus is positioned at an endogenous immunoglobulin heavy chain locus. In some embodiments, the immunoglobulin heavy chain locus replaces all or part of the endogenous immunoglobulin heavy chain locus. In certain embodiments, the modified IgH locus is located on a transgene positioned outside of the endogenous locus. In some embodiments, the endogenous IgH locus is inactivated (e.g., through the deletion, relocation and / or inversion of all or part of the endogenous Ig heavy chain locus).

[0211] Thus, in some embodiments, one or more immunoglobulin heavy chain constant regions (or portion thereof) of an immunoglobulin heavy chain locus are not deleted (i.e., intact). In some embodiments, one or more CH gene segments of an immunoglobulin heavy chain locus are altered, disrupted, deleted or replaced with, among other things, an immunoglobulin heavy chain constant region sequence as described herein (e.g., a sequence encoding a human IgG CH1-H-CH2-CH3 polypeptide) operably linked to a transmembraneand cytoplasmic encoding sequence(s) of a non-human immunoglobulin heavy chain IgG constant region gene (e.g., an Ml and / or M2 encoding sequence) and, in some embodiments, an immunoglobulin heavy chain constant region sequence (e.g., a sequence encoding a human IgE CH1-CH2-CH3-CH4 polypeptide) operably linked to a transmembrane and cytoplasmic encoding sequence(s) an IgE constant region gene. In some embodiments, all or substantially all of an immunoglobulin heavy chain constant region is replaced with a heterologous immunoglobulin heavy chain constant region. In some embodiments, a heterologous immunoglobulin heavy chain constant region sequence is operably linked to a transmembrane and cytoplasmic encoding sequence (e.g., Ml and M2 exons) of one or more IgG constant region gene. In some embodiments, a heterologous immunoglobulin heavy chain constant region sequence is operably linked to a transmembrane and cytoplasmic encoding sequence (e.g., Ml and M2 exons) of an IgE constant region gene. In some embodiments, a heterologous immunoglobulin heavy chain constant region sequence is operably linked to a transmembrane and cytoplasmic encoding sequence (e.g., an M exonfs]) of an IgA constant region gene. In some certain embodiments, one or more CH gene segments (e.g., Cg, Cg, etc.) are not deleted or replaced in an immunoglobulin heavy chain constant region that includes a heterologous immunoglobulin heavy chain constant region sequence operably linked to a transmembrane and cytoplasmic encoding sequence of one or more constant region genes as described herein. In some embodiments, a heterologous immunoglobulin heavy chain constant region sequence is a human immunoglobulin heavy chain constant region sequence. In some embodiments, an immunoglobulin heavy chain constant region that is altered, disrupted, deleted, replaced or engineered with one or more heterologous immunoglobulin heavy chain constant region sequences is a murine immunoglobulin heavy chain constant region. In some embodiments, a heterologous immunoglobulin heavy chain constant region sequence is inserted into one copy (i.e., allele) of an IgG constant region gene (e.g., Cyi, Cy2a, Cy2b, Cy2c or Cy?) of the two copies of said IgG constant region gene of an immunoglobulin heavy chain constant region, giving rise to a non-human animal that is heterozygous with respect to the heterologous immunoglobulin heavy chain constant region sequence. In some embodiments, a non-human animal is provided that is homozygous for an immunoglobulin heavy chain constant region that includes a heterologous immunoglobulin heavy chain constant region sequence as described herein.

[0212] In some embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises one or more IgG encoding CH gene segments that eachcomprise a human extracellular domain encoding sequence (e.g., a human IgG CH1-H-CH2-CH3) operably linked to a non-human transmembrane and cytoplasmic domain encoding sequence (e.g., a non-human IgG M1-M2) of the same or different IgG subclasses.

[0213] In some embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises one or more engineered IgG encoding CH gene segments as described herein and further comprises a wild-type (e.g., unmodified non-human such as rat or mouse) Cu constant region gene.

[0214] In some embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises one or more engineered IgG encoding CH gene segments as described herein and further comprises wild-type (e.g., unmodified non-human such as rat or mouse) Cu and Cg constant region genes.

[0215] In various embodiments, an engineered immunoglobulin heavy chain constant region as described herein comprises one or more further modifications including engineering constant region genes (i.e., isotypes) other than one or more IgG constant regions that comprise a sequence encoding human IgG CH1-H-CH2-CH3 or human IgG CH1-H-CH2-CH3-M1-M2 (e.g., IgGl and / or IgG2a) to be altered, modified, replaced, engineered, etc. via insertion of a human immunoglobulin heavy chain constant region sequence as described herein into one or more immunoglobulin constant region genes for IgD, IgE, IgA and an IgG that does not itself contain a sequence encoding human IgG CH1-H-CH2-CH3 or human IgG CH1-H-CH2-CH3-M1-M2 as described herein (e.g., IgG2b and / or IgG3).

[0216] In some certain embodiments, an engineered immunoglobulin heavy chain constant region provided herein comprises a wild-type Cugene segment, a wild-type Cg gene segment, a Cy3 gene segment comprising a sequence encoding human IgG3 CH1-H-CH2-CH3 in the place of CH1-H-CH2-CH3 exons and operably linked to Ml -M2 exons of said Cy3 gene segment, a Cyigene segment comprising a sequence encoding human IgG4 CH1-H-CH2-CH3 in the place of CH1-H-CH2-CH3 exons and operably linked to Ml -M2 exons of said Cyigene segment, a Cy2b gene segment comprising a sequence encoding human IgG2 CH1-H-CH2-CH3 in the place of CH1-H-CH2-CH3 exons and operably linked to Ml -M2 exons of said Cy2b gene segment, a Cy2a gene segment comprising a sequence encoding human IgGl CH1-H-CH2-CH3 in the place of CH1-H-CH2-CH3 exons and operably linked to Ml -M2 exons of said Cy2a gene segment (and / or a Cy2c gene segment comprising a sequence encoding human IgGl CH1-H-CH2-CH3 in the place of CH1-H-CH2-CH3 exons and operably linked to Ml -M2 exons of said Cy2c gene segment), a Cegene segment comprising a sequence encoding human IgE CHI-CH2-CH3-CH4 in the place of CH1-CH2-CH3-CH4 exons and operably linked to Ml -M2 exons of said an Cegene segment, and a Cagene segment comprising a sequence encoding human IgAl or IgA2 CH1-H-CH2-CH3 in the place of CH1-H-CH2-CH3 exons and operably linked to M exon(s) of said Cagene segment.

[0217] In some certain embodiments, an engineered immunoglobulin heavy chain constant region provided herein comprises a wild-type Cu gene segment, a wild-type Cg gene segment, a Cy3 gene segment comprising a sequence encoding human IgG3 CH1-H-CH2-CH3-M1-M2 in the place of CH1-H-CH2-CH3-M1-M2 exons and operably linked to the switch region of said Cy3 gene segment, a Cyigene segment comprising a sequence encoding human IgG4 CH1-H-CH2-CH3-M1-M2 in the place of CH1-H-CH2-CH3-M1-M2 exons and operably linked to the switch region of said a Cyigene segment, a Cy2b gene segment comprising a sequence encoding human IgG2 CH1-H-CH2-CH3-M1-M2 in the place of CH1-H-CH2-CH3-M1-M2 exons and operably linked to the switch region of said Cy2b gene segment, a Cy2a gene segment comprising a sequence encoding human IgGl CH1-H-CH2-CH3-M1-M2 in the place of CH1-H-CH2-CH3-M1-M2 exons and operably linked to the switch region of said Cy2a gene segment (and / or a Cy2c gene segment comprising a sequence encoding human IgGl CH1-H-CH2-CH3-M1-M2 in the place of CH1-H-CH2-CH3-M1-M2 exons and operably linked to the switch region of said Cy2c gene segment), a Cegene segment comprising a sequence encoding human IgE CH1-CH2-CH3-CH4 in the place of CH1-CH2-CH3-CH4 exons and operably linked to Ml -M2 exons of said an Cegene segment, and a Cagene segment comprising a sequence encoding human IgAl or IgA2 CH1-H-CH2-CH3 in the place of CH1-H-CH2-CH3 exons and operably linked to M exon(s) of said Cagene segment.

[0218] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin heavy chain locus (e.g., an engineered endogenous rodent immunoglobulin heavy chain locus) comprising one or more unrearranged human VL gene segments and one or more unrearranged human JL gene segments that are upstream of (e.g., operably linked to) one or more rodent (e.g., rat or mouse) immunoglobulin heavy chain constant region genes (e.g., one or more endogenous rodent (e.g., rat or mouse) immunoglobulin heavy chain constant region genes). In other words, such a genetically modified rodent comprises a hybrid heavy chain locus with both light chain (e.g., light chain variable region) and heavy chain (e.g., heavy chain constant region) sequences. Such an engineered immunoglobulin heavy chain locus isreferred to herein as an “LoH locus.” Rodents including an LoH locus are exemplified in, e.g., U. S. Patent Nos. 9,686,970 and U. S. Patent Publication No. 2013 / 0212719, each of which is incorporated by reference in its entirety. In some embodiments, one or more unrearranged human VL gene segments and one or more unrearranged human JL gene segments are one or more unrearranged human VK gene segments and one or more unrearranged human JK gene segments. Such engineered immunoglobulin heavy chain locus is referred to herein as a “KoH” locus. Rodents including a KoH locus are exemplified in, e.g., PCT Publication No. WO 2012018764. In some embodiments, one or more unrearranged human VL gene segments and one or more unrearranged human JL gene segments are one or more unrearranged human Vl gene segments and one or more unrearranged human Jl gene segments. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is homozygous at an LoH locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is heterozygous at an LoH locus.

[0219] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises an LoH locus, produces an antibody comprising, inter alia, immunoglobulin chains, where each immunoglobulin chain comprises a human light chain variable domain operably linked to a rodent (e.g., rat or mouse) heavy chain constant domain, e.g., in response to antigenic stimulation.

[0220] In some embodiments, a genetically modified rodent (e.g., rat or mouse) as provided herein has a genome (e.g., a germline genome) comprising a modification including a deletion of a nucleic acid sequence encoding a CHI domain of an endogenous IgG constant region gene, referred to herein as a “CHI delete modification.” In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a CHI delete modification, produces an IgG heavy chain antibody comprising, inter alia, immunoglobulin heavy chains, where each immunoglobulin heavy chain lacks a CHI domain, in whole or in part. In other words, a genetically modified rodent (e.g., rat or mouse) as provided herein has a genome (e.g., a germline genome) comprising a heavy chain only immunoglobulin encoding sequence comprising an unrearranged human heavy chain variable region in operable linkage to an endogenous heavy chain constant region, wherein the endogenous heavy chain constant region comprises (1) an intact endogenous IgM gene that encodes an IgM isotype that associates with light chain and (2) a non-IgM gene, e.g., an IgG gene, lacking a sequence that encodes a functional CHI domain, wherein the non-IgM gene encodes a non-IgM isotype lacking a CHI domain capable of covalently associating with a light chain constant domain.In some embodiments, an IgG antibody produced also lacks a cognate light chain and secretes an IgG heavy chain only antibody into its serum. Exemplary rodents comprising a CHI delete modification are described, e.g., in US Patent No. 8,754,287 US Patent Appln. No. 2015 / 0289489, and PCT Publication No. WO2022126113, each incorporated herein by reference in its entirety.

[0221] In some embodiments, a genetically modified rodent (e.g., rat or mouse) as provided herein has a genome (e.g., a germline genome) comprising an engineered immunoglobulin heavy chain (e.g., HoH, UHC, LoH) locus (e.g., an engineered endogenous rodent immunoglobulin heavy chain locus) lacking a functional endogenous rodent Adam6 gene.

[0222] In some embodiments, a genetically modified rodent (e.g., rat or mouse) as provided herein has a genome (e.g., a germline genome) comprising one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, one or more rodent ADAM6 polypeptides is or comprises mouse ADAM6a. In some embodiments, one or more rodent ADAM6 polypeptides is or comprises mouse ADAM6b. In some embodiments, one or more rodent ADAM6 polypeptides is or comprises mouse ADAM6a and mouse ADAM6b. In some embodiments, one or more rodent ADAM6 polypeptides comprises only one mouse ADAM6 polypeptide (i.e., either a mouse ADAM6a polypeptide or a mouse ADAM6b polypeptide). Rodents including one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are exemplified in, e.g., U. S. Patent Nos. 8,642,835; 8,697,940; 9,706,759; 10,130,081; 10,238,093, U. S. Patent Publication No. 2013 / 0212719, and US Publication No. 2021 / 0059229, each of which is incorporated by reference in its entirety. In some embodiments, a genetically modified rodent (e.g., rat or mouse) as provided expresses one or more rodent (e.g., rat or mouse) ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, a genetically modified rodent (e.g., rat or mouse) as provided has a genome (e.g., a germline genome) comprising one or more nucleotide sequences encoding one or more rodent (e.g., rat or mouse) ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof that are included on the same chromosome as an engineered immunoglobulin heavy chain (e.g., HoH, UHC, LoH) locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) as provided has a genome (e.g., a germlinegenome) comprising an engineered immunoglobulin heavy chain (e.g., HoH, UHC, LoH) locus comprising one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof. In some embodiments, a genetically modified rodent (e.g., rat or mouse) as provided has a genome (e.g., a germline genome) comprising one or more nucleotide sequences encoding one or more rodent (e.g., rat or mouse) ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof in place of a human Adam6 pseudogene. In some embodiments, a genetically modified rodent (e.g., rat or mouse) as provided has a genome (e.g., germline genome) comprising one or more nucleotide sequences encoding one or more rodent (e.g., rat or mouse) ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof that replace a human Adam6 pseudogene.

[0223] In some embodiments, a genetically modified rodent as provided has a genome (e.g., a germline genome) comprising one or more human VH gene segments comprising a first and a second human VH gene segment, and one or more nucleotide sequences encoding one or more rodent (e.g., rat or mouse) ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof between the first human VH gene segment and the second human VH gene segment. In some embodiments, a first human VH gene segment is VHI-2 and a second human VH gene segment is VH6-1.

[0224] In some embodiments, a genetically modified rodent as provided has a genome (e.g., a germline genome) comprising one or more human VH gene segments, and one or more nucleotide sequences encoding one or more rodent (e.g., rat or mouse) ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof upstream (i.e., 5') of the one or more human VH gene segments.

[0225] In some embodiments, one or more nucleotide sequences encoding one or more rodent (e.g., a rat or mouse) ADAM6 polypeptides, functional orthologs, functional homologs, or functional fragments thereof are between a human VH gene segment and a human DH gene segment.

[0226] In some embodiments, one or more nucleotide sequences encoding one or more rodent ADAM6 polypeptides restore or enhance fertility in a male rodent.

[0227] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin light chain locus (e.g., an engineered endogenous rodent immunoglobulin light chain locus)comprising one or more unrearranged human VL gene segments and one or more unrearranged human JL gene segments that are upstream of (e.g., operably linked to) one or more immunoglobulin light chain constant region genes. In some embodiments, one or more unrearranged human VL gene segments and one or more unrearranged human JL gene segments are one or more unrearranged human VK gene segments and one or more unrearranged human JK gene segments. In some embodiments, one or more unrearranged human VL gene segments and one or more unrearranged human JL gene segments are one or more unrearranged human Vl gene segments and one or more unrearranged human Jl gene segments. In some embodiments, one or more unrearranged immunoglobulin light chain constant region genes is or comprises a CK. In some embodiments, one or more unrearranged immunoglobulin light chain constant region genes is or comprises a Cl.

[0228] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin light chain locus (e.g., an engineered endogenous rodent immunoglobulin light chain locus) comprising one or more unrearranged human VK gene segments and one or more unrearranged human JK gene segments that are upstream of (e.g., operably linked to) a CK gene. Such an engineered immunoglobulin light chain locus is referred to herein as a “KoK locus.” Rodents including a KoK locus are exemplified in, e.g., U. S. Patent Nos. 6,596,541; 8,642,835; and 8,697,940, each of which is incorporated by reference in its entirety. In some embodiments, an immunoglobulin K light chain constant region gene of a KoK locus is a rodent (e.g., rat or mouse) CK gene. In some embodiments, an immunoglobulin K light chain constant region gene of a KoK locus is an endogenous rodent (e.g., rat or mouse) CK gene. In some embodiments, an immunoglobulin K light chain constant region gene of a KoK locus is an endogenous rodent (e.g., rat or mouse) CK gene at an endogenous immunoglobulin K light chain locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is homozygous at a KoK locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is heterozygous at a KoK locus.

[0229] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a KoK locus, produces an antibody comprising, inter alia, K light chains, where each K light chain comprises a human K light chain variable domain operably linked to a rodent (e.g., rat or mouse) K light chain constant domain, e.g., in response to antigenic stimulation.

[0230] In some embodiments, one or more unrearranged human VK gene segments includes at least six human VK gene segments. In some embodiments, one or more unrearranged human VK gene segments includes at least 16 human VK gene segments. In some embodiments, one or more unrearranged human VK gene segments includes at least 30 human VK gene segments. In some embodiments, one or more unrearranged human VK gene segments includes at least 40 human VK gene segments. In some embodiments, one or more unrearranged human JK gene segments includes at least five human JK gene segments.

[0231] In some embodiments, one or more unrearranged human VK gene segments includes at least 16 human VK gene segments, and one or more unrearranged human JK gene segments includes at least five human JK gene segments. Such an engineered immunoglobulin light chain locus is referred to herein as a “Veloclmmune® 1 KoK locus.” In some embodiments, one or more unrearranged human VK gene segments includes at least 30 human VK gene segments, and one or more unrearranged human JK gene segments includes at least five human JK gene segments. Such an engineered immunoglobulin light chain locus is referred to herein as a “Veloclmmune® 2 KoK locus.” In some embodiments, one or more unrearranged human VK gene segments includes at least 40 human VK gene segments, and one or more unrearranged human JK gene segments includes at least five human JK gene segments. Such an engineered immunoglobulin light chain locus is referred to herein as a “Veloclmmune® 3 KoK locus.”

[0232] In some embodiments, one or more unrearranged human VK gene segments includes all human VK gene segments. In some embodiments, one or more unrearranged human VK gene segments includes all functional human VK gene segments. In some embodiments, one or more unrearranged human VK gene segments includes a contiguous (e.g., unmodified) human sequence including human IGKV3D-7 to human IGKV4-1 from a human kappa light immunoglobulin gene locus.

[0233] In some embodiments, one or more unrearranged human JK gene segments includes all human JK gene segments. In some embodiments, one or more unrearranged human JK gene segments includes a contiguous (e.g., unmodified) human sequence including human IGKJ1 to human IGKJ5 from a human kappa light immunoglobulin gene locus.

[0234] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin light chain locus (e.g., an engineered endogenous rodent immunoglobulin light chain locus) that comprises a deletion of all endogenous mouse immunoglobulin K variable region genesegments (e.g., deletion of a contiguous mouse immunoglobulin K gene sequence from mouse IGKV2-137 to IGKJ5 on mouse chromosome 6). In some embodiments, an endogenous mouse immunoglobulin K sequence is replaced (e.g., by homologous recombination) with a contiguous sequence of human immunoglobulin K sequence. In some embodiments, the human immunoglobulin K sequence comprises all human immunoglobulin K variable gene segment sequence (i.e., a contiguous sequence from human IGKV3D-7 to IGKJ5 on chromosome 2). In some embodiments, the human immunoglobulin K sequence is at least 500 kb in size (i.e., at least 500kb, at least lOOkb, at least l,000kb). In some embodiments, the replacement of the endogenous mouse immunoglobulin light chain locus is performed with a single recombination step. Exemplary embodiments can be found, e.g., in PCT Publication No. W02020169022, which is hereby incorporated by reference in its entirety).

[0235] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin light chain locus (e.g., an engineered endogenous rodent immunoglobulin light chain locus) comprising one or more unrearranged human VI gene segments upstream of (e.g., operably linked to) one or more unrearranged human JI gene segments and one or more Ck genes. Such an engineered immunoglobulin light chain locus is referred to herein as an “LoL locus.” Mice including an LoL locus are exemplified in, e.g., U. S. Patent Nos. 9,012,717; 9,226,484; 9,029,628, and U. S. Patent Publication No. 2018 / 0125043, each of which is incorporated by reference in its entirety. In some embodiments, the one or more unrearranged human JI gene segments and one or more Ck genes of an LoL locus are present in Jl-Cl clusters. In some embodiments, one or more Ck genes of an LoL locus comprise one or more human Ck genes. In some embodiments, one or more Ck genes of an LoL locus comprise one or more mouse Ck genes. In some embodiments, one or more Ck genes of an LoL locus comprise one or more human Ck genes and one or more mouse Ck genes. In some embodiments, one or more mouse Ck genes of an LoL locus comprise a mouse Cll gene. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is homozygous at an LoL locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is heterozygous at an LoL locus.

[0236] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises an LoL locus, produces an antibody comprising, inter alia, k light chains, where each k light chain comprises a human k light chain variable domain operably linked to a rodent (e.g., rat or mouse) k light chain constant domain, e.g., in response to antigenicstimulation. In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises an LoL locus, produces an antibody comprising, inter alia, light chains, where each 1 light chain comprises a human 1 light chain variable domain operably linked to a human 1 light chain constant domain, e.g., in response to antigenic stimulation.

[0237] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin light chain locus comprising one or more unrearranged human VI gene segments and one or more unrearranged human JI gene segments upstream of (e.g., operably linked to) a CK gene. Such an engineered immunoglobulin light chain locus is referred to herein as an “LoK locus.” Rodents including an LoK locus are exemplified in, e.g., U. S. Patent Nos. 9,006,511 and 9,035,128, each of which is incorporated by reference in its entirety. In some embodiments, a CK gene of an LoK locus is a rodent (e.g., rat or mouse) CK gene. In some embodiments, a CK gene of an LoK locus is an endogenous rodent (e.g., rat or mouse) CK gene. In some embodiments, a CK gene of an LoK locus is an endogenous rodent (e.g., rat or mouse) CK gene at an endogenous immunoglobulin K light chain locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is homozygous at an LoK locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is heterozygous at an LoK locus.

[0238] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises an LoK locus, produces an antibody comprising, inter alia, light chains, where each light chain comprises a human A light chain variable domain operably linked to a rodent (e.g., rat or mouse) K light chain constant domain, e.g., in response to antigenic stimulation.

[0239] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin K light chain locus (e.g., an engineered endogenous rodent immunoglobulin K light chain locus) comprising one or more unrearranged human VI gene segments and one or more unrearranged human JI gene segments upstream of (e.g., operably linked to) a Ck gene. Such an engineered immunoglobulin light chain locus is referred to herein as an “LiK locus.” Rodents including an LiK locus are exemplified in, e.g., U. S. Patent Publication No.2019 / 0223418, which is incorporated by reference in its entirety. In some embodiments, a Ck gene of an LiK locus is a rodent (e.g., rat or mouse) Ck gene. In some embodiments, a Ck gene of an LiK locus is a mouse Cll gene. In some embodiments, a genetically modifiedrodent (e.g., rat or mouse) is homozygous at an LiK locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is heterozygous at an LiK locus.

[0240] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises an LiK locus, produces an antibody comprising, inter alia, light chains, where each 1 light chain comprises a human A light chain variable domain operably linked to a rodent (e.g., rat or mouse) 1 light chain constant domain, e.g., in response to antigenic stimulation.

[0241] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin K light chain locus (e.g., an engineered endogenous rodent immunoglobulin K light chain locus) comprising one or more unrearranged human VI gene segments upstream of (e.g., operably linked to) one or more unrearranged human JI gene segments and one or more human Ck genes. In some embodiments, the one or more unrearranged human JI gene segments and one or more Ck genes of such an engineered immunoglobulin K light chain locus are present in Jl-Cl clusters. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is homozygous for such an engineered immunoglobulin K light chain locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is heterozygous for such an engineered immunoglobulin K light chain locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises such an engineered immunoglobulin K light chain locus, produces an antibody comprising, inter alia, k light chains, where each k light chain comprises a human k light chain variable domain operably linked to a human k light chain constant domain, e.g., in response to antigenic stimulation.

[0242] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin k light chain locus comprising all human VI gene segments, human JI gene segments, and human Ck gene segments (e.g., at the endogenous rodent K or k light chain locus). In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) a deletion of all or part of its endogenous k light chain locus.

[0243] In some embodiments, an endogenous mouse immunoglobulin k light chain locus sequence is replaced (e.g., by homologous recombination) with a contiguous sequence of human immunoglobulin k light chain locus sequence. In some embodiments, the human immunoglobulin k light chain locus sequence comprises all of the human immunoglobulin k light chain locus (i.e., a contiguous sequence). In some embodiments, the humanimmunoglobulin 1 light chain locus sequence is at least 500 kb in size (i.e., at least 500kb, at least lOOkb, at least l,000kb). In some embodiments, the replacement of the endogenous mouse immunoglobulin lambda light chain locus is performed with a single recombination step. Exemplary embodiments can be found, e.g., in PCT Publication No. W02020169022, which is hereby incorporated by reference in its entirety).

[0244] In some embodiments, a genetically modified rodent (e.g., rat or mouse) has a germline genome comprising a limited human light chain variable region repertoire.Exemplary genetically modified rodents, comprising human V(D)J gene segments having a germline genome comprising a limited human light chain variable region repertoire are described in, e.g., U. S. Patent Nos. 9,796,788; 10,130,081; 10,143,186; 10,167,344;10,412,940; and 10,130,081; as well as W02019008123, WO2020247623, WO2020132557, WO2017035274, WO2019236670, WO2019236671, and WO2021244522, each of which is hereby incorporated by reference in its entirety. In some embodiments, a limited human light chain variable region repertoire comprises a limited number of human VL gene segments. In some embodiments, a limited number of human VL gene segments comprises two human VL gene segments. In some embodiments, a limited number of human Vi.gene segments is one human VL gene segment. For example, in some embodiments a limited number of human VL gene segments is one human VK gene segment. One human VK gene segment can be, e.g., a human VK1-39 gene segment, a human VK3-15 gene segment, a human VK 3-11 gene segment, or a human VK3-20 gene segment. In some embodiments a limited number of human VL gene segments is one human Vl gene segment. One human Vl gene segment can be, e.g., a human Vll-51 gene segment, a human V15-45 gene segment, a human Vll-44 gene segment, a human Vll-40 gene segment, a human V13-21 gene segment, or a human V12-14 gene segment.

[0245] In some embodiments, a limited human light chain variable region repertoire comprises one or more JL gene segments. In some embodiments, a limited human light chain variable region repertoire comprises one JL gene segment. In some embodiments, one JL gene segment is a JK gene segment. In some embodiments, one JL gene segment is a Jl gene segment. In some embodiments, one JL gene segment is a human JL gene segment. In some embodiments, one JL gene segment is a mouse JL gene segment.

[0246] In some embodiments, a limited human light chain variable region repertoire comprises (i) a human VK gene segment and a human JK gene segment, (ii) a human VK genesegment and a mouse JK gene segment, (iii) a human VK gene segment and a human JI gene segment, or (iv) a human VK gene segment and a mouse JI gene segment.

[0247] In some embodiments, a limited human light chain variable region repertoire comprises (i) a human VI gene segment and a human JI gene segment, (ii) a human VI gene segment and a mouse JI gene segment, (iii) a human VI gene segment and a human JK gene segment, or (iv) a human VI gene segment and a mouse JK gene segment.

[0248] In some embodiments, a limited human light chain variable region repertoire comprises (i) a human VK1-39 gene segment and a human JK5 gene segment, (ii) a human VK1-39 gene segment and a human JKI gene segment, (iii) a human VK3-20 gene segment and a human JKI gene segment, (iv) a human VK3-20 gene segment and a human JK5 gene segment, (v) a human VK1-39 gene segment and a human JK4 gene segment, or (vi) a human VK3-11 gene segment and a human JKI gene segment.

[0249] In some embodiments, a limited human light chain variable region repertoire comprises (i) a human VK1-39 gene segment and a mouse JK2 gene segment, (ii) a human VK3-20 gene segment and a mouse JK2 gene segment, or (iii) a human VK3-15 gene segment and a mouse JK2 gene segment.

[0250] In some embodiments, a limited human light chain variable region repertoire comprises (i) a human Vll-51 gene segment and a human J12 gene segment, (ii) a human VZ.5-45 gene segment and a human J12 gene segment, (iii) a human Vz.1-44 gene segment and a human J12 gene segment, (iv) a human Vz.1-40 gene segment and a human J12 gene segment, (v) a human VA3-21 gene segment and a human J12 gene segment, or (vi) a human V 2-14 gene segment and a human J12 gene segment.

[0251] In some embodiments, a limited human light chain variable region repertoire is operably linked to a CK gene segment. In some embodiments, a CK gene segment is human. In some embodiments, a CK gene segment is mouse. In some embodiments, a mouse CK gene segment is an endogenous mouse CK gene segment, e.g., at an endogenous mouse immunoglobulin K light chain locus. In some embodiments, a mouse CK gene segment is at an endogenous mouse immunoglobulin 1 light chain locus.

[0252] In some embodiments, a limited human light chain variable region repertoire is operably linked to a Ck gene segment. In some embodiments, a Ck gene segment is human. In some embodiments, a Ck gene segment is mouse. In some embodiments, a mouse Ck gene segment is an endogenous mouse Ck gene segment, e.g., at an endogenous mouseimmunoglobulin 1 light chain locus. In some embodiments, a mouse C gene segment is at an endogenous mouse immunoglobulin K light chain locus.

[0253] In some embodiments, a genetically modified mouse is heterozygous for a limited human light chain variable region repertoire. In some embodiments, a genetically modified mouse is homozygous for a limited human light chain variable region repertoire.

[0254] In some embodiments, a genetically modified rodent comprises an engineered immunoglobulin light chain locus (e.g., an engineered endogenous rodent immunoglobulin light chain locus) comprising a restricted light chain variable region sequence, comprising a limited human light chain variable region repertoire. In some embodiments, a limited human light chain variable region repertoire comprises one or two human light chain V gene segments and one or more human light chain J gene segments. In some embodiments, a limited human light chain variable region repertoire is operably linked to a light chain constant region gene segment. In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprising a limited human light chain variable region repertoire comprises in its genome (e.g., its germline genome) exactly two unrearranged human light chain V gene segments and one or more unrearranged human light chain J gene segments operably linked to a light chain constant region sequence. Such an engineered immunoglobulin light chain locus is referred to herein as a “DLC locus.” In some embodiments, a genetically modified rodent comprising a limited human light chain variable region repertoire comprises in its genome (e.g., its germline genome) a single rearranged light chain variable region locus comprising a single human light chain V gene segment rearranged to a single human light chain J gene segment. In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprising a limited human light chain variable region repertoire comprises in its genome (e.g., its germline genome) a single rearranged light chain variable region locus operably linked to a light chain constant region sequence, where the single rearranged light chain variable region locus comprises a single human light chain V gene segment rearranged to a single human light chain J gene segment. Such an engineered immunoglobulin light chain locus is referred to herein as “ULC locus.” As used herein, the phrase “ULC locus” is interchangeable with “universal light chain locus” or “common light chain locus.”

[0255] In some embodiments, a genetically modified rodent (e.g., a rat or mouse) comprises a limited light chain antibody repertoire that is at least 90% pure for a single human VL domain (e.g,, a single rearranged human VL domain) or a somatically hypermutated version thereof. In some embodiments, a genetically modified rodent (e.g., arat or mouse) comprises a limited light chain antibody repertoire that is at least 95% pure for a single human VL domain (e.g,, a single rearranged human VL domain) or a somatically hypermutated version thereof. In some embodiments, a genetically modified rodent (e.g., a rat or mouse) comprises a limited light chain antibody repertoire that is at least 99% pure for a single human VL domain (e.g,, a single rearranged human VL domain) or a somatically hypermutated version thereof.

[0256] In some embodiments, a genetically modified rodent (e.g., rat or mouse) has a germline genome comprising a limited human K light chain variable region repertoire. In some embodiments, a genetically modified rodent comprises an engineered immunoglobulin K light chain locus (e.g., an engineered endogenous rodent immunoglobulin K light chain locus) comprising a limited human K light chain variable region repertoire. In some embodiments, a limited human K light chain variable region repertoire comprises one or two human VK gene segments and one or more human JK gene segments. In some embodiments, a limited human K light chain variable region repertoire operably linked to a light chain constant region gene segment. In some embodiments, a genetically modified rodent as provided comprises a limited human K light chain variable region repertoire operably linked to a CK gene segment.

[0257] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) a limited human K light chain variable region repertoire, wherein the limited human K light chain variable region repertoire comprises a single rearranged human K light chain variable region (VK / JK). A single rearranged human K light chain variable region comprises a human VK gene segment joined to a human JK gene segment. In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin K light chain locus (e.g., an engineered endogenous rodent immunoglobulin K light chain locus) comprising a single rearranged human K light chain variable region upstream of (e.g., operably linked to) a CK gene. Such an engineered immunoglobulin light chain locus is referred to as a “KULC locus” and is an example of a ULC locus. Rodents including a KULC locus are exemplified in, e.g., U. S. Patent Nos. 10,130,081 and 10,143,186, each of which is incorporated by reference in its entirety.

[0258] In some embodiments, a single rearranged human K light chain variable region comprises a human VK gene segment and a human JK gene segment. In some embodiments, a human VK gene segment is a human VK1-39 gene segment, a human VK3-11 gene segment,or a human VK3-20 gene segment. In some embodiments, a human JK gene segment is a human JKI gene segment, a human JK2 gene segment, a human JK3 gene segment, a human JK4 gene segment, or a human JK5 gene segment. In some embodiments, a human VK gene segment is a human VK1-39 gene segment, and a human JK gene segment is a human JK5 gene segment. In some embodiments, a single rearranged human K light chain variable region is a human VK1-39 / JK5. In some embodiments, a human VK gene segment is a human VK3-20 gene segment, and a human JK gene segment is a human JKI gene segment. In some embodiments, a human VK gene segment is a human VK1-39 gene segment, and a human JK gene segment is a human JK4 gene segment. In some embodiments, a human VK gene segment is a human VK3-11 gene segment, and a human JK gene segment is a human JKI gene segment. In some embodiments, a single rearranged human K light chain variable region is a human VK3-20 / JK1.

[0259] In some embodiments, a CK gene of a KULC locus is a rodent (e.g., rat or mouse) CK gene. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is homozygous at a KULC locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is heterozygous at a KULC locus.

[0260] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a KULC locus, lacks endogenous VK and / or JK gene segments that are capable of rearranging to form an endogenous K light chain variable region. In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a KULC locus, lacks endogenous VI and / or Jk gene segments that are capable of rearranging to form an endogenous 1 light chain variable region.

[0261] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a KULC locus, produces an antibody comprising, inter alia, K light chains, where each K light chain comprises a human K light chain variable domain operably linked to a rodent (e.g., rat or mouse) K light chain constant domain, e.g., in response to antigenic stimulation. In some embodiments, all K light chains expressed by B cells of a genetically modified rodent (e.g., rat or mouse), which comprises a KULC locus, comprise human K light chain variable domains expressed from the single rearranged human K light chain variable region or a somatically hypermutated version thereof.

[0262] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises in its genome (e.g., its germline genome) an engineered immunoglobulin K light chain locus (e.g., an engineered endogenous rodent immunoglobulin K light chain locus)comprising exactly two unrearranged human VK gene segments and one or more unrearranged human JK gene segments operably linked to a K light chain constant region sequence of (e.g., operably linked to) a CK gene. Such an engineered immunoglobulin K light chain locus is referred to herein as a “KDLC locus,” and is an example of a DLC locus.Rodents including a KDLC locus are exemplified in, e.g., U. S. Patent Nos. 9,796,788;10,167,344; 10,412,940; and 10,130,081, each of which is incorporated by reference in its entirety.

[0263] In some embodiments, exactly two unrearranged human VK gene segments comprise a human VK1-39 gene segment and a human VK3-20 gene segment. In some embodiments, one or more unrearranged human JK gene segments comprises two human JK gene segments. In some embodiments, one or more unrearranged human JK gene segments comprises three human JK gene segments. In some embodiments, one or more unrearranged human JK gene segments comprises four human JK gene segments. In some embodiments, one or more unrearranged human JK gene segments comprises five human JK gene segments. In some embodiments, one or more unrearranged human JK gene segments comprises a human JKI gene segment, a human JK2 gene segment, a human JK3 gene segment, a human JK4 gene segment, a human JK5 gene segment, or a combination thereof.

[0264] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a KDLC locus, comprises in its genome (e.g., germline genome) exactly two unrearranged human VK gene segments and five unrearranged human JK gene segments. In some embodiments, exactly two unrearranged human VK gene segments comprises a human VK1-39 gene segment and a human VK3-20 gene segment, and five unrearranged human JK gene segments comprise a human JKI gene segment, a human JK2 gene segment, a human JK3 gene segment, a human JK4 gene segment, and a human JK5 gene segment.

[0265] In some embodiments, a CK gene of a KDLC locus is a rodent (e.g., rat or mouse) CK gene. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is homozygous at a KDLC locus. In some embodiments, a genetically modified rodent (e.g., rat or mouse) is heterozygous at a KDLC locus.

[0266] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a KDLC locus, lacks endogenous immunoglobulin VK and / or JK gene segments that are capable of rearranging to form an endogenous immunoglobulin K light chain variable region. In some embodiments, a genetically modified rodent (e.g., rat ormouse), which comprises a KDLC locus, lacks endogenous VI and / or JI gene segments that are capable of rearranging to form an endogenous 1 light chain variable region.

[0267] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a KDLC locus, produces an antibody comprising, inter alia, K light chains, where each K light chain comprises a human K light chain variable domain operably linked to a rodent (e.g., rat or mouse) K light chain constant domain, e.g., in response to antigenic stimulation.

[0268] In some embodiments, a genetically modified rodent (e.g., rat or mouse) has a genome (e.g., germline genome) comprising a limited human X light chain variable region repertoire. In some embodiments, a genetically modified rodent (e.g., rat or mouse) has a genome (e.g., germline genome) comprising an engineered immunoglobulin K light chain locus (e.g., an engineered endogenous rodent immunoglobulin K light chain locus) comprising a limited human X light chain variable region repertoire. In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises an engineered immunoglobulin K light chain locus (e.g., an engineered endogenous rodent immunoglobulin K light chain locus) comprising a limited human X light chain variable region repertoire, wherein the limited human X light chain variable region repertoire comprises one or two human VX gene segments and one or more human JX gene segments. In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises a limited human X light chain variable region repertoire operably linked to a light chain constant region gene segment. In some embodiments, a genetically modified rodent (e.g., rat or mouse) as provided comprises a limited human X light chain variable region repertoire operably linked to a rodent (e.g., rat or mouse) CK gene segment. In some embodiments, a genetically modified rodent as provided comprises a limited human X light chain variable region repertoire operably linked to a rodent (e.g., rat or mouse) CX gene segment.

[0269] In some embodiments, a genetically modified rodent (e.g., rat or mouse) has a genome (e.g., germline genome) comprising an engineered immunoglobulin K light chain locus (e.g., an engineered endogenous rodent immunoglobulin K light chain locus) that comprises a limited human X light chain variable region repertoire, wherein the limited human X light chain variable region repertoire comprises a single rearranged human immunoglobulin X light chain variable region (VX / JX). A single rearranged human X light chain variable region comprises a human VX gene segment joined to a human JX genesegment. In some embodiments, a genetically modified rodent comprises a limited human X light chain variable region repertoire operably linked to a rodent (e.g., rat or mouse) CK or CX gene segment (e.g., a mouse CXI gene segment). Such an engineered immunoglobulin light chain locus is an example of a ULC locus and is referred to herein as a “ULCiK locus.” Rodents including a ULCiK locus are exemplified in, e.g., WO2020 / 247623, which is incorporated by reference in its entirety.

[0270] In some embodiments, a human VX gene segment is selected from a group consisting of: VX4-69, VX8-61, VX4-60, VX6-57, VX10-54, VX5-52, VX1-51, VX9-49, VX1-47, VX7-46, VX5-45, VX1-44, VX7-43, VX1-40, VX5-37, VX1-36, VX3-27, VX3-25, VX2-23, VX3-22, VX3-21, VX3-19, VX2-18, VX3-16, VX2-14, VX3-12, VX2-11, VX3-10, VX3-9, VX2-8, VX4-3, and VX3-1. In some embodiments, a human VX gene segment is selected from a group consisting of: VX5-52, VX1-51, VX9-49, VXL47, VX7-46, VX5-45, VX1-44, VX7-43, VX1-40, VX5-37, VX1-36, VX3-27, VX3-25, VX2-23, VX3-22, VX3-21, VX3-19, VX2-18, VX3-16, VX2-14, VX3-12, VX2-11, VX3-10, VX3-9, VX2-8, VX4-3, and VX3-1. In some embodiments, a human VX gene segment is selected from a group consisting of: VX1-51, VX5-45, VX1-44, VX1-40, VX3-21, and VX2-14. In some embodiments, a human VX gene segment is VX1-51 or VX2-14. In some embodiments, a human JX gene segment is selected from a group consisting of: JX1, JX2, JX3, JX6, and JX7. In some embodiments, a human JX gene segment is selected from a group consisting of: JX1, JX2, JX3, and JX7. In some embodiments, a human JX gene segment is JX2.

[0271] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a ULCiK locus, lacks endogenous VK and / or JK gene segments that are capable of rearranging to form an endogenous K light chain variable region. In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a ULCiK locus, lacks endogenous VX and / or JX gene segments that are capable of rearranging to form an endogenous X light chain variable region.

[0272] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a ULCiK locus, produces an antibody comprising, inter alia, light chains, wherein each light chain comprises a human X light chain variable domain operably linked to a (e.g., rat or mouse) light chain constant domain (e.g., a CX or CK domain), e.g., in response to antigenic stimulation. In some embodiments, all light chains expressed by B cells of a genetically modified rodent (e.g., rat or mouse), which comprises a ULCiK locus, comprisehuman 1 light chain variable domains expressed from the single rearranged human X light chain variable region or a somatically hypermutated version thereof.

[0273] In some embodiments, a genetically modified rodent (e.g., rat or mouse) has a genome (e.g., germline genome) comprising an engineered immunoglobulin K light chain locus (e.g., an engineered endogenous rodent immunoglobulin K light chain locus) that comprises a limited human X light chain variable region repertoire, wherein the limited human 1 light chain variable region repertoire comprises two unrearranged human VX gene segments and one or more unrearranged human JX gene segments. In some embodiments, a limited human X light chain variable region repertoire comprises two unrearranged human VX gene segments and four unrearranged human JX gene segments. In some embodiments, a limited human X light chain variable region repertoire comprises two unrearranged human VX gene segments and five unrearranged human JX gene segments. In some embodiments, a genetically modified rodent comprises a limited human X light chain variable region repertoire operably linked to a rodent (e.g., rat or mouse) CX gene segment (e.g., a mouse CXI gene segment). Such an engineered immunoglobulin light chain locus is an example of a DLC locus and is referred to herein as a “DLCiK locus.” Rodents including a DLCiK locus are exemplified in, e.g., WO2020 / 247623, which is incorporated by reference in its entirety.

[0274] In some embodiments, a germline genome of the genetically modified rodent is homozygous for a engineered immunoglobulin K light chain locus comprising a limited human X light chain variable region repertoire. In some embodiments, a germline genome of the genetically modified rodent is heterozygous for a engineered immunoglobulin K light chain locus comprising a limited human X light chain variable region repertoire.

[0275] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a DLCiK locus, lacks endogenous immunoglobulin VK and / or JK gene segments that are capable of rearranging to form an endogenous immunoglobulin K light chain variable region. In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a DLCiK locus, lacks endogenous VX and / or JX gene segments that are capable of rearranging to form an endogenous X light chain variable region.

[0276] In some embodiments, a genetically modified rodent (e.g., rat or mouse), which comprises a DLCiK locus, produces an antibody comprising, inter alia, light chains, where each light chain comprises a human X light chain variable domain operably linked to arodent (e.g., rat or mouse) light chain constant domain (e.g., a Ck or CK domain), e.g., in response to antigenic stimulation.

[0277] In some embodiments, a genetically modified rodent comprises a ULC locus that is modified to reduce or eliminate somatic hypermutation (SHM). In some embodiments, a ULC locus is modified to increase the distance of the human rearranged V / J gene sequence from the transcription start site to reduce or eliminate SHM. In some embodiments, a ULC locus is modified to comprise an intronic enhancer (e.g., EiK enhancer) upstream (i.e., 5') of the rearranged V / J gene sequence to reduce or eliminate SHM.

[0278] In some embodiments, a genetically modified rodent (e.g., rat or mouse) comprises an exogenous terminal deoxynucleotidyl transferase (TdT) gene. Rodents including an exogenous TdT are exemplified in, e.g., U. S. Patent Publication No.2019 / 0223418 and PCT Publication No. WO 2017 / 210586, each of which is incorporated by reference in its entirety. In some embodiments, a rodent (e.g., rat or mouse) that comprises an exogenous TdT gene can have increased antigen receptor diversity when compared to a rodent without an exogenous TdT gene.

[0279] In some embodiments, a rodent as described herein has a genome comprising an exogenous TdT gene operably linked to a transcriptional control element.

[0280] In some embodiments, a transcriptional control element includes a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin K light chain transcriptional control element, an immunoglobulin k light chain transcriptional control element, or any combination thereof.

[0281] In some embodiments, an exogenous TdT is located at an immunoglobulin K light chain locus, an immunoglobulin k light chain locus, an immunoglobulin heavy chain locus, a RAG1 locus, or a RAG2 locus.

[0282] In some embodiments, a TdT is a human TdT. In some embodiments, a TdT is a short isoform of TdT (TdTS).

[0283] In some embodiments, a rodent as described herein has a genome comprising an engineered or recombinant diversity cluster with an immunoglobulin heavy chain variable region.

[0284] In some embodiments, an engineered or recombinant heavy chain diversity (DH) cluster comprises an insertion of one or more DH segments that are each operablylinked to a 23-mer recombination signal sequence (RSS). In some embodiments, an engineered or recombinant DH cluster comprises an engineered DH region comprising at least one DH gene segment operably linked to a 23-mer RSS and an unrearranged DH gene segment flanked on one side by a 12-mer RSS and on the other side by another 12-mer RSS, which DH gene segments are operably linked such that they are able to join in a DH-DH recombination event according to the 12 / 23 rule. In some embodiments, the engineered or recombinant DH gene segments are human. Exemplary engineered or recombinant DH clusters are shown in e.g., WO 2019241692).

[0285] In some embodiments, an engineered or recombinant DH cluster comprises at least two DH gene segments that are fused (i.e., a D-D fusion), wherein the DH gene segments, when recombined with V and J gene segments in the heavy chain locus, generate a rearranged VDDJ transcript that encodes an immunoglobulin heavy chain variable domain. Exemplary recombinant DH gene segments are shown in e.g., U. S. Patent Pub. No.2019 / 0127757.

[0286] In some embodiments, a rodent as described herein has a genome comprising an engineered or recombinant diversity cluster with an immunoglobulin heavy chain variable region such that the rodent produces human antibodies that comprise HCDR3 sequences that are least 10 amino acids in length (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more amino acids in length).

[0287] In some embodiments, a rodent described herein comprises an immunoglobulin heavy chain variable region that includes an engineered DH region, wherein the engineered DH region includes one or more nucleotide sequences that each encode a nonimmunoglobulin polypeptide of interest, or portion thereof (e.g., a chemokine receptor (e.g., atypical chemokine receptor (ACKR), e.g., a D6 chemokine decoy receptor), a conotoxin, or a tarantula toxin), wherein the immunoglobulin heavy chain variable region is operably linked to an immunoglobulin heavy chain constant region. Exemplary engineered DH regions are shown in e.g., WO 2017123804.

[0288] In some embodiments, rodents described herein comprise an engineered immunoglobulin locus capable of making anchor-modified immunoglobulin polypeptides. In some embodiments, an immunoglobulin locus comprises a nucleic acid sequence comprising a modified immunoglobulin variable (V) gene segment that encodes an anchor-modified immunoglobulin polypeptide. In some embodiments, a modified immunoglobulin V genesegment comprises a nucleic acid sequence encoding an anchor between a nucleic acid sequence encoding an immunoglobulin signal peptide and a nucleic acid sequence encoding the framework region (FR)1, complementarity determining region (CDR)l, FR2, CDR2, FR3, and CDR3 of a germline immunoglobulin V gene segment, or a variant thereof. In some embodiments, an anchor modified immunoglobulin polypeptide comprises in operable linkage: (i) an immunoglobulin signal peptide, (ii) an anchor, and (iii) an FR1, CDR1, FR2, CDR2, FR3, and CDR3 of the germline immunoglobulin V segment, or a variant thereof. In some embodiments, an anchor comprises a receptor-binding portion of a nonimmunoglobulin polypeptide of interest that binds a cognate receptor. Exemplary engineered V regions are shown in e.g., WO 2022140219.Human Immune System Rodents with Humanized plgRImmunodeficient Rodents

[0289] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein further have been genetically modified to be immunodeficient. In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein further have been genetically modified to knock-out the endogenous Rag2 gene and / or knock-out the endogenous IL2rg gene.

[0290] In some embodiments, a rodent (e.g., mouse) provided herein may include at least one null allele for the Rag2 gene (“recombination activating gene 2”, wherein the coding sequence for the mouse gene may be found at Genbank Accession No.NM_009020.3). In some embodiments, a rodent (e.g., mouse) includes two null alleles for Rag2. In other words, the rodent (e.g., mouse) is homozygous null for Rag2. As another example, a rodent (e.g., mouse) includes at least one null allele for the IL2rg gene (“interleukin 2 receptor, gamma”, also known as the common gamma chain, or yC, wherein the coding sequence for the mouse gene may be found at Genbank Accession No. NM 013563.3). In some embodiments, the rodent (e.g., mouse) includes two null alleles for IL2rg. In other words, the rodent (e.g., mouse) is homozygous null for IL2rg, i.e., it is IL2rg / _(or IL2rgY / _where the IL2rg gene is located on the X chromosome as in mouse). In some embodiments, the rodent (e.g., mouse) includes a null allele for both Rag2 and IL2rg, i.e., it is Rag2 / _IL2rg / _(or Rag2 / _IL2rgY / _where the IL2rg gene is located on the X chromosome as in mouse).

[0291] In some embodiments, provided genetically modified rodents are SRG rodents. In some embodiments, provided genetically modified rodents lack mature rodent B cells, T cells, and / or NK cells.Humanized SIRPa Loci

[0292] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein have been further genetically modified to express a human or humanized SIRPa protein encoded by a nucleic acid operably linked to a SIRPa promoter.

[0293] Signal regulatory proteins (SIRPs) constitute a family of cell surface glycoproteins which are expressed on lymphocytes, myeloid cells (including macrophages, neutrophils, granulocytes, myeloid dendritic cells, and mast cells) and neurons (e.g., see Barclay and Brown, 2006, Nat Rev Immunol 6, 457-464, which is incorporated herein by reference). The reported SIRP genes include at least SIRPa, SIRP3, SIRPp, SIRPy, and SIRP8 and can be categorized by their respective ligands and types of signaling in which they are involved. SIRPa (also referred to as CD172A, SHPS1, P84, MYD-1, BIT and PTPNS1) is expressed on immune cells of the myeloid lineage and functions as an inhibitory receptor via an immunoreceptor tyrosine-based inhibitory motif (ITIM). SIRPa expression has also been observed on neurons. Reported ligands for SIRPa include, most notably, CD47, but also include surfactant proteins A and D. The role of SIRPa, in particular, has been investigated in respect of its inhibitory role in the phagocytosis of host cells by macrophages. For example, CD47 binding to SIRPa on macrophages, triggers inhibitory signals that negatively regulates phagocytosis. Alternatively, positive signaling effects mediated through SIRPa binding have been reported (Shultz et al., 1995, J Immunol 154, 180-91, which is incorporated herein by reference). SIRPa has been shown to improve cell engraftment in immunodeficient mice (Strowig et al. Proc Natl Acad Sci USA 2011; 108: 13218-13223, which is incorporated herein by reference).

[0294] Polypeptide sequences for wild-type human SIRPa and the nucleic acid sequences that encode wild-type human SIRPa may be found at Genbank Accession Nos. NP_001035111.1 and NM_001040022.1 (isoform 1 and transcript variant 1);NP_001035112.1 and NM_001040023.2 (isoform 1 and transcript variant 2);NP_001317657.1 and NM_001330728.1 (isoform 2 and transcript variant 4); and NP_542970.1 and NM_080792.3 (isoform 1 and transcript variant 3). The SIRPa gene isconserved in at least chimpanzee, Rhesus monkey, dog, cow, mouse, rat, and chicken. The genomic locus encoding the wild-type human SIRPa protein may be found in the human genome at Chromosome 20; NC_000020.ll (1894167-1940592). In some embodiments, human SIRPa protein is encoded by exons 2 through 9 at this locus. As such, in some embodiments, a nucleic acid sequence including coding sequence for human SIRPa includes one or more of exons 2-9 of the human SIRPa gene. In some instances, the nucleic acid sequence also includes aspects of the genomic locus of the human SIRPa, e.g., introns, 3' and / or 5' untranslated sequence (UTRs). In some instances, the nucleic acid sequence includes whole regions of the human SIRPa genomic locus. In some instances, the nucleic acid sequence includes exons 2-4 of the human SIRPa genomic locus.

[0295] Exemplary humanized SIRPa sequences are set forth below. For protein sequences, signal peptides are underlined and transmembrane and cytoplasmic sequences are italicized. Representative mouse SIRPa cDNA, mouse SIRPa protein, human SIRPa cDNA, and human SIRPa protein sequences are described in U. S. Pat. No. 11,019,810, which is incorporated by reference herein in its entirety.

[0296] Humanized SIRPa Protein (SEP ID NO: 33) MEPAGPAPGRLGPLLLCLLLSASCFCTGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFR GAGPGRELI YNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGT ELSVRAKPSAPWSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIH STAKWLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYP QRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVS ARPKEQGSYITAADNNATHNWNVFIGVGVACALLWLLMAALYLLRIKQKKAKGSTSSTRLHEPEKNAR EITQIQDTNDINDITYADLNLPKEKKPAPRAPEPNNHTEYASIETGKVPRPEDTLTYADLDMVHLSRAQP APKPEPSFSEYASVQVQRK

[0297] In some embodiments, rodents (e.g., mice) provided herein express humanized SIRPa proteins on the surface of immune cells (e.g., myeloid cells) resulting from a genetic modification of an endogenous locus that encodes a SIRPa protein. Suitable examples described herein include rodents, in particular, mice.

[0298] A humanized SIRPa gene, in some embodiments, comprises genetic material from a heterologous species (e.g., humans), wherein the humanized SIRPa gene encodes a SIRPa protein that comprises the encoded portion of the genetic material from the heterologous species. In some embodiments, a humanized SIRPa gene of the present disclosure comprises genomic DNA of a heterologous species that corresponds to the extracellular portion of a SIRPa protein that is expressed on the plasma membrane of a cell.rodents, embryos, cells and targeting constructs for making rodents, rodent embryos, and cells containing said humanized SIRPa gene are also provided.

[0299] In some embodiments, an endogenous SIRPa gene is deleted. In some embodiments, an endogenous SIRPa gene is altered, wherein a portion of the endogenous SIRPa gene is replaced with a heterologous sequence (e.g., a human SIRPa sequence in whole or in part). In some embodiments, all or substantially all of an endogenous SIRPa gene is replaced with a heterologous gene (e.g., a human SIRPa gene). In some embodiments, a portion of a heterologous SIRPa gene is inserted into an endogenous rodent SIRPa gene at an endogenous SIRPa locus. In some embodiments, the heterologous gene is a human gene. In some embodiments, the modification or humanization is made to one of the two copies of the endogenous SIRPa gene, giving rise to a rodent which is heterozygous with respect to the humanized SIRPa gene. In other embodiments, a rodent is provided that is homozygous for a humanized SIRPa gene.

[0300] In some embodiments, a rodent (e.g., mouse) of the present disclosure contains a human SIRPa gene in whole or in part at an endogenous rodent SIRPa locus. Thus, such rodents (e.g., mice) can be described as having a heterologous SIRP gene. The replaced, inserted or modified SIRPa gene at the endogenous SIRPa locus can be detected using a variety of methods including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or a gain or loss of allele assay. In some embodiments, the rodent is heterozygous with respect to the humanized SIRPa gene.

[0301] In various embodiments, a humanized SIRPa gene according to the present disclosure includes a SIRPa gene that has a second, third and fourth exon each having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a second, third and fourth exon that appear in a human SIRPa gene.

[0302] In various embodiments, a humanized SIRPa gene according to the present disclosure includes a SIRPa gene that has a nucleotide coding sequence (e.g., a cDNA sequence) at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to nucleotides 352-1114 that appear in a human SIRPa cDNA sequence.

[0303] In various embodiments, a humanized SIRPa protein produced by a rodent (e.g., mouse) of the present disclosure has an extracellular portion having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to an extracellular portion of a human SIRPa protein.

[0304] In various embodiments, a humanized SIRPa a protein produced by a rodent (e.g., mouse) of the present disclosure has an extracellular portion having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 28-362 that appear in a human SIRPa protein.

[0305] In various embodiments, a humanized SIRPa protein produced by a rodent (e.g., mouse) of the present disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an amino acid sequence of a humanized SIRPa protein as set forth in SEQ ID NO: 33.

[0306] Compositions and methods for making rodents (e.g., mice) that express a humanized SIRPa protein, including specific polymorphic forms or allelic variants (e.g., single amino acid differences), are provided, including compositions and methods for making rodents that express such proteins from a human promoter and a human regulatory sequence. In some embodiments, compositions and methods for making rodents that express such proteins from an endogenous promoter and an endogenous regulatory sequence are also provided. The methods include inserting the genetic material encoding a human SIRPa protein in whole or in part at a precise location in the genome of a rodent (e.g., mouse) that corresponds to an endogenous SIRPa gene thereby creating a humanized SIRPa gene that expresses a SIRPa protein that is human in whole or in part. In some embodiments, the methods include inserting genomic DNA corresponding to exons 2-4 of a human SIRPa gene into an endogenous SIRPa gene of the rodent (e.g., mouse) thereby creating a humanized gene that encodes a SIRPa protein that contains a human portion containing amino acids encoded by the inserted exons.

[0307] A humanized SIRPa gene approach employs a relatively minimal modification of the endogenous gene and results in natural SIRPa-mediated signal transduction in the rodent, in various embodiments, because the genomic sequence of the SIRPa sequences are modified in a single fragment and therefore retain normal functionality by including necessary regulatory sequences. Thus, in such embodiments, the SIRPa gene modification does not affect other surrounding genes or other endogenous SIRP genes. Further, in various embodiments, the modification does not affect the assembly of a functional receptor on theplasma and maintains normal effector functions via binding and subsequent signal transduction through the cytoplasmic portion of the receptor which is unaffected by the modification.

[0308] In addition to mice having humanized SIRPa genes as described herein, also provided herein are other genetically modified non-human animals that comprise humanized SIRPa genes. In some embodiments, such non-human animals comprise a humanized SIRPa gene operably linked to an endogenous SIRPa promoter. In some embodiments, such non-human animals express a humanized SIRPa protein from an endogenous locus, wherein the humanized SIRPa protein comprises amino acid residues 28-362 of a human SIRPa protein.

[0309] Humanized SIRPa polypeptides, loci encoding humanized SIRPa polypeptides and non-human animals expressing humanized SIRPa polypeptides are described in U. S. Pat. No. 11,019,810, WO 2014 / 039782, WO 2014 / 071397, and WO 2016 / 168212, each of which is incorporated by reference herein in its entirety.Humanized CD47 Loci

[0310] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein have been further genetically modified to express a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0311] CD47, originally named integrin-associated protein (IAP) for its role in signal transduction from integrins on immune cells, is a transmembrane protein that includes an N-terminal immunoglobulin V (IgV) domain, five transmembrane domains, and a short C-terminal intracytoplasmic tail. The intracytoplasmic tail differs in length according to four alternatively spliced isoforms that have been identified. CD47 (or IAP) was initially described as being expressed on all tissues (isoform 2), neurons (isoform 4) and keratinocytes and macrophages (isoform 1; see Reinhold et al. (1995) J. Cell Sci. 108:3419-3425). In addition to integrins, CD47 is known to interact with several other cell surface proteins such as, for example, thrombospondin and members of the SIRP family. Most notably, CD47 interacts with SIRPa and leads to bidirectional signaling that regulates a variety of cell-to-cell responses such as, for example, inhibition of phagocytosis and T cell activation. Indeed, CD47-SIRPa interaction has come into focus in recent years for its role in providing tumor cells with the capacity to evade immune surveillance. CD47 binding to SIRPa normally provides protection through anti-phagocytic signals (“don't eat me”) for normal cells.However, it has been discovered that tumors also express anti-phagocytic signals, including CD47, to evade destruction by phagocytosis. Interestingly, CD47 is known to be upregulated in several hematologic cancers and contribute to both the growth and dissemination of tumors (Chao et al. (2012) Curr Opin Immunol. 24(2): 225-232, which is incorporated herein by reference).

[0312] Exemplary polypeptide sequences for wild-type human CD47 and the nucleic acid sequence that encodes wild-type human CD47 may be found at Genbank Accession Nos. NP_001369235.1 and NM_001382306.1 (isoform 3 and transcript variant 3); NP_001768.1 and NM_001777.4 (isoform 1 and transcript variant 1); NP_942088.1 and NM_198793.3 (isoform 2 and transcript variant 2); and XP_005247966.1 and XM_005247909.3 (isoform XI and transcript variant XI). The CD47 gene is conserved in at least chimpanzee, Rhesus monkey, dog, cow, mouse, rat, and chicken. The genomic locus encoding the wild-type human CD47 protein may be found in the human genome at Chromosome 3; NC_000003.12 (c 108091031-108043091). In some embodiments, human CD47 protein is encoded by exons 1 through 11 at this locus. As such, in some embodiments, a nucleic acid sequence including coding sequence for human CD47 includes one or more of exons 1-11 of the human CD47 gene. In some instances, the nucleic acid sequence also includes aspects of the genomic locus of the human CD47, e.g., introns, 3' and / or 5' untranslated sequence (UTRs). In some instances, the nucleic acid sequence includes whole regions of the human CD47 genomic locus. In some instances, the nucleic acid sequence includes exons 2-7 of the human CD47 genomic locus.

[0313] Exemplary humanized CD47 sequences are set forth below. For humanized protein sequences, non-human (e.g., mouse) sequences are indicated in regular font, human sequences are indicated in bold font, and signal peptides are underlined. Representative mouse CD47 cDNA, mouse CD47 protein, human CD47 cDNA, and human CD47 protein sequences are described in U. S. Pat. Pub. No. 2021 / 0161112 Al, which is incorporated by reference herein in its entirety.Humanized CD47 amino acid isoform 1 (SEP ID NO: 34) MWPLAAALLLGSCCCGSAQLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNT TEVYVKWKFKGRD I YTFDGALNKS TVP TDF S S AKI EVSQLLKGDAS LKMDK SDAVSHTGNYTCEVTELTREGETIIELKYRWSWFSPNENILIVIFPIFAILLF WGQFGI KTLKYRSGGMDEKT I ALLVAGLVI TVI VI VGAI LFVPGE Y S LKNAT GLGLI VT S TGI LI LLHY YVF S TAI GLT SFVI Al LVI QVI AY I LAWGLS LC I AAC IPMHGPLLI SGLS I LALAQLLGLVYMKFVEHumanized CD47 amino acid isoform 2 (SEP ID NO: 35) MWPLAAALLLGSCCCGSAQLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNT TEVYVKWKFKGRD I YTFDGALNKS TVP TDF S S AKI EVSQLLKGDAS LKMDK SDAVSHTGNYTCEVTELTREGETIIELKYRWSWFSPNENILIVIFPIFAILLF WGQFGI KTLKYRSGGMDEKT I ALLVAGLVI TVI VI VGAI LFVPGE Y S LKNAT GLGLI VT S TGI LI LLHY YVF S TAI GLT SFVI Al LVI QVI AY I LAWGLS LC I AAC IPMHGPLLI SGLS I LALAQLLGLVYMKFVAS'MQfi TIQPPRNRHumanized CD47 amino acid isoform 3 (SEP ID NO: 36) MWPLAAALLLGSCCCGSAQLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNT TEVYVKWKFKGRD I YTFDGALNKS TVP TDF S S AKI EVSQLLKGDAS LKMDK SDAVSHTGNYTCEVTELTREGETIIELKYRWSWFSPNENILIVIFPIFAILLF WGQFGI KTLKYRSGGMDEKT I ALLVAGLVI TVI VI VGAI LFVPGE Y S LKNAT GLGLI VT S TGI LI LLHY YVF S TAI GLT SFVI Al LVI QVI AY I LAWGLS LC I AAC IPMHGPLLI SGLS I LALAQLLGLVYMKFVAS'MQP TIQPPRKAVEEPLNEHumanized CD47 amino acid isoform 4 (SEP ID NO: 37) MWPLAAALLLGSCCCGSAQLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNT TEVYVKWKFKGRD I YTFDGALNKS TVP TDF S S AKI EVSQLLKGDAS LKMDK SDAVSHTGNYTCEVTELTREGETIIELKYRWSWFSPNENILIVIFPIFAILLF WGQFGI KTLKYRSGGMDEKT I ALLVAGLVI TVI VI VGAI LFVPGE Y S LKNAT GLGLI VT S TGI LI LLHY YVF S TAI GLT SFVI Al LVI QVI AY I LAWGLS LC I AAC IPMHGPLLI SGLSILALAQLLGLVYMKFVASNQRTTQPPRKAVEEPLNAFKESKG MMNDE

[0314] In some embodiments, rodents (e.g., mice) provided herein express humanized CD47 proteins on the surface of cells resulting from a genetic modification of an endogenous locus that encodes a CD47 protein.

[0315] A humanized CD47 gene, in some embodiments, comprises genetic material from a heterologous species (e.g., humans), wherein the humanized CD47 gene encodes a CD47 protein that comprises the encoded portion of the genetic material from the heterologous species. In some embodiments, a humanized CD47 gene of the present disclosure comprises genomic DNA of a heterologous species that encodes the extracellular portion of a CD47 protein that is expressed on the plasma membrane of a cell. In some embodiments, a humanized CD47 gene of the present disclosure comprises genomic DNA of a heterologous species that encodes the extracellular portion and the transmembrane portion of a CD47 protein that is expressed on the plasma membrane of a cell. Rodents, embryos, cells and targeting constructs for making rodents, rodent embryos, and cells containing said humanized CD47 gene are also provided.

[0316] In some embodiments, an endogenous CD47 gene is deleted. In some embodiments, an endogenous CD47 gene is altered, wherein a portion of the endogenousCD47 gene is replaced with a heterologous sequence (e.g., a human CD47 sequence, in whole or in part). In some embodiments, all or substantially all of an endogenous CD47 gene is replaced with a heterologous gene (e.g., a human CD47 gene). In some embodiments, a portion of a heterologous CD47 gene is inserted into an endogenous CD47 gene at an endogenous CD47 locus. In some embodiments, the heterologous gene is a human gene. In some embodiments, the modification or humanization is made to one of the two copies of the endogenous CD47 gene, giving rise to a rodent (e.g., mouse) that is heterozygous with respect to the humanized CD47 gene. In other embodiments, a rodent (e.g., mouse) is provided that is homozygous for a humanized CD47 gene.

[0317] In some embodiments, a rodent (e.g., mouse) of the present disclosure contains a human CD47 gene, in whole or in part, at an endogenous CD47 locus. Thus, such rodents (e.g., mice) can be described as having a heterologous CD47 gene. The replaced, inserted, modified or altered CD47 gene at the endogenous CD47 locus can be detected using a variety of methods including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or a gain or loss of allele assay. In some embodiments, the rodent (e.g., mouse) is heterozygous with respect to the humanized CD47 gene. In some embodiments, the rodent (e.g., mouse) is homozygous for the humanized CD47 gene.

[0318] In various embodiments, a humanized CD47 gene according to the present disclosure includes a CD47 gene that has a second, third, fourth, fifth, sixth and seventh exon each having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a second, third, fourth, fifth, sixth and seventh exon that appear in a human CD47 gene.

[0319] In various embodiments, a humanized CD47 gene according to the present disclosure includes a CD47 gene that has a first exon and exon(s) downstream of exon 7 (e.g., eighth and ninth exons of isoform 2) each having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a respective exon that appears in a mouse CD47 gene.

[0320] In various embodiments, a humanized CD47 gene according to the present disclosure includes a CD47 gene that has a 5' untranslated region and a 3' untranslated region each having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a 5' untranslated region and a 3' untranslated region that appear in a mouse CD47 gene.

[0321] In various embodiments, a humanized CD47 gene according to the present disclosure includes a CD47 gene that has a nucleotide coding sequence (e.g., a cDNA sequence) at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a nucleotide coding sequence that appears in a human CD47 nucleotide coding sequence.

[0322] In various embodiments, a humanized CD47 protein produced by a rodent (e.g., mouse) of the present disclosure has an extracellular portion having an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an extracellular portion of a human CD47 protein.

[0323] In various embodiments, a humanized CD47 protein produced by a rodent (e.g., mouse) of the present disclosure has an extracellular portion having an amino acid sequence that is identical to amino acid residues 19-141 that appear in a human CD47 protein.

[0324] In various embodiments, a humanized CD47 protein produced by a rodent (e.g., mouse) of the present disclosure has an N-terminal immunoglobulin V domain having an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an N-terminal immunoglobulin V domain of a human CD47 protein.

[0325] In various embodiments, a humanized CD47 protein produced by a rodent (e.g., mouse) of the present disclosure has an N-terminal immunoglobulin V domain having an amino acid sequence that is identical to amino acid residues 19-127 that appear in a human CD47 protein.

[0326] In various embodiments, a humanized CD47 protein produced by a rodent (e.g., mouse) 1 of the present disclosure has an N-terminal immunoglobulin V domain and five transmembrane domains each having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an N-terminal immunoglobulin V domain and five transmembrane domains of a human CD47 protein.

[0327] In various embodiments, a humanized CD47 protein produced by a rodent (e.g., mouse) of the present disclosure has an intracytoplasmic tail having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to an intracytoplasmic tail of a mouse CD47 protein.

[0328] In various embodiments, a humanized CD47 protein produced by a rodent (e.g., mouse) of the present disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 16-292 that appear in a human CD47 protein.

[0329] In various embodiments, a humanized CD47 protein produced by a rodent (e.g., mouse) of the present disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 19-292 that appear in a human CD47 protein.

[0330] In various embodiments, a humanized CD47 protein produced by a rodent (e.g., mouse) of the present disclosure has an amino acid sequence that is identical to amino acid residues 19-292 (or 16-292) that appear in a human CD47 protein.

[0331] In various embodiments, a humanized CD47 protein produced by a rodent (e.g., mouse) of the present disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an amino acid sequence of a humanized CD47 protein comprising a sequence of any one of SEQ ID NOs: 34-37.

[0332] In various embodiments, a humanized CD47 protein produced by a rodent (e.g., mouse) of the present disclosure has an amino acid sequence that is identical to an amino acid sequence of a humanized CD47 protein comprising a sequence of any one of SEQ ID NOs: 34-37.

[0333] Compositions and methods for making rodents that express a humanized CD47 protein, including specific polymorphic forms, allelic variants (e.g., single amino acid differences) or alternatively spliced isoforms, are provided, including compositions and methods for making rodents (e.g., mice) that express such proteins from a human promoter and a human regulatory sequence. In some embodiments, compositions and methods for making rodents (e.g., mice) that express such proteins from an endogenous promoter and an endogenous regulatory sequence are also provided. The methods include inserting the genetic material encoding a human CD47 protein in whole or in part at a precise location in the genome of a rodent (e.g., mouse) that corresponds to an endogenous CD47 gene therebycreating a humanized CD47 gene that expresses a CD47 protein that is human in whole or in part. In some embodiments, the methods include inserting genomic DNA corresponding to exons 2-7 of a human CD47 gene into an endogenous CD47 gene of the rodent (e.g., mouse) thereby creating a humanized gene that encodes a CD47 protein that contains a human portion containing amino acids encoded by the inserted exons.

[0334] Where appropriate, the coding region of the genetic material or polynucleotide sequence(s) encoding a human CD47 protein in whole or in part may be modified to include codons that are optimized for expression in the non-human animal (e.g., see U. S. Pat. Nos.5,670,356 and 5,874,304). Codon optimized sequences are synthetic sequences, and preferably encode the identical polypeptide (or a biologically active fragment of a full-length polypeptide which has substantially the same activity as the full length polypeptide) encoded by the non-codon optimized parent polynucleotide. In some embodiments, the coding region of the genetic material encoding a human CD47 protein, in whole or in part, may include an altered sequence to optimize codon usage for a particular cell type (e.g., a rodent cell). For example, the codons of the genomic DNA corresponding to exons 2-7 of a human CD47 gene to be inserted into an endogenous CD47 gene of a rodent (e.g., mouse) may be optimized for expression in a cell of the rodent. Such a sequence may be described as a codon-optimized sequence.

[0335] A humanized CD47 gene approach employs a relatively minimal modification of the endogenous gene and results in natural CD47-mediated signal transduction in the rodent (e.g., mouse), in various embodiments, because the genomic sequence of the CD47 sequences are modified in a single fragment and therefore retain normal functionality by including necessary regulatory sequences. Thus, in such embodiments, the CD47 gene modification does not affect other surrounding genes or other endogenous CD47-interacting genes (e.g., thrombospondin, SIRPs, integrins, etc.). Further, in various embodiments, the modification does not affect the assembly of a functional CD47 transmembrane protein on the plasma membrane and maintains normal effector functions via binding and subsequent signal transduction through the cytoplasmic portion of the protein which is unaffected by the modification.

[0336] Although embodiments employing a humanized CD47 gene in a mouse (i.e., a mouse with a CD47 gene that encodes a CD47 protein that includes a human portion and a mouse portion) are extensively discussed herein, other non-human animals that comprise a humanized CD47 gene are also provided. In some embodiments, such non-human animalscomprise a humanized CD47 gene operably linked to an endogenous CD47 promoter. In some embodiments, such non-human animals express a humanized CD47 protein from an endogenous locus, wherein the humanized CD47 protein comprises amino acid residues 16-292 (or 19-141 or 19-127) of a human CD47 protein.

[0337] Humanized CD47 polypeptides, loci encoding humanized CD47 polypeptides and rodents expressing humanized CD47 polypeptides are described in U. S. Pat. Publication No. 2021 / 0161112, which is incorporated by reference herein.Humanized M-CSF

[0338] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein have been further genetically modified to express a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter. By a human M-CSF protein, it is a meant a protein that is human M-CSF or is substantially identical to human M-CSF, e.g., it is 80% or more identical, 85% or more identical, 90% or more identical, or 95% or more identical to human M-CSF, for example, 97%, 98%, or 99% identical to human M-CSF. A nucleic acid sequence that encodes a human M-CSF protein is, therefore, a polynucleotide that comprises coding sequence for a human M-CSF protein, i.e., human M-CSF or a protein that is substantially identical to human M-CSF.

[0339] M-CSF (also known as CSF-1, for “colony stimulating factor 1”) is a cytokine that controls the production, differentiation, and function of macrophages. Polypeptide sequence for human M-CSF and the nucleic acid sequence that encodes for human M-CSF may be found at Genbank Accession Nos. NP_000748.4 and NM_000757.6 (isoform a and transcript variant 1); NP_757349.2 and NM_172210.3 (isoform b and transcript variant 2); NP_757350.2 and NM_172211.4 (isoform c and transcript variant 3) and NP_757351.2; and NM 172212.3 (isoform a and transcript variant 4). The genomic locus encoding the human M-CSF protein may be found in the human genome at Chromosome 1; NC_000001.ll (109910506-109930992). Protein sequence is encoded by exons 1 through 8 at this locus, while exon 9 comprises untranslated sequence. As such, a nucleic acid sequence comprising coding sequence for human M-CSF comprises one or more of exons 1-8 of the human M-CSF gene. In some instances, the nucleic acid sequence also comprises aspects of the genomic locus of the human M-CSF, e.g., introns, 3' and / or 5' untranslated sequence (UTRs). In some instances, the nucleic acid sequence comprises whole regions of the human M-CSFgenomic locus. In some instances, the nucleic acid sequence comprises exon 2 of the human M-CSF genomic locus to 633 nucleotides downstream of noncoding exon 9.

[0340] In some embodiments, in the genetically modified rodents (e.g., mice) provided herein, the nucleic acid sequence that encodes a human M-CSF protein is operably linked to one or more regulatory sequences of the rodent (e.g., mouse) M-CSF gene. Rodent (e.g., mouse) M-CSF regulatory sequences are those sequences of the rodent (e.g., mouse) M-CSF genomic locus that regulate rodent (e.g., mouse) M-CSF expression, for example, 5' regulatory sequences, e.g., the M-CSF promoter, M-CSF 5' untranslated region (UTR), etc.; 3' regulatory sequences, e.g., the 3'UTR; and enhancers, etc. For example, mouse M-CSF is located on chromosome 3, NC_000069.7, at about positions C107668048-107648364, and the mouse M-CSF coding sequence may be found at Genbank Accession Nos. NM_007778.4 (transcript variant 1 encoding isoform 1), NM_001113529.1 (transcript variant 2 encoding isoform 2), and NM 001113530.1 (transcript variant 3 encoding isoform 1). The regulatory sequences of mouse M-CSF are well defined in the art, and may be readily identified using in silico methods, e.g., by referring to the above Genbank Accession Nos. on the UCSC Genome Browser, on the world wide web at genome.ucsc.edu, or by experimental methods as described in the art, e.g., Abboud et al. (2003) Analysis of the Mouse CSF-1 Gene Promoter in a Transgenic Mouse Model. J. Histochemistry and Cytochemistry 51 (7):941 -949, the disclosure of which is incorporated herein by reference. In some instances, e.g., when the nucleic acid sequence that encodes a human M-CSF protein is located at the rodent (e.g., mouse) M-CSF genomic locus, the regulatory sequences operably linked to the human CSF coding sequence are endogenous, or native, to the rodent (e.g., mouse) genome, i.e., they were present in the rodent (e.g., mouse) genome prior to integration of human nucleic acid sequences.

[0341] In some instances, the genetically modified rodent (e.g., mouse) expressing a human M-CSF protein comprises one copy of the nucleic acid sequence encoding a human M-CSF protein. For example, the rodent (e.g., mouse) may be heterozygous for the nucleic acid sequence. In other words, one allele at a locus will comprise the nucleic acid sequence, while the other will be the endogenous allele. For example, as discussed above, in some instances, human M-CSF nucleic acid sequence is integrated into the rodent (e.g., mouse) M-CSF locus such that it creates a null allele for rodent (e.g., mouse) M-CSF. In some such embodiments, the humanized M-CSF mouse may be heterozygous for the nucleic acid sequence encoding, i.e., the humanized M-CSF mouse comprises one null allele for rodent(e.g., mouse) M-CSF (the allele comprising the nucleic acid sequence) and one endogenous M-CSF allele (wild type or otherwise). In other instances, the genetically modified rodent (e.g., mouse) expressing a human M-CSF protein comprises two copies of the nucleic acid sequence encoding a human M-CSF protein. For example, the rodent (e.g., mouse) may be homozygous for the nucleic acid sequence, i.e., both alleles for a locus in the diploid genome will comprise the nucleic acid sequence, i.e., the genetically modified rodent (e.g., mouse) expressing a human M-CSF protein comprises two null alleles for the mouse M-CSF (the allele comprising the nucleic acid sequence).

[0342] Human M-CSF polypeptides, loci encoding human M-CSF polypeptides and rodents expressing human M-CSF polypeptides are described in WO 2012 / 112544, WO 2014 / 039782, and WO 2014 / 071397, each of which is incorporated by reference herein.Humanized GM-CSF Loci

[0343] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein have been further genetically modified to express a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter. By a human GM-CSF protein, it is meant a protein that is human GM-CSF or is substantially identical to human GM-CSF, e.g., it is 80% or more identical, 85% or more identical, 90% or more identical, or 95% or more identical to human GM-CSF, for example, 97%, 98%, or 99% identical to human GM-CSF. A nucleic acid sequence that encodes a human GM-CSF protein is, therefore, a polynucleotide that comprises coding sequence for a human GM-CSF protein, i.e., human GM-CSF or a protein that is substantially identical to human GM-CSF.

[0344] GM-CSF is a cytokine crucial for myeloid cell development and function. GM-CSF is not cross-reactive between human and mouse. GM-CSF is highly expressed in the lung and important for lung homeostasis in vivo, as demonstrated by the fact that GM-CSF KO mice develop pulmonary alveolar proteinosis (PAP) which is characterized by protein accumulation in the lung due to defective surfactant clearance. Alveolar macrophages from GM-CSF KO mice have a defect in terminal differentiation, which leads to impaired innate immunity to pathogens in the lung. GM-CSF also stimulates the proliferation of human alveolar macrophages (AM) in vitro. GM-CSF is largely dispensable for steady-state hematopoiesis. In contrast, GM-CSF is required for inflammatory responses such as the production of proinflammatory cytokines by macrophages and the mobilization and recruitment of monocytes. GM-CSF is also essential for protective immunity against a rangeof pathogens, including M. tuberculosis. In particular, GM-CSF KO mice infected with M. tuberculosis do not develop granulomas, a hallmark of tuberculosis.

[0345] Polypeptide sequence for human GM-CSF and the nucleic acid sequence that encodes for human GM-CSF may be found at Genbank Accession Nos. NP_000749.2 and NM_000758.4, respectively. The genomic locus encoding the human GM-CSF protein may be found in the human genome at Chromosome 5; NG_033024.1 (4998-7379). Protein sequence is encoded by exons 1 through 4 at this locus. As such, a nucleic acid sequence comprising coding sequence for human GM-CSF comprises one or more of exons 1-4 of the human GM-CSF gene. In some instances, the nucleic acid sequence also comprises aspects of the genomic locus of the human GM-CSF, e.g., introns, 3' and / or 5' untranslated sequence (UTRs). In some instances, the nucleic acid sequence comprises whole regions of the human GM-CSF genomic locus.

[0346] In some embodiments, in the genetically modified rodents (e.g., mice) provided herein, the nucleic acid sequence that encodes a human GM-CSF protein is operably linked to one or more regulatory sequences of the rodent (e.g., mouse) GM-CSF gene. Rodent (e.g., mouse) GM-CSF regulatory sequences are those sequences of the rodent (e.g., mouse) GM-CSF genomic locus that regulate rodent (e.g., mouse) GM-CSF expression, for example, 5' regulatory sequences, e.g., the GM-CSF promoter, GM-CSF 5' untranslated region (UTR), etc.; 3' regulatory sequences, e.g., the 3'UTR; and enhancers, etc. For example, mouse GM-CSF is located on chromosome 11, GRCm39, NC_000077.7, at about positions c54140725-54138096, and the mouse GM-CSF coding sequence may be found at Genbank Accession No. NM_009969.4. The regulatory sequences of mouse GM-CSF are well defined in the art, and may be readily identified using in silico methods, e.g., by referring to the above Genbank Accession Nos. on the UCSC Genome Browser, on the world wide web at genome.ucsc.edu, or by experimental methods as described in the art. In some instances, e.g., when the nucleic acid sequence that encodes a human GM-CSF protein is located at the rodent (e.g., mouse) GM-CSF genomic locus, the regulatory sequences operably linked to the human GM-CSF coding sequence are endogenous, or native, to the rodent (e.g., mouse) genome, i.e., they were present in the rodent (e.g., mouse) genome prior to integration of human nucleic acid sequences.

[0347] In some instances, the genetically modified rodent (e.g., mouse) expressing a human GM-CSF protein comprises one copy of the nucleic acid sequence encoding a human GM-CSF protein. For example, the rodent (e.g., mouse) may be heterozygous for the nucleicacid sequence. In other words, one allele at a locus will comprise the nucleic acid sequence, while the other will be the endogenous allele. For example, as discussed above, in some instances, human GM-CSF nucleic acid sequence is integrated into the rodent (e.g., mouse) GM-CSF locus such that it creates a null allele for rodent (e.g., mouse) GM-CSF. In some such embodiments, the humanized GM-CSF mouse may be heterozygous for the nucleic acid sequence encoding, i.e., the humanized GM-CSF mouse comprises one null allele for rodent (e.g., mouse) GM-CSF (the allele comprising the nucleic acid sequence) and one endogenous GM-CSF allele (wild type or otherwise). In other instances, the genetically modified rodent (e.g., mouse) expressing a human GM-CSF protein comprises two copies of the nucleic acid sequence encoding a human GM-CSF protein. For example, the rodent (e.g., mouse) may be homozygous for the nucleic acid sequence, i.e., both alleles for a locus in the diploid genome will comprise the nucleic acid sequence, i.e., the genetically modified rodent (e.g., mouse) expressing a human GM-CSF protein comprises two null alleles for the mouse GM-CSF (the allele comprising the nucleic acid sequence).

[0348] Human GM-CSF polypeptides, loci encoding human GM-CSF polypeptides and rodents expressing human GM-CSF polypeptides are described in WO2011 / 044050, WO 2014 / 039782 and WO 2014 / 071397, each of which is incorporated by reference herein.Humanized TPO Loci

[0349] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein have been further genetically modified to express a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter. By a human TPO protein, it is a meant a protein that is human TPO or is substantially identical to human TPO, e.g., it is 80% or more identical, 85% or more identical, 90% or more identical, or 95% or more identical to human TPO, for example, 97%, 98%, or 99% identical to human TPO. A nucleic acid sequence that encodes a human TPO protein is, therefore, a polynucleotide that comprises coding sequence for a human TPO protein, i.e., human TPO or a protein that is substantially identical to human TPO.

[0350] Thrombopoietin (TPO) was initially identified as a growth factor that promotes the development of megakaryocytes and platelets. TPO is constitutively produced by the liver and the kidneys and released into the blood circulation. The receptor for TPO, c-Mpl, is expressed by hematopoietic stem and progenitor cells in the bone marrow. C-Mpl is also expressed on circulating platelets. However, the binding of TPO on platelets does notactivate any signaling pathway. Thus, thrombocytes act as a sink or scavengers for TPO and via this mechanism contribute to negative regulation of thrombopoiesis. Subsequently, TPO has been recognized for its important function to support the expansion and self- renewal of HSCs. TPO deficiency leads to reduced numbers of HSCs in adult mice, and the presence of TPO is needed to maintain adult HSCs in quiescence. Furthermore, TPO is required to support post-transplantation expansion of HSCs, necessary to replenish the hematopoietic compartment of irradiated hosts. Interestingly, it has been demonstrated that osteoblastic cells involved in forming the HSC niche in the bone marrow produce TPO, critical for HSC function and maintenance.

[0351] Polypeptide sequence for human TPO and the nucleic acid sequence that encodes for human TPO may be found at Genbank Accession Nos. NM_000547.6 and NP_000538.3 (transcript variant 1 and isoform a); NM_001206744.2 and NP_001193673.1 (transcript variant 6 and isoform a); NM_001206745.2 and NP_001193674.1 (transcript variant 7 and isoform b); NM_175719.4 and NP_783650.1 (transcript variant 2 and isoform b); NM_175721.3 and NP_783652.1 (transcript variant 4 and isoform d); and NM_175722.3 and NP_783653.1 (transcript variant 5 and isoform e). The genomic locus encoding the human TPO protein may be found in the human genome at Chromosome 2; NG_011581.1 (4999-134265). Protein sequence is encoded by exons 2 through 17 at this locus. As such, a nucleic acid sequence comprising coding sequence for human TPO comprises one or more of exons 2-17 of the human TPO gene. In some instances, the nucleic acid sequence also comprises aspects of the genomic locus of the human TPO, e.g., introns, 3' and / or 5' untranslated sequence (UTRs). In some instances, the nucleic acid sequence comprises whole regions of the human TPO genomic locus.

[0352] In some embodiments, in the genetically modified rodents (e.g., mice) provided herein, the nucleic acid sequence that encodes a human TPO protein is operably linked to one or more regulatory sequences of the rodent (e.g., mouse) TPO gene. Rodent (e.g., mouse) TPO regulatory sequences are those sequences of the rodent (e.g., mouse) TPO genomic locus that regulate rodent (e.g., mouse) TPO expression, for example, 5' regulatory sequences, e.g., the TPO promoter, TPO 5' untranslated region (UTR), etc.; 3' regulatory sequences, e.g., the 3'UTR; and enhancers, etc. For example, mouse TPO is located on chromosome 12, GRCm39, NC_000078.7, at about positions c30182983-30104658, and the mouse TPO coding sequence may be found at Genbank Accession No. NM_009417.3. The regulatory sequences of mouse TPO are well defined in the art, and may be readily identifiedusing in silico methods, e.g., by referring to the above Genbank Accession Nos. on the UCSC Genome Browser, on the world wide web at genome.ucsc.edu, or by experimental methods as described in the art. In some instances, e.g., when the nucleic acid sequence that encodes a human TPO protein is located at the rodent (e.g., mouse) TPO genomic locus, the regulatory sequences operably linked to the human TPO coding sequence are endogenous, or native, to the rodent (e.g., mouse) genome, i.e., they were present in the rodent (e.g., mouse) genome prior to integration of human nucleic acid sequences.

[0353] In some instances, the genetically modified rodent (e.g., mouse) expressing a human TPO protein comprises one copy of the nucleic acid sequence encoding a human TPO protein. For example, the rodent (e.g., mouse) may be heterozygous for the nucleic acid sequence. In other words, one allele at a locus will comprise the nucleic acid sequence, while the other will be the endogenous allele. In some embodiments, human TPO nucleic acid sequence is integrated into the rodent (e.g., mouse) TPO locus such that it creates a null allele for rodent (e.g., mouse) TPO. In some such embodiments, the humanized TPO mouse may be heterozygous for the nucleic acid sequence encoding, i.e., the humanized TPO mouse comprises one null allele for rodent (e.g., mouse) TPO (the allele comprising the nucleic acid sequence) and one endogenous TPO allele (wild type or otherwise). In other instances, the genetically modified rodent (e.g., mouse) expressing a human TPO protein comprises two copies of the nucleic acid sequence encoding a human TPO protein. For example, the rodent (e.g., mouse) may be homozygous for the nucleic acid sequence, i.e., both alleles for a locus in the diploid genome will comprise the nucleic acid sequence, i.e., the genetically modified rodent (e.g., mouse) expressing a human TPO protein comprises two null alleles for the mouse TPO (the allele comprising the nucleic acid sequence).

[0354] Human TPO polypeptides, loci encoding human TPO polypeptides and rodents expressing human TPO polypeptides are described in WO2011 / 044050, WO 2014 / 039782 and WO 2014 / 071397, each of which is incorporated by reference herein.Humanized IL-3 Loci

[0355] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein have been further genetically modified to express a human IL-3 protein encoded by a nucleic acid operably linked to an IL-3 promoter. By a human IL-3 protein, it is a meant a protein that is human IL-3 or is substantially identical to human IL-3, e.g., it is 80% or more identical, 85% or more identical, 90% or more identical,or 95% or more identical to human IL-3, for example, 97%, 98%, or 99% identical to human IL-3. A nucleic acid sequence that encodes a human IL-3 protein is, therefore, a polynucleotide that comprises coding sequence for a human IL-3 protein, i.e., human IL-3 or a protein that is substantially identical to human IL-3.

[0356] Like GM-CSF, IL-3 is a cytokine crucial for myeloid cell development and function. IL-3 is not cross -reactive between human and mouse. IL-3 stimulates early hematopoietic progenitors in vitro, but is dispensable for steady-state hematopoiesis in vivo. However, together with GM-CSF it is required for effective DTH responses in vivo. IL-3 also specifically stimulates the proliferation of alveolar macrophages (AM) in vitro.

[0357] Polypeptide sequence for human IL-3 and the nucleic acid sequence that encodes for human IL-3 may be found at Genbank Accession Nos. NP_000579.2 and NM_000588.4. The genomic locus encoding the human IL-3 protein may be found in the human genome at chromosome 5, GRCh38.pl4; NC_000005.10 (132060655-132063204). Protein sequence is encoded by exons 1 through 5 at this locus. As such, a nucleic acid sequence comprising coding sequence for human IL-3 comprises one or more of exons 1-5 of the human IL-3 gene. In some instances, the nucleic acid sequence also comprises aspects of the genomic locus of the human IL-3, e.g., introns, 3' and / or 5' untranslated sequence (UTRs). In some instances, the nucleic acid sequence comprises whole regions of the human IL-3 genomic locus.

[0358] In some embodiments, in the genetically modified rodents provided herein, the nucleic acid sequence that encodes a human IL-3 protein is operably linked to one or more regulatory sequences of the rodent (e.g., mouse) IL-3 gene. Rodent (e.g., mouse) IL-3 regulatory sequences are those sequences of the rodent (e.g., mouse) IL-3 genomic locus that regulate rodent (e.g., mouse) IL-3 expression, for example, 5' regulatory sequences, e.g., the IL-3 promoter, IL-3 5' untranslated region (UTR), etc.; 3' regulatory sequences, e.g., the 3'UTR; and enhancers, etc. For example, mouse IL-3 is located on chromosome 11, GRCm39, NC_000077.7, at about positions c54158105-54155911, and the mouse IL-3 coding sequence may be found at Genbank Accession No. NM_010556.4. The regulatory sequences of mouse IL-3 are well defined in the art, and may be readily identified using in silico methods, e.g., by referring to the above Genbank Accession Nos. on the UCSC Genome Browser, on the world wide web at genome.ucsc.edu, or by experimental methods as described in the art. In some instances, e.g., when the nucleic acid sequence that encodes a human IL-3 protein is located at the rodent (e.g., mouse) IL-3 genomic locus, the regulatorysequences operably linked to the human IL-3 coding sequence are endogenous, or native, to the rodent (e.g., mouse) genome, i.e., they were present in the rodent (e.g., mouse) genome prior to integration of human nucleic acid sequences.

[0359] In some instances, the genetically modified rodent (e.g., mouse) expressing a human IL-3 protein comprises one copy of the nucleic acid sequence encoding a human IL-3 protein. For example, the rodent (e.g., mouse) may be heterozygous for the nucleic acid sequence. In other words, one allele at a locus will comprise the nucleic acid sequence, while the other will be the endogenous allele. For example, as discussed above, in some instances, human IL-3 nucleic acid sequence is integrated into the rodent (e.g., mouse) IL-3 locus such that it creates a null allele for rodent (e.g., mouse) IL-3. In some such embodiments, the humanized IL-3 mouse may be heterozygous for the nucleic acid sequence encoding, i.e., the humanized IL-3 mouse comprises one null allele for rodent (e.g., mouse) IL-3 (the allele comprising the nucleic acid sequence) and one endogenous IL-3 allele (wild type or otherwise). In other instances, the genetically modified rodent (e.g., mouse) expressing a human IL-3 protein comprises two copies of the nucleic acid sequence encoding a human IL-3 protein. For example, the rodent (e.g., mouse) may be homozygous for the nucleic acid sequence, i.e., both alleles for a locus in the diploid genome will comprise the nucleic acid sequence, i.e., the genetically modified rodent (e.g., mouse) expressing a human IL-3 protein comprises two null alleles for the mouse IL-3 (the allele comprising the nucleic acid sequence).

[0360] Human IL-3 polypeptides, loci encoding human IL-3 polypeptides and rodents expressing human IL-3 polypeptides are described in WO2011 / 044050, WO 2014 / 039782 and WO 2014 / 071397, each of which is incorporated by reference herein.Humanized IL15 Loci

[0361] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein have been further genetically modified to express a human IL- 15 protein encoded by a nucleic acid operably linked to a IL- 15 promoter. As used herein, “human IL-15 protein”, means a protein that is a wild-type (or native) human IL-15 protein or a variant of a wild-type (or native) human IL- 15 protein, which retains one or more signaling functions of a wild-type (or native) human IL-15 protein, e.g., which allows for stimulation of (or signaling via) the human IL- 15 receptor, and / or which is capable of binding to the human IL- 15 receptor alpha subunit of the human IL- 15 receptor, and / or which iscapable of binding to IL-2R beta / IL-15R beta and the common y-chain (yc). Also encompassed by the term “human IL- 15 protein” are fragments of a wild-type human IL- 15 protein (or variants thereof), which retain one or more signaling functions of a wild-type human IL-15 protein, e.g., a fragment of a human IL-15 protein, which allows for stimulation of (or signaling via) the human IL- 15 receptor, and / or which is capable of binding to the human IL- 15 receptor alpha subunit of the human IL- 15 receptor, and / or which is capable of binding to IL-2R beta / IL-15R beta and the common y-chain (yc).

[0362] The term “human IL- 15 protein” also encompasses fusion proteins, i.e., chimeric proteins, which include one or more fragments of a wild-type human IL- 15 protein (or a variant thereof) and which retain one or more signaling functions of a wild-type human IL- 15 protein, e.g., as described above. A fusion protein which includes one or more fragments of a wild-type human IL- 15 protein (or a variant thereof) may also be referred to herein as a humanized IL- 15 protein.

[0363] A nucleic acid sequence that encodes a human IL- 15 protein is, therefore, a polynucleotide that includes a coding sequence for a human IL-15 protein, i.e., a wild-type human IL- 15 protein, a variant of a wild-type human IL- 15 protein, a fragment of a wild-type human IL- 15 protein (or a variant thereof) which retains one or more signaling functions of a wild-type human IL-15 protein, or fusion proteins, i.e., chimeric proteins, which include one or more fragments of a wild-type human IL- 15 protein (or a variant thereof) and which retain one or more signaling functions of a wild-type human IL- 15 protein, e.g., as described above.

[0364] IL- 15 (also known as “Interleukin 15”) is a cytokine that stimulates the proliferation of T lymphocytes. Polypeptide sequence for wild-type human IL- 15 and the nucleic acid sequence that encodes wild-type human IL- 15 may be found at Genbank Accession Nos. NP_000576.1 and NM_000585.5 (isoform 1 and transcript variant 3), NP 751915.1 and NM_ 172175.3 (isoform 2 and transcript variant 2). The genomic locus encoding the wild-type human IL- 15 protein may be found in the human genome at Chromosome 4; NC 000004.12 (141636583-141733987) or NG_029605.2 (4988-102392). In some embodiments, the human IL- 15 locus (e.g., NM_000585.5) includes 8 exons, with exons 3-8 being coding exons. As such, in some embodiments, a nucleic acid sequence including coding sequence for human IL-15 includes one or more of exons 3-8 of the human IL- 15 gene. In some instances, the nucleic acid sequence also includes aspects of the genomic locus of the human IL- 15, e.g., introns, 3' and / or 5' untranslated sequence (UTRs). In some instances, the nucleic acid sequence includes whole regions of the human IL- 15 genomiclocus. In some instances, the nucleic acid sequence includes exons 5-8 of the human IL-15 genomic locus (i.e., coding exons 3-6).

[0365] In the humanized IL- 15 rodents described herein, the nucleic acid sequence that encodes a human IL- 15 protein is operably linked to one or more regulatory sequences of an IL-15 gene, e.g., a regulatory sequence of an IL-15 gene of the rodent (e.g., mouse).Rodent, e.g., mouse, IL-15 regulatory sequences are those sequences of the rodent IL-15 genomic locus that regulate the rodent IL- 15 expression, for example, 5' regulatory sequences, e.g., the IL-15 promoter, IL-155' untranslated region (UTR), etc.; 3' regulatory sequences, e.g., the 3 'UTR; and enhancers, etc. Mouse IL-15 is located on Chromosome 8, NC_000074.7 (c83129883-83058253), and the mouse IL- 15 coding sequence may be found at Genbank Accession Nos. NM_008357.3 (transcript variant 1); NM_001254747.2 (transcript variant 2). The regulatory sequences of mouse IL- 15 are well defined in the art, and may be readily identified using in silico methods, e.g., by referring to the above Genbank Accession Nos. on the UCSC Genome Browser, on the world wide web at genome.ucsc.edu, or by experimental methods as described in the art. In some instances, e.g., when the nucleic acid sequence that encodes a human IL- 15 protein is located at the mouse IL- 15 genomic locus, the regulatory sequences operably linked to the human IL-15 coding sequence are endogenous, or native, to the mouse genome, i.e., they were present in the mouse genome prior to integration of human nucleic acid sequences.

[0366] In some instances, the humanized IL- 15 rodent, e.g., mouse, includes one copy of the nucleic acid sequence encoding a human IL- 15 protein. For example, the rodent may be heterozygous for the nucleic acid sequence. In other words, one allele at a locus will include the nucleic acid sequence, while the other will be the endogenous allele. For example, as discussed above, in some instances, a human IL- 15 nucleic acid sequence is integrated into the rodent, e.g., mouse, IL- 15 locus such that it creates a null allele for the rodent IL- 15. In some such embodiments, the humanized IL- 15 rodent may be heterozygous for the nucleic acid sequence encoding human IL- 15, i.e., the humanized IL- 15 rodent includes one null allele for the rodent IL- 15 (the allele including the nucleic acid sequence) and one endogenous IL- 15 allele (wild-type or otherwise). In other instances, the humanized IL- 15 includes two copies of the nucleic acid sequence encoding a human IL- 15 protein. For example, the rodent, e.g., mouse, may be homozygous for the nucleic acid sequence, i.e., both alleles for a locus in the diploid genome will include the nucleic acid sequence, i.e., thehumanized IL- 15 rodent includes two null alleles for the rodent IL- 15 (the allele including the nucleic acid sequence).

[0367] Human IL- 15 polypeptides, loci encoding human IL- 15 polypeptides and rodents expressing human IL-15 polypeptides are described in WO 2016 / 168212, which is incorporated by reference herein.Humanized EPO Loci

[0368] In some embodiments, provided genetically modified rodents (e.g., mice) and cells (e.g., ES cells) described herein have been further genetically modified to express a human erythropoietin (hEPO) protein encoded by a nucleic acid operably linked to an EPO promoter. By a human EPO protein, it is a meant a protein that is human EPO or is substantially identical to human EPO, e.g., it is 80% or more identical, 85% or more identical, 90% or more identical, or 95% or more identical to human EPO, for example, 97%, 98%, or 99% identical to human EPO. A nucleic acid sequence that encodes a human EPO protein is, therefore, a polynucleotide that comprises coding sequence for a human EPO protein, i.e., human EPO or a protein that is substantially identical to human EPO.

[0369] Erythropoietin (EPO) encodes a secreted, glycosylated cytokine composed of four alpha helical bundles. The encoded EPO protein is mainly synthesized in the kidney, secreted into the blood plasma, and binds to the erythropoietin receptor to promote red blood cell production, or erythropoiesis, in the bone marrow. Expression of EPO gene is upregulated under hypoxic conditions, in turn leading to increased erythropoiesis and enhanced oxygen-carrying capacity of the blood. Expression of EPO gene has also been observed in brain and in the eye, and elevated expression levels have been observed in diabetic retinopathy and ocular hypertension. Recombinant forms of the encoded EPO protein exhibit neuroprotective activity against a variety of potential brain injuries, as well as antiapoptotic functions in several tissue types, and have been used in the treatment of anemia and to enhance the efficacy of cancer therapies.

[0370] Polypeptide sequence for human EPO and the nucleic acid sequence that encodes for human EPO may be found at Genbank Accession Nos. NP_000790.2 and NM_000799.4, respectively. The genomic locus encoding the human EPO protein may be found in the human genome at Chromosome 7, NG_021471.2 (4669-7901) or NC_000007.14 (100720468-100723700). Protein sequence is encoded by exons 1 through 5 at this locus. As such, a nucleic acid sequence comprising coding sequence for human EPO comprises one ormore of exons 1-5 of the human EPO gene. In some instances, the nucleic acid sequence also comprises aspects of the genomic locus of the human EPO, e.g., introns, 3' and / or 5' untranslated sequence (UTRs). In some instances, the nucleic acid sequence comprises whole regions of the human EPO genomic locus.

[0371] In some embodiments, in the genetically modified rodents (e.g., mice) provided herein, the nucleic acid sequence that encodes a human EPO protein is operably linked to one or more regulatory sequences of the rodent (e.g., mouse) EPO gene. Rodent (e.g., mouse) EPO regulatory sequences are those sequences of the rodent (e.g., mouse) EPO genomic locus that regulate rodent (e.g., mouse) EPO expression, for example, 5' regulatory sequences, e.g., the EPO promoter, EPO 5' untranslated region (UTR), etc.; 3' regulatory sequences, e.g., the 3'UTR; and enhancers, etc. For example, mouse EPO is located on chromosome 5, GRCm39, NC_000071.7, at about positions C137484078-137481282, and the mouse EPO coding sequence may be found at Genbank Accession Nos. NM_007942.2 (transcript variant 1 encoding isoform 1), and NM_001312875.1 (transcript variant 2 encoding isoform 2). The regulatory sequences of mouse EPO are well defined in the art, and may be readily identified using in silico methods, e.g., by referring to the above Genbank Accession Nos. on the UCSC Genome Browser, on the world wide web at genome.ucsc.edu, or by experimental methods as described in the art. In some instances, e.g., when the nucleic acid sequence that encodes a human EPO protein is located at the rodent (e.g., mouse) EPO genomic locus, the regulatory sequences operably linked to the human EPO coding sequence are endogenous, or native, to the rodent (e.g., mouse) genome, i.e., they were present in the rodent (e.g., mouse) genome prior to integration of human nucleic acid sequences.

[0372] In some instances, the genetically modified rodent (e.g., mouse) expressing a human EPO protein comprises one copy of the nucleic acid sequence encoding a human EPO protein. For example, the rodent (e.g., mouse) may be heterozygous for the nucleic acid sequence. In other words, one allele at a locus will comprise the nucleic acid sequence, while the other will be the endogenous allele. For example, as discussed above, in some instances, human EPO nucleic acid sequence is integrated into the rodent (e.g., mouse) EPO locus such that it creates a null allele for rodent (e.g., mouse) EPO. In some such embodiments, the humanized EPO mouse may be heterozygous for the nucleic acid sequence encoding, i.e., the humanized EPO mouse comprises one null allele for rodent (e.g., mouse) EPO (the allele comprising the nucleic acid sequence) and one endogenous EPO allele (wild type or otherwise). In other instances, the genetically modified rodent (e.g., mouse) expressing ahuman EPO protein comprises two copies of the nucleic acid sequence encoding a human EPO protein. For example, the rodent (e.g., mouse) may be homozygous for the nucleic acid sequence, i.e., both alleles for a locus in the diploid genome will comprise the nucleic acid sequence, i.e., the genetically modified rodent (e.g., mouse) expressing a human EPO protein comprises two null alleles for the mouse EPO (the allele comprising the nucleic acid sequence).

[0373] Human EPO polypeptides, loci encoding human EPO polypeptides and rodents expressing human EPO polypeptides are described in WO 2015 / 179317, which is incorporated by reference herein.Genetically modified mice and mouse ES cells

[0374] In some embodiments, the present disclosure provides genetically modified mice and / or mouse ES cells comprising a humanized PIGR locus. In some embodiments, provided are genetically modified mice comprising a humanized PIGR locus and one or more of the additional humanized loci and / or additional modifications disclosed herein. In some embodiments, genetically modified mice comprising a humanized PIGR locus and one or more of the additional humanized loci are combined together by breeding or retargeting ES cells in accordance with the known techniques in the art.

[0375] In some embodiments, a genetically modified mouse is of a C57BL strain. In some embodiments, the C57BL strain is selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / 01a. In some embodiments, the mouse is a mouse of a 129 strain. In some embodiments, the 129 strain is selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129Sl / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2. In some embodiments, the genetically modified mouse is a mix of a 129 strain and a C57BL strain. In some embodiments, the mouse is a mix of 129 strains and / or a mix of C57BL / 6 strains. In some embodiments, the 129 strain of the mix is a 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse is a BALB strain (e.g., BALB / c). In some embodiments, the mouse is a mix of a BALB strain and another strain (e.g., a C57BL strain and / or a 129 strain). In some embodiments, mice provided herein can be a mouse derived from any combination of the aforementioned strains.

[0376] Genetically modified mice and ES cells can be generated using any appropriate method known in the art. For example, such genetically modified mouse ES cells can be generated using VELOCIGENE® technology, which is described in U. S. Patent Nos.6,586,251, 6,596,541, 7,105,348, and Valenzuela et al. (2003) “High-throughput engineering of the mouse genome coupled with high-resolution expression analysis” Nat. Biotech. 21(6): 652-659, each of which is hereby incorporated by reference. Modifications can also be made using a genome targeted nuclease system, such as a CRISPR / Cas system, a transcription activator-like effector nuclease (TALEN) system or a zinc finger nuclease (ZFN) system. In some embodiments, modifications are made using a CRISPR / Cas system, as described, for example, in U. S. Pat. App. Nos. 14 / 314,866, 14 / 515,503, 14 / 747,461 and 14 / 731,914, each of which is incorporated by reference. Exemplary methods of making such genetically modified mice and ES cells are also provided herein in Example 1.

[0377] ES cells described herein can then be used to generate a mouse using methods known in the art. For example, the mouse ES cells described herein can be used to generate genetically modified mice using the VELOCIMOUSE® method, as described in U. S. Pat. No. 7,294,754 and Poueymirou et al., Nature Biotech 25:91-99 (2007), each of which is hereby incorporated by reference. Resulting mice can be bred to homozygosity.Nonlimiting Applications of Rodents with Humanized PIGR

[0378] Genetically modified rodents (e.g., mice) and cells (e.g., ES cells) comprising a humanized PIGR locus described herein find many uses in the art. For example, rodents (e.g., mice) of the present disclosure may be useful for studying human IgA immune responses and diseases associated therewith.

[0379] In some embodiments, provided herein are methods of screening agents. In screening assays for biologically active agents, a genetically modified rodent of the present disclosure, is contacted with or administered a candidate agent of interest and the effect of the candidate agent is assessed by monitoring one or more output parameters.

[0380] In some embodiments, provided herein are methods of testing an agent in a rodent as described herein. In some embodiments, an agent is a protein comprising a human Fea domain (e.g., a human antibody or an Fea fusion protein). In some embodiments, provided methods of testing an agent comprise administering the agent to a rodent (e.g., mouse) provided herein.- Ill -

[0381] In some embodiments, provided herein are uses and methods for assessing and / or characterizing delivery of a payload in a rodent as described herein. In some embodiments, provided are uses and methods for characterizing delivery of a payload conjugated to an agent (e.g., an antibody agent). In some embodiments, provided herein are use and methods for characterizing delivery of a payload conjugated to an antibody. In some embodiments, provided herein are uses and methods for assessing and / or characterizing delivery of a payload conjugated to a PIGR-targeting agent. In some embodiments, a PIGR-targeting agent is a polypeptide that specifically binds PIGR (e.g., an anti-PIGR antibody or antigen binding antibody fragment).

[0382] In some embodiments, provided herein are uses and methods for assessing and / or characterizing delivery of a payload to mucosa of a rodent as described herein. In some embodiments, a payload is conjugated to an agent. In some embodiments, a payload is conjugated to a PIGR-targeting agent. In some embodiments, a PIGR-targeting agent is a polypeptide that specifically binds PIGR (e.g., an anti-PIGR antibody or antigen binding antibody fragment). In some embodiments, the mucosa is selected from mucosa of the respiratory tract, mucosa of the gastrointestinal tract, and mucosa of the urogenital tract.

[0383] In some embodiments, provided herein are uses and methods for assessing and / or characterizing delivery of a payload to the respiratory tract, gastrointestinal tract, and / or urogenital tract of a rodent as described herein. In some embodiments, provided herein are uses and methods for assessing and / or characterizing delivery of a payload to the mucosa of the respiratory tract, mucosa of the gastrointestinal tract, and / or mucosa of the urogenital tract. In some embodiments, a payload is conjugated to an agent. In some embodiments, a payload is conjugated to a PIGR-targeting agent. In some embodiments, a PIGR-targeting agent is a polypeptide that specifically binds PIGR (e.g., an anti-PIGR antibody or antigen binding antibody fragment).

[0384] In some embodiments, provided herein are uses and methods for assessing and / or characterizing transcytosis activity in a rodent as described herein. In some embodiments, provided herein are methods for assessing and / or characterizing removal of molecules (e.g., from the bloodstream). In some embodiments, provided herein are methods for assessing and / or characterizing transcytosis of a molecule conjugated to a PIGR-targeted agent.

[0385] In some embodiments, provided herein are methods and / or uses of a humanized PIGR rodent as described herein for removing autoantibodies from circulation. Insome embodiments, autoantibodies to be removed from circulation are human IgA autoantibodies and / or human IgM autoantibodies.

[0386] In some embodiments, provided herein are methods and / or uses for assessing and / or characterizing delivery of cell therapies to target cells and / or tissues. In some embodiments, provided herein are methods for assessing and / or characterizing delivery of cell therapies to mucosa. In some embodiments, the mucosa is selected from mucosa of the respiratory tract, mucosa of the gastrointestinal tract, and mucosa of the urogenital tract. In some embodiments, a cell therapy is targeted to PIGR. In some embodiments, a cell therapy is chimeric antigen receptor (CAR)-T cell therapy. In some embodiments, a cell therapy is CAR-T regulatory (Treg) cell therapy.

[0387] In some embodiments, provided herein are methods and / or uses for assessing and / or characterizing targeted immune modulation.

[0388] In some embodiments, provided are methods comprising: (a) administering a payload to a rodent as described herein; and (b) assessing delivery of the payload to the mucosa of the rodent. In some embodiments, the mucosa is respiratory mucosa, gastrointestinal mucosa, and / or urogenital mucosa. In some embodiments, the payload conjugated to an agent. In some embodiments, the agent is a PIGR-targeting agent. In some embodiments, a PIGR-targeting agent is a polypeptide that specifically binds PIGR (e.g., an anti-PIGR antibody or antigen binding antibody fragment).

[0389] In some embodiments, provided are methods comprising: (a) administering a cell therapy to a rodent as described herein; and (b) assessing delivery of the cell therapy to the mucosa of the rodent. In some embodiments, the mucosa is respiratory mucosa, gastrointestinal mucosa, and / or urogenital mucosa. In some embodiments, the cell therapy is CAR-T cell therapy. In some embodiments, the cell therapy is CAR-Treg cell therapy.

[0390] In some embodiments, provided methods and / or uses comprise measuring and / or detecting an administered agent and / or cell therapy in one or more tissues of the rodent (e.g., mucosal tissue).

[0391] In some embodiments, provided methods comprise measuring one or more pharmacokinetic properties of an administered agent and / or cell therapy. In some embodiments, the animal model used to determine the pharmacokinetic properties of the administered agent and / or cell therapy in a genetically modified rodent as described herein.

[0392] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art,determining the effective concentration of an agent typically uses a range of concentrations resulting from 1:10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.

[0393] In some embodiments, the one or more pharmacokinetic parameters include, but are not limited to, area under the plasma concentration versus time (AUC), in vivo recovery (IVR), clearance rate (CL), mean residence time (MRT), agent half-life (t½), and volume of distribution at steady state (Vss). In general, the pharmacokinetic properties of the administered agent is determined by administering a selected dose of the agent (e.g., 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / mg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg or more) and then determining how the plasma concentration of the agent changes over time (e.g., 0 hr, 6 hr, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 or more days).

[0394] In some embodiments, provided methods comprise measuring the therapeutic efficacy of an administered therapeutic agent (e.g., the ability of an administered dose of the therapeutic agent to reduce or eliminate one or more disease symptoms in the animal model). In some embodiments, the animal model is a cancer model and disease symptoms can include, for example, tumor size, tumor metastasis, and / or animal survival. In some embodiments, the animal model is an autoimmune or inflammation model, and the disease symptoms can include, for example, levels of cytokine expression, proliferation of immune cells, tissue damage, and / or animal survival. In some embodiments, the animal model is an infectious disease model, and the disease symptoms can include, for example, levels of the infectious agent, tissue damage, and / or animal survival.

[0395] In some embodiments, provided methods comprise measuring the safety and dosing of an administered therapeutic agent (e.g., the extent to which an administered dose of the therapeutic agent produces one or more adverse effects in the animal model). Adverse effects include, but are not limited to, allergic reactions, alopecia, anaphylaxis, anemia, lack of appetite, loss of balance, bleeding, blood clots, difficulty breathing, bronchitis, bruising, low white blood cell count, low red blood cell count, low platelet count, cardiotoxicity, conjunctivitis, constipation, coughing, dehydration, diarrhea, electrolyte imbalance, loss offertility, fever, hair loss, heart failure, infection, injection site reactions, iron deficiency, kidney failure, leukopenia, liver dysfunction, pneumonia, rapid heartbeat, rectal bleeding, seizures, weight loss, and weight gain. In some embodiments, provided herein is a method of measuring allergic reactions induced by an agent using passive cutaneous anaphylaxis (PC A) and / or passive systemic anaphylaxis (PSA) models.

[0396] In some embodiments, the agent is administered to a rodent (e.g., mouse) provided herein as part of a pharmaceutical composition e.g., a pharmaceutical composition, containing a human IgA antibody or Fc fusion protein formulated together with a pharmaceutically acceptable carrier. Such agents may be formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

[0397] In methods provided herein, an agent and / or pharmaceutical composition may be delivered by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually. In some embodiments, the pharmaceutical compositions are delivered generally (e.g., via oral or parenteral administration).

[0398] In various embodiments, rodents (e.g., mice) as described herein are used to measure the therapeutic effect of blocking or modulating the activity of a polypeptide of interest and the effect on gene expression as a result of cellular changes or, in the context of a receptor polypeptide, and / or the density of a receptor polypeptide on the surface of cells in the rodent. In various embodiments, a rodent (e.g., mouse) as described herein or cells isolated therefrom are exposed to a candidate therapeutic that binds a polypeptide of interest and, after a subsequent period of time, analyzed for effects on specific cellular processes that are associated with said polypeptide of interest, for example, ligand-receptor interactions or signal transduction.

[0399] Rodents (e.g., mice) as described herein provide an improved in vivo system for development and selection of human IgA antibodies or Fc fusion polypeptides.Alternatively and / or additionally, the immune response may be monitored in such mice so as to characterize and select potential human antibodies that may be developed as a therapeutic.

[0400] Additional applications of the genetically modified mice described in this disclosure will be apparent to those skilled in the art upon reading this disclosure.Methods of Making Genetically Modified Rodents and ES cells

[0401] In certain aspects, provided herein are methods of making rodents (e.g., mice) and ES cells that comprise one or more of the genetically modified loci provided here. The exemplary methods of making genetically modified rodents (e.g., mice) and ES cells provided herein are described in the description, examples, and / or figures herein. For example, in some embodiments, provided herein are methods of making rodents (e.g., mice) and ES cells that comprise a humanized PIGR locus provided herein. In some embodiments, a humanized PIGR locus is inserted into an endogenous rodent PIGR locus.

[0402] Any of various methods may be used to introduce a human nucleic acid sequence into an animal cell to produce a genetically modified animal that expresses a human gene. Such techniques are well-known in the art and include, but are not limited to, pronuclear microinjection, transformation of embryonic stem cells, homologous recombination and knock-in techniques. Methods for generating genetically modified animals that can be used include, but are not limited to, those described in Sundberg and Ichiki (2006, Genetically Engineered Mice Handbook, CRC Press), Hofker and van Deursen (2002, Genetically modified Mouse Methods and Protocols, Humana Press), Joyner (2000, Gene Targeting: A Practical Approach, Oxford University Press), Turksen (2002, Embryonic stem cells: Methods and Protocols in Methods Mol Biol, Humana Press), Meyer et al. (2010, Proc. Nat. Acad. Sci. USA 107: 15022-15026), and Gibson (2004, A Primer Of Genome Science 2nd ed. Sunderland, Massachusetts: Sinauer), U. S. Pat. No. 6,586,251, Rathinam et al. (2011, Blood 118:3119-28), Willinger et al, (2011, Proc Natl Acad Sci USA, 108:2390-2395), Rongvaux et al, (2011, Proc Natl Acad Sci USA, 108:2378-83) and Valenzuela et al. (2003, Nat Biot 21:652-659).

[0403] For example, genetically modified rodents can be created by introducing a desired nucleic acid into an oocyte, e.g., by microinjection, and allowing the oocyte to develop in a female foster animal. In preferred embodiments, the expression is injected into fertilized oocytes. Fertilized oocytes can be collected from superovulated females the day after mating and injected with the expression construct. The injected oocytes are either cultured overnight or transferred directly into oviducts of 0.5-day p.c. pseudopregnant females. Methods for superovulation, harvesting of oocytes, expression construct injection and embryo transfer are known in the art and described in Manipulating the Mouse Embryo (2002, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press). Offspringcan be evaluated for the presence of the introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or by protein analysis (e.g., ELISA, Western blot, etc.).

[0404] As another example, a construct comprising a nucleic acid sequence comprising a desired genetic modification (e.g., encoding a human protein) may be transfected into stem cells (e.g., ES cells or iPS cells) using well-known methods, such as electroporation, calcium-phosphate precipitation, lipofection, etc. The cells can be evaluated for the presence of the introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or by protein analysis (e.g., ELISA, Western blot, etc.). Cells determined to have incorporated the expression construct can then be introduced into preimplantation embryos. For a detailed description of methods known in the art useful for the compositions and methods of the invention, see Nagy et al, (2002, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press), Nagy et al. (1990, Development 110:815-821), U. S. Pat. No. 7,576,259, U. S. Pat. No.7,659,442, U. S. Pat. No. 7,294,754, and Kraus et al. (2010, Genesis 48:394-399).

[0405] Additionally, a nucleic acid construct may be constructed using VELOCIGENE® genetic engineering technology (see, e.g., Valenzuela et al. (2003) High throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6): 652-59 and U. S. Patent No. 6,586,251), introduced into stem cells (e.g., ES cells), and correctly targeted clones determined using loss-of-allele and gain-of-allele assays (Valenzuela et al, supra); correctly targeted ES cells may be used as donor ES cells for introduction into an 8-cell stage mouse embryo using the VELOCIMOUSE® method (see, e.g., U. S. Pat. No. 7,294,754 and Poueymirou et al. 2007, F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25(1):91-99). In addition, genetically modified rat ES cells and rats can be made according to US 2014 / 0235933 Al (Regeneron Pharmaceuticals, Inc.), US 2014 / 0310828 Al (Regeneron Pharmaceuticals, Inc.), Tong et al. (2010) Nature 467:211-215, and Tong et al. (2011) NatProtoc. 6(6): doi:10.1038 / nprot.2011.338 (all of which are incorporated herein by reference in their entireties).

[0406] In some embodiments, genetically modified founder animals can be bred to additional animals carrying one or more genetic modifications.

[0407] In some embodiments, stem cells, e.g., ES cells, may be generated such that they comprise several genetic modifications, e.g., humanizations or gene deletions describedherein, and such stem cells may be introduced into an embryo to generate genetically modified animals with several genetic modifications.

[0408] In some embodiments, provided herein are methods of making rodents (e.g., mice) and ES cells that comprise a humanize PIGR locus and further comprise a humanized FcaR locus provided herein. In some embodiments, provided herein are methods of making rodents (e.g., mice) and ES cells that further comprise a human or humanized FcRn locus provided herein and / or a human or humanized P2M locus provided herein. In some embodiments, provided herein are methods of making rodents (e.g., mice) and ES cells that further comprise a human or humanized FCERIOC locus provided herein. In certain embodiments, provided herein are methods of making rodents (e.g., mice) and ES cells that further comprise a human or humanized FcyRla locus provided herein. In some embodiments, provided herein are methods of making rodents (e.g., mice) and ES cells that further comprise a human or humanized FcyR2a locus provided herein, a human or humanized FcyR2b locus provided herein, a human or humanized FcyR2c locus provided herein, a human or humanized FcyR3a locus provided herein, and / or a human or humanized FcyR3b locus provided herein. In some embodiments, provided herein are methods of making rodents (e.g., mice) and ES cells that further comprise a human or humanized heavy and / or light chain locus provided herein.

[0409] In some embodiments, provided herein are methods of making rodents (e.g., mice) and ES cells that comprise a humanize PIGR locus and further comprise a humanized heavy chain locus and / or a humanized light chain locus as described herein.

[0410] In some embodiments, provided herein are methods of making rodents (e.g., mice) and ES cells that comprise a humanize PIGR locus and further comprise: (i) a Ragl and / or Rag2 gene knock-out; (ii) a IL2rg gene knock-out. In some embodiments, provided herein are methods of making rodents (e.g., mice) and ES cells that further comprise human or humanized SIRPA locus. In some embodiments, provided herein are methods of making rodents (e.g., mice) and ES cells whose genome further comprises one or more of: a nucleic acid encoding a human TPO protein operably linked to a TPO promoter; a nucleic acid encoding a human GM-CSF protein operably linked to a GM-CSF promoter; a nucleic acid encoding a human IL3 protein operably linked to a IL3 promoter; a nucleic acid encoding a human IL 15 protein operably linked to a IL 15 promoter; a nucleic acid encoding a human M-CSF protein operably linked to an M-CSF promoter; a nucleic acid encoding a human orhumanized CD47 protein operably linked to a CD47 promoter; and a nucleic acid encoding a human EPO protein operably linked to an EPO promoter.

[0411] Additional methods of generating genetically modified rodents (e.g., mice) comprising a genome that includes a nucleic acid encoding one or more human proteins, e.g., hSIRPa, hIL-3, hGM-CSF, hM-CSF, hEPO, hCD47, hIL-15, and / or hTPO, are described in US Pat. No. US 11019810, US Pat. Publ. ...

Claims

1. What is claimed is:

1. A genetically modified rodent whose genome comprises a humanized polymeric immunoglobulin receptor (PIGR) locus encoding a plgR polypeptide comprising: human plgR Ig domains 1-5.

2. The genetically modified rodent of claim 1, wherein the humanized PIGR locus encodes a plgR polypeptide comprising a human, rodent, or human-rodent chimeric plgR signal peptide.

3. The genetically modified rodent of claim 1 or 2, wherein the humanized PIGR locus encodes a plgR polypeptide comprising a human or rodent plgR cleavage region.

4. The genetically modified rodent of any one of claims 1-3, wherein the humanized PIGR locus encodes a plgR polypeptide comprising a human or rodent plgR transmembrane domain.

5. The genetically modified rodent of any one of claims 1-4, wherein the humanized PIGR locus encodes a plgR polypeptide comprising a human plgR cleavage region and a human plgR transmembrane domain.

6. The genetically modified rodent of claim 1-5, wherein the humanized PIGR locus encodes a plgR polypeptide comprising a human, rodent, or human-rodent chimeric plgR cytoplasmic domain.

7. The genetically modified rodent of claim 6, wherein the humanized PIGR locus encodes a plgR polypeptide comprising a human plgR cytoplasmic domain.

8. The genetically modified rodent of claim 6, wherein the humanized PIGR locus encodes a plgR polypeptide comprising a human-rodent chimeric plgR cytoplasmic domain.

9. The genetically modified rodent of claim 1, wherein the humanized PIGR locus comprises:(a) rodent plgR exon 1 and rodent plgR exon 2;11.(b) human or rodent- human chimeric plgR exon 3;12.(c) human plgR exons 4-8; and13.(d) human or rodent plgR exons 9-11.

10. The genetically modified rodent of claim 1 or 9, wherein the humanized PIGR locus comprises rodent plgR exons 9-11.

11. The genetically modified rodent of any one of claims 1-10, wherein the humanized PIGR locus encodes a plgR polypeptide comprising any of the following:16.(a) a signal peptide comprising an amino acid sequence that is at least 95% identical to a sequence of SEQ ID NO: 1 or 2;17.(b) a plgR Ig domain comprising an amino acid sequence that is at least 95% identical to a sequence of one or more of SEQ ID NOs: 3-7,18.(c) a transmembrane domain comprising an amino acid sequence that is at least 95% identical to a sequence of SEQ ID NO: 8 or 9; and19.(d) a cytoplasmic domain comprising an amino acid sequence that is at least 95% identical to a sequence of any one of SEQ ID NOs: 10-12.

12. The genetically modified rodent of any one of claims 1-10, wherein the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence of any of the following:21.(a) a signal peptide sequence of SEQ ID NO: 1 or 2;22.(b) a plgR Ig domain sequence of one or more of SEQ ID NOs: 3-7,23.(c) a transmembrane domain of SEQ ID NO: 8 or 9; and24.(d) a cytoplasmic domain of any one of SEQ ID NOs: 10-12.

13. The genetically modified rodent of any one of claims 1-12, wherein the humanized PIGR locus encodes a plgR polypeptide comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13 or 14.

14. The genetically modified rodent of any one of claims 1-13, wherein the humanized PIGR locus comprises a nucleic acid sequence that is at least 95% identical to any of the following:26.(a) an exon 1 sequence of SEQ ID NO: 1527.(b) an exon 2 sequence of SEQ ID NO: 16;28.(c) an exon 3 sequence of any one of SEQ ID NOs: 17-19;29.(d) an exon 4 sequence of SEQ ID NO: 20;30.(e) an exon 5 sequence of SEQ ID NO: 21;31.(f) an exon 6 sequence of SEQ ID NO: 22;32.(g) an exon 7 sequence of SEQ ID NO: 23 or 24;33.(h) an exon 8 sequence of SEQ ID NO: 25 or 26;34.(i) an exon 9 sequence of SEQ ID NO: 27 or 28;35.(j) an exon 10 sequence of SEQ ID NO: 29 or 30;36.(k) an exon 11 sequence of SEQ ID NO: 31 or 32.

15. The genetically modified rodent of any one of claims 1-13, wherein the humanized PIGR locus comprises a nucleic acid sequence of any of the following:38.(a) an exon 1 sequence of SEQ ID NO: 1539.(b) an exon 2 sequence of SEQ ID NO: 16;40.(c) an exon 3 sequence of any one of SEQ ID NOs: 17-19;41.(d) an exon 4 sequence of SEQ ID NO: 20;42.(e) an exon 5 sequence of SEQ ID NO: 21;43.(f) an exon 6 sequence of SEQ ID NO: 22;44.(g) an exon 7 sequence of SEQ ID NO: 23 or 24;45.(h) an exon 8 sequence of SEQ ID NO: 25 or 26;46.(i) an exon 9 sequence of SEQ ID NO: 27 or 28;47.(j) an exon 10 sequence of SEQ ID NO: 29 or 30;48.(k) an exon 11 sequence of SEQ ID NO: 31 or 32.

16. The genetically modified rodent of any one of claims 1-15, wherein the humanized PIGR locus comprises:50.(i) a genomic sequence found between coordinates 130,826,684 and 130,841,507 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,931,955 and 206,939,452 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,849,332 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly; or51.(ii) a genomic sequence found between coordinates 130,826,684 and 130,841,507 on mouse chromosome 1 (+ strand, GRCm38 assembly), a genomic sequence found between coordinates 206,931,955 and 206,939,452 on human chromosome 1 (- strand, GRCh38 assembly), and / or a genomic sequence found between coordinates 130,849,332 and 130,852,249 on mouse chromosome 1 (+ strand, GRCm38 assembly).

17. The genetically modified rodent of any one of claims 1-16, wherein the humanized PIGR locus is at an endogenous PIGR locus.

18. The genetically modified rodent of any one of claims 1-17, wherein the humanized PIGR locus is under the control of a PIGR promoter.

19. The genetically modified rodent of claim 18, wherein the humanized PIGR locus is under the control of an endogenous rodent PIGR promoter or a human PIGR promoter.

20. The genetically modified rodent of any one of claims 1-19, wherein the rodent genome does not encode a functional rodent plgR polypeptide.

21. The genetically modified rodent of any one of claims 1-20, wherein the humanized PIGR locus replaces all or part of the endogenous rodent PIGR locus.

22. The genetically modified rodent of any one of claims 1-30, further comprising in its genome a human or humanized Fc alpha receptor (FcaR) locus, wherein the FcaR locus is positioned in the rodent leukocyte receptor complex (LRC), wherein the FcaR locus comprises a nucleic acid sequence encoding a FcaR polypeptide comprising a human extracellular domain, a human or rodent transmembrane domain, and a human or rodent cytoplasmic domain.

23. The genetically modified rodent of claim 22, wherein the FcaR locus is positioned in an intergenic region between the gene loci for the Pira6 protein and the Ncrl protein, such as between coding nucleic acid sequences for the Pira6 protein and the Ncrl protein.

24. The genetically modified rodent of claim 22 or 23, wherein the FcaR locus comprises a nucleic acid sequence encoding a fully human FcaR polypeptide.

25. The genetically modified rodent of any one of claims 22-24, wherein the FcaR locus comprises human exons 1-5 of the human Fc alpha receptor gene.

26. The genetically modified rodent of claim 22 or 23, wherein the FcaR locus comprises a non-coding portion of non-mouse rodent FcaR exon 1, a coding portion of human FcaR exons 1 and 2, human FcaR exons 3 and 4, and non-mouse rodent FcaR exon 5.

27. The genetically modified rodent of any one of claims 22-26, wherein the FcaR locus further comprises a nucleic acid sequence present in a human KIR3DL2 gene, and / or nucleic acid sequence present in the 5’UTR of the human NCR1 gene.

28. The genetically modified rodent of any one of claims 22-27, wherein the rodent expresses the FcaR polypeptide on neutrophils, monocytes, macrophages, eosinophils, and dendritic cells (e.g., plasmacytoid dendritic cells).

29. The genetically modified rodent of any one of claims 22-28, further comprising in its genome a human or humanized Fc gamma receptor (FcyR) locus, a human or humanized IgH locus, a human or humanized IgK locus, a human or humanized Igl locus, a human or humanized FcRn locus, a human or humanized P2M locus, and / or a human or humanized FcsRla locus.

30. The genetically modified rodent of any one of claims 1-29, wherein the rodent is immunodeficient.

31. The genetically modified rodent of any one of claims 1-30, further comprising one or more of:(i) a Rag2 gene knock-out;66.(ii) a IL2rg gene knock-out; and67.(iii) a human or humanized SIRPa knock-in.

32. The genetically modified rodent of claim 31, comprising a human or humanized SIRPa knock-in, wherein the rodent:69.(i) expresses a human or humanized SIRPa protein encoded by a nucleic acid operably linked to a SIRPa promoter;70.(ii) expresses a humanized SIRPa protein, and the humanized SIRPa protein comprises a functional fragment of a full-length human SIRPa protein human or humanized SIRPa protein, optionally wherein the functional fragment comprises an extracellular domain of a human SIRPa protein; and / or71.(iii) expresses a humanized SIRPa protein, and the humanized SIPRa protein comprises an extracellular portion of a human SIRPa protein and an intracellular portion of an endogenous rodent SIRPa protein.

33. The genetically modified rodent of any one of claims 1-32, wherein the genetically modified rodent further expresses one or more of:73.(i) a human or humanized thrombopoietin (TPO);74.(ii) a human or humanized GM-CSF;75.(iii) a human or humanized interleukin 3 (IL3);76.(iv) a human or humanized interleukin 15 (IL 15);77.(v) a human or humanized M-CSF;78.(vi) a human or humanized CD47; and79.(vii) a human or humanized Erythropoietin (EPO).

34. The genetically modified rodent of claim 1-32, wherein the genome of the genetically modified rodent comprises one or more of:81.(i) a human or humanized TPO gene operably linked to a TPO promoter;82.(ii) a human or humanized GM-CSF gene operably linked to a GM-CSF promoter;83.(iii) a human or humanized IL3 gene operably linked to a IL3 promoter;84.(iv) a human or humanized IL 15 gene operably linked to a IL 15 promoter; (v) a human or humanized M-CSF gene operably linked to a M-CSF promoter; (vi) a human or humanized CD47 gene operably linked to a CD47 promoter; and (vii) a human or humanized EPO gene operably linked to an EPO promoter.

35. The genetically modified rodent of claim 34, wherein the nucleic acids that encode the human or humanized proteins replace the corresponding endogenous rodent gene.

36. The genetically modified rodent of any one of claims 1-35, further comprising in its genome an immunoglobulin heavy chain variable region comprising one or more human heavy chain V gene segments, one or more human D gene segments, and one or more human heavy chain J gene segments, wherein the immunoglobulin heavy chain variable region is operably linked to a heavy chain constant region.

37. The genetically modified rodent of claim 36, wherein the heavy chain constant region is a human heavy chain constant region or a rodent heavy chain constant region.

38. The genetically modified rodent of claim 36 or 37, further comprising in its genome an immunoglobulin light chain variable region comprising one or more unrearranged human VL gene segments and one or more unrearranged human JL gene segments that are operably linked to one or more immunoglobulin light chain constant region genes.

39. The genetically modified rodent of claim 38, wherein the immunoglobulin light chain variable region comprises90.(a) one or more human VK gene segments, and one or more human JK gene segments; and / or91.(b) one or more human VI gene segments, and one or more human Jl gene segments.

40. The genetically modified rodent of claim 38 or 39, wherein one or more immunoglobulin light chain constant region genes are human immunoglobulin light chain constant region genes or rodent immunoglobulin light chain constant region genes.

41. The genetically modified rodent of any one of claims 1-40, wherein the rodent is a mouse or rat.

42. The genetically modified rodent of any one of claims 1-40, wherein the rodent is a mouse.

43. A cell isolated from a genetically modified rodent of any one of claims 1-42.

44. An isolated rodent cell whose genome comprises the same genetic modifications as a genetically modified rodent of any one of claims 1-42.

45. The isolated rodent cell of claim 44, wherein the cell is an ES cell.

46. A method of making a genetically modified rodent of any one of claims 1-42.

47. Use of a genetically modified rodent of any one of claims 1-42 for assessing and / or characterizing delivery of a payload to mucosa.

48. The use of claim 47, wherein100.(a) the mucosa is respiratory mucosa, gastrointestinal mucosa, and / or urogenital mucosa;101.(b). the payload conjugated to a PIGR-targeting agent.

49. Use of a genetically modified rodent of any one of claims 1-42 for assessing and / or characterizing transcytosis of a molecule conjugated to a PIGR-targeting agent.

50. Use of a genetically modified rodent of any one of claims 1-42 for removing autoantibodies from circulation.

51. The use of claim 50, wherein the autoantibodies are human IgA autoantibodies and / or human IgM autoantibodies.

52. Use of a genetically modified rodent of any one of claims 1-42 for assessing and / or characterizing delivery of a cell therapy to a target cell, target tissue, and / or mucosa.

53. The use of claim 52, wherein the cell therapy is a CAR-Treg cell therapy.

54. Use of a genetically modified rodent of any one of claims 1-42 for testing an agent, wherein the agent is or comprises and IgA antibody or Fea fusion polypeptide.

55. The use of claim 54, further comprising:108.(a) measuring the immune response generated by the mouse against the human antibody; and / or109.(b) measuring the therapeutic efficacy of the administered human antibody or Fea fusion polypeptide.

56. A method of testing a human IgA antibody or an Fea fusion polypeptide, comprising administering an IgA antibody or Fea fusion polypeptide to a rodent of any one of claims 1-42.

57. The method of claim 56, further comprising:112.(a) measuring the immune response generated by the rodent against the IgA antibody;113.and / or114.(b) measuring the therapeutic efficacy of the administered IgA antibody or Fea fusion polypeptide.

58. A method comprising:116.administering a payload or cell therapy to a rodent of any one of claims 1-42; and assessing delivery of the payload or cell therapy to mucosa of the rodent.

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