Non-human animals with humanized FC epsilon receptors that lack functional alpha-1, 3-galactosyltransferase (GGTA1)

Abstract

Provided herein are genetically modified rodents (e.g., mice or rats) with a human or humanized Fc epsilon receptor 1 alpha (FcεR1α) that do not express a functional α-1,3, -galactosyltransferase (GGTA1). Also provided herein are methods of making and / or using such rodents, cells from such rodents, and ES cells comprising the same genetic modifications.

Description

Attorney Docket No. RPB-03125 (11689WO)NON-HUMAN ANIMALS WITH HUMANIZED FC EPSILON RECEPTORS THAT LACK FUNCTIONAL ALPHA-1, 3-GALACTOSYLTRANSFERASE (GGTA1)CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 683,421, filed August 15, 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 July 18, 2025, is identified as RPB-03125_SL.xml and is 54,015 bytes in size.BACKGROUND

[0003] Due to their small size and well-characterized physiology, rodents have long been used as animal models for the preclinical testing of therapeutic agents. Further, rodents are highly amenable to genetic modification using well-established techniques and, thus, there are many disease models available in rodents that are not available in larger mammals. Complex therapeutic agents, however, such as antibodies and Fc fusion proteins, often behave differently in rodents than they do in humans. This in turn reduces the usefulness of such animal models in preclinical testing. Thus, there is an ongoing need for new animal models and methods that allow for precise pre-clinical testing of complex therapeutic agents in rodents that produce results that are more predictive of the properties of such therapeutic agents in human patients.SUMMARY

[0004] The present disclosure relates to genetically modified non-human animals (e.g., rodents, e.g., mice or rats) comprising a humanized Fc epsilon receptor (FcsR) that do not express a functional a-l,3-galactosyltransferase (GGTA1). Provided herein are methods and compositions related to rodents (e.g., mice or rats) that lack GGTA1 activity and express a human or humanized FcsR, cells from such rodents, as well as methods and uses of such rodents. The present disclosure provides the insight that such rodents may be useful as a model for galactose-a- 1,3, -galactose (alpha-gal) syndrome and / or sensitization. For example, such rodents may be used in methods for characterizing, evaluating and / or measuring allergic response to alpha-gal and / or treatments of the same.Attorney Docket No. RPB-03125 (11689WO)

[0005] In some embodiments, provided herein is a genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) model of alpha-gal syndrome. In some embodiments, provided non-human animals express a human or humanized FcsR and do not express a functional GGTA1. In some embodiments, provided herein are genetically modified rodents (e.g., mice or rats) that comprise a GGTA1 mutation and a human or humanized Fc epsilon receptor 1 alpha (FcsRla) locus. In some embodiments, a GGTA1 mutation is a GGTA1 knock-out. In some embodiments, a GGTA1 mutation is or comprises a deletion of at least part of the endogenous rodent GGTA1 locus. In some embodiments, a GGTA1 mutation is or comprises a deletion of all of the endogenous GGTA1 exons. In some embodiments, a human or humanized FcsRla locus comprises a nucleic acid sequence encoding FcsRla polypeptide comprising a human extracellular domain, a rodent or human transmembrane domain and a rodent or human cytoplasmic domain. In some embodiments, the nucleic acid sequence encoding the FcsRla polypeptide is positioned at an endogenous rodent FcsRla locus.

[0006] In some embodiments, provided are methods for characterizing or measuring allergic response to alpha-gal in a rodent as described herein. In some embodiments, provided are methods for testing agents (e.g., human antibodies and / or Fc fusion proteins) for treating an alpha-gal allergic response using a rodent as described herein.

[0007] In some embodiments, provided herein is a rodent whose genome (a) does not encode a functional a a- 1,3, -galactosyltransferase (GGTA1), and (b) comprises a human or a humanized Fc epsilon receptor 1 alpha (FcsRla) locus, wherein the human or the humanized FcsRla locus comprises a nucleic acid sequence encoding a FcsRla polypeptide comprising a human extracellular domain.

[0008] In some embodiments, the rodent genome comprises a deletion of at least part of the endogenous rodent GGTA1 locus. In some embodiments, the rodent comprises a deletion of a sequence that is 10,000 bp to 64,087 bp, wherein the deletion is within genomic coordinates chr2: 35,399,144 to 35,463,231 (GRCm38 / mml0, - strand). In some embodiments, the rodent comprises a deletion of a sequence that is 10,000 bp to 24,247 bp, wherein the deletion is within genomic coordinates chr2: 35,401,275 to 35,425,521 (GRCm38 / mml0, - strand).

[0009] In some embodiments, the rodent genome comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to the sequence corresponding to SEQ ID NO: 32. In some embodiments, the rodent genome comprises the sequence corresponding to SEQ ID NO: 32.Attorney Docket No. RPB-03125 (11689WO)

[0010] In some embodiments, the rodent genome comprises a knock-out of a GGTA1 gene. In some embodiments, the rodent genome comprises a deletion of the entire GGTA1 encoding region. In some embodiments, the rodent genome is heterozygous for a GGTA1 deletion. In some embodiments, the rodent genome is homozygous for a GGTA1 deletion.

[0011] In some embodiments, the FcsRla polypeptide further comprises a rodent transmembrane domain. In some embodiments, the FcsRla polypeptide further comprises a human transmembrane domain. In some embodiments, the FcsRla polypeptide further comprises a rodent cytoplasmic domain. In some embodiments, the FcsRl a polypeptide further comprises a human cytoplasmic domain.

[0012] In some embodiments, the nucleic acid sequence encoding the FcsRla polypeptide is positioned at an endogenous rodent FcsRla locus. In some embodiments, the nucleic acid sequence encoding the FcsRla polypeptide replaces all or part of an endogenous rodent FcsRla gene. In some embodiments, in the nucleic acid sequence encoding the human FcsRla extracellular domain replaces an endogenous nucleic acid sequence encoding a rodent FcsRla extracellular domain. In some embodiments, the rodent does not express a rodent FcsRla.

[0013] In some embodiments, the rodent is heterozygous for the human or humanized FcsRla locus. In some embodiments, the rodent is homozygous for the human or humanized FcsRla locus.

[0014] In some embodiments, the rodent is a mouse.

[0015] In some embodiments, the present disclosure provides a cell or population of cells isolated from a genetically modified rodent described herein. In some embodiments, the present disclosure provides a cell or population of cells whose genome comprises a a- 1,3,- galactosyltransferase (GGTA1) knock-out and a human or humanized Fc epsilon receptor 1 alpha (FcsRla) locus.

[0016] In some embodiments, the cell(s) are mouse cell(s). In some embodiments, the cell(s) are ES cell(s).

[0017] In some embodiments, the present disclosure provides an ES cell whose genome comprises a a- 1,3, -galactosyltransferase (GGTA1) knock-out and a human or humanized Fc epsilon receptor 1 alpha (FcsRla) locus.

[0018] In some embodiments, the present disclosure provides methods of making a genetically modified rodent described herein. In some embodiments, the present disclosure provides methods of making a genetically modified rodent, the method comprising: deleting at least a portion of an endogenous a- 1,3, -galactosyltransferase (GGTA1) locus in theAttorney Docket No. RPB-03125 (11689WO) genome of a rodent, wherein the rodent genome comprises a humanized Fc epsilon receptor 1 alpha (FcsRla) locus at the endogenous rodent FcsRla locus.

[0019] In some embodiments, the present disclosure provides methods of making a genetically modified rodent, the method comprising inserting a humanized Fc epsilon receptor 1 alpha (FcsRla) locus in the endogenous FcsRla locus of the rodent genome, wherein rodent genome does not encode a functional endogenous a- 1,3, -galactosyltransferase (GGTA1).

[0020] In some embodiments, the present disclosure provides methods of making a genetically modified rodent, comprising: (a) generating a rodent ES cell whose genome comprises a a- 1,3, -galactosyltransferase (GGTA1) knock-out and a human or humanized Fc epsilon receptor 1 alpha (FcsRla) locus; and (b) generating a rodent from said ES cell.

[0021] In some embodiments, the present disclosure provides methods of making a genetically modified rodent, the method comprising: (a) breeding rodent comprising a GGTA1 mutation with a rodent comprising a humanized FcsRIa locus, and (b) selecting for rodents that are homozygous for the GGTA1 mutation and are heterozygous or homozygous for the humanized FcsRIa locus. In some embodiments, the present disclosure provides methods of making a genetically modified rodent, the method comprising: (a) breeding rodent comprising a GGTA1 mutation with a rodent comprising a humanized FcsRIa locus, and (b) selecting for rodents that are homozygous for the GGTA1 mutation and the humanized FcsRIa locus.

[0022] In some embodiments, the present disclosure provides methods of making a rodent model of alpha-gal allergy, the method comprising: (a) genetically modifying a rodent such that its genome comprises a humanized FcsRIa locus and does not encode a functional GGTA1.

[0023] In some embodiments, the present disclosure provides uses of genetically modified rodents described herein. In some embodiments, provided genetically modified rodents are useful in a method for testing an agent. In some embodiments, the agent is tested for its ability to prevent and / or treat alpha-gal allergy. In some embodiments, provided uses comprise administering the agent to a rodent described herein.

[0024] In some embodiments, the present disclosure provides methods of testing an agent for preventing and / or treating alpha-gal allergy, the method comprising administering the agent to a rodent described herein. In some embodiments, provided methods further comprise measuring one or more pharmacokinetic properties of the administered agent. In some embodiments, the one or more pharmacokinetic properties are selected from one orAttorney Docket No. RPB-03125 (11689WO) more of area under the plasma concentration versus time (AUC), in vivo recovery (IVR), clearance rate (CL), mean residence time (MRT), agent half-life (d / 2), and volume of distribution at steady state (Vss).

[0025] In some embodiments, provided methods further comprise measuring the therapeutic efficacy of the administered agent. In some embodiments, provided methods further comprise administering a plurality of doses of the human antibody and determining a therapeutic efficacy of each dose of the agent. In some embodiments, provided methods further comprise administering a plurality of doses of the human antibody and determining the safety of each dose the agent. In some embodiments, provided methods further comprise administering a plurality of doses of the human antibody and determining the tolerability of each dose the agent. In some embodiments, provided methods further comprise measuring the immune response generated by the rodent against the agent.

[0026] In some embodiments, the agent used in methods provided herein is an antibody or Fc fusion. In some embodiments, the agent targets alpha-gal. In some embodiments, the agent is an anti-alpha-gal antibody or Fc fusion protein.

[0027] In some embodiments, provided methods further comprise measuring mast cell degranulation observed in the rodent. In some embodiments, provided methods further comprise measuring passive cutaneous anaphylaxis (PCA) response in rodents administered the agent. In some embodiments, provided methods further comprise measuring passive systemic anaphylaxis (PSA) in rodents administered the agent.

[0028] These, and other aspects of the disclosure, as described in more detail below and in the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The Drawings included herein, which is composed of the following Figures, are provided for exemplary, illustrative purposes only and not for limitation.

[0030] FIG. 1 is shows a schematic summary, not to scale, of an exemplary targeting strategy for the creation of mice comprising an exemplary modified FcsRla locus as described in Example 1. Unless otherwise indicated, mouse sequences are represented by dashed lines and shapes that are empty or have horizontal shading; human sequences are represented by solid lines and shapes with vertical or diagonal shading.

[0031] FIG. 2 shows expression of humanized FcsRla is expressed on the surface of splenic basophils in exemplary mice comprising the humanized FcsRla locus disclosed herein. Splenic basophils were defined as a population of lymphocytes and basophils,Attorney Docket No. RPB-03125 (11689WO) excluding large granulocytes, singlet cells, Live, TCRb7CD45R (B220)', and CD49b+ / FCERlA+(either human or mouse). Panel A shows representative flow cytometry plots showing basophils from either WT or Fcerlahu / humice stained for human FCER1A. Panel B shows combined data from all WT or Fcerlahll / humice (n = 5) expressed as the % of living spleen cells staining for human FCER1A. Panel C shows representative flow cytometry plots showing basophils from either WT or Fcerlahll / humice stained for mouse FCER1A. Panel D shows combined data from all WT or Fcerlahu / humice (n = 5) expressed as the % of living spleen cells staining for mouse FCER1A. Each black, filled square symbol represents 1 mouse. Statistical analyses were performed using a non-parametric Mann- Whitney test in GraphPad Prism. Statistical significance (p<0.01) is denoted with the symbol **

[0032] FIGs. 3A-B show that an exemplary humanized FcsRla locus provided herein is functional. FIG. 3A shows a schematic for sensitization and challenge of mice. Mice were sensitized by intradermal ear injections with either human allergen- specific IgEs or IgG as a negative control. After 1 day, mice were challenged IV with allergen diluted in Evans blue dye. FIG. 3B shows mast cell degranulation in wild-type (“WT”) and humanized FcsRla mice. Evans blue dye extravasation in the ear was measured as a readout of mast cell degranulation. As human IgE does not bind to mouse FcsRl, the response in the mouse comprising the humanized FcsRla locus indicates that a functional FcsRla was produced.

[0033] FIGs. 4A-B show that an exemplary humanized FcsRla locus provided herein is functional. FIG. 4A shows a schematic for sensitization and challenge of mice. Mice were sensitized with an IV injection of human allergen- specific IgE (“DO”). After 1 day (“DI”), mice were challenged with an IV injection of allergen and temperature changes were monitored over a 4 hour time period after the injection. FIG. 4B shows temperature change over time after IV challenge. Temperature drop is a readout of systemic anaphylaxis.

[0034] FIG. 5 shows a schematic of an example deletion strategy for knock-out of the GGTA1 locus in mice. Guides direct SpCas9 cleavage upstream of the Ggtal start ATG (guide mGU, cut site lOlObp upstream from the ATG; guide mGU2, cut site 2184bp upstream of the ATG) and close to the stop codon (guide mGD, cut site 295bp upstream of the stop; guide mGD2, cut site 830bp downstream of the stop).

[0035] FIG. 6A provides a table describing antibodies used for flow cytometry analysis of spleens of humanized FcsRla mice with GGTA1-KO or a GGTA1-WT locus.Attorney Docket No. RPB-03125 (11689WO)

[0036] FIG. 6B shows validation of GGTA1 KO mice using flow cytometry to confirm absence of alpha-gal epitopes on splenocytes.

[0037] FIG. 7A shows an example gating strategy for isolation of different immune cells in mice with a GGTA1-WT locus and GGTA1-KO mice.

[0038] FIG. 7B shows alpha-gal expression in mouse immune cells. Alpha-gal is expressed on most immune cells with a GGTA1-WT locus, including B cells, neutrophils, macrophages, and monocytes; no / low expression was observed on T cells. Alpha-gal was not expressed on immune cells of GGTA1-KO mice.

[0039] FIG. 8 shows experimental design for optimization of sensitization and challenge for Passive Cutaneous Anaphylaxis (PC A) response in GGTAl-KO / humanized FcsRla mice to a first example alpha-gal allergen, BSA-alpha-gal trisaccharide (NGP0334, Dextra Labs). Mice were sensitized at a first timepoint (“DI”) by intradermal (“ID”) injection of human donor plasma with alpha-gal- specific IgE (“AGS Donor”) or control plasma from a non AGS control donor (“No AGS Donor”). Mice were then challenged with an IV injection of allergen diluted in Evan’s Blue dye at a second time point (“D2”).

[0040] FIG. 9 shows results of a PCA response analysis of GGTAl-KO / humanized FcsRla mice compared to humanized FcsRla mice (having a GGTA1-WT locus) for a first example alpha-gal allergen, BSA-alpha-gal trisaccharide (NGP0334, Dextra Labs). Mast cell degranulation observed in GGTA1-KO / humanized FcsRla mice with 20 pg challenge; no PCA response was observed in humanized FcsRla mice with a GGTA1-WT locus under any of the tested conditions.

[0041] FIG. 10 shows mast cell degranulation in GGTA1-KO / humanized FcsRla mice sensitized with plasma from multiple AGS donors and then challenged with increasing doses of a first example alpha-gal allergen, BSA-alpha-gal trisaccharide (NGP0334, Dextra Labs): 10 pg, 20 pg, and 50 pg alpha-gal allergen. Dose-dependent alpha-gal induced mast cell degranulation was observed in mice sensitized with AGS plasma with Donor 1 or Donor 2 and a consistent mast cell degranulation response across all challenge concentrations for Donor 3. No alpha-gal induced mast cell degranulation response was observed in samples sensitized with non-AGS plasma under any of the tested doses.

[0042] FIG. 11 shows experimental design for sensitization and challenge for PCA response in GGTAl-KO / humanized FcsRla mice to a second example alpha-gal allergen, bovine thyroglobulin (InBio Catalog AGAL-1, lot 47399), which is a natural alpha-gal containing challenge reagent. Mice were sensitized at a first timepoint (“DI”) by ID injection of human donor plasma with alpha-gal- specific IgE (“AGS Donor”) or control plasma from aAttorney Docket No. RPB-03125 (11689WO) non AGS control donor (“No AGS Donor”). Mice were then challenged with an IV injection of allergen diluted in Evan’s Blue dye at a second time point (“D2”), in increasing amounts: 25 pg, 50 pg, and 100 pg bovine thyroglobulin.

[0043] FIG. 12 shows results of PCA response analysis of GGTAl-KO / humanized FcsRla mice in response to a second example alpha-gal allergen, bovine thyroglobulin (InBio Catalog AGAL-1, lot 47399) in increasing doses of 25 pg, 50 pg, and 100 pg. Dosedependent alpha-gal induced mast cell degranulation was observed in mice sensitized with AGS plasma. No alpha-gal induced mast cell degranulation response was observed in samples sensitized with non- AGS plasma under any of the tested doses.DEFINITIONS

[0044] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

[0045] In this application, unless otherwise clear from context, (i) the terms “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and "including" may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) where ranges are provided, endpoints are included.

[0046] 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.

[0047] As used herein, the term “ antibody” 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 domains of hypervariability, termed complementarity determining regions (CDR),Attorney Docket No. RPB-03125 (11689WO) interspersed with regions that are more conserved, termed framework regions (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-terminus in 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.

[0048] 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 “antigenbinding 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.

[0049] 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.

[0050] A “coding region” 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).

[0051] As used herein, the term “correspond to” refers to exons that encode the same or homologous functional domain or portion of a protein.

[0052] For example, “exons that correspond to mouse FcsRla exons 1-3” 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 1-3.Attorney Docket No. RPB-03125 (11689WO)These could be exons 1-3 of the genetically modified non-human animal, or other exons due to the difference in exon configuration among different non-human animal species.

[0053] The phrase “derived from” when used concerning a rearranged variable region gene “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 back to a set of unrearranged variable region gene segments that were rearranged to form a 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 is still derived from the unrearranged variable region gene segments. In some embodiments, where the endogenous locus is replaced with a universal light chain or heavy chain locus, the term “derived from” indicates the ability to trace origin of the sequence to said rearranged locus even though the sequence may have undergone somatic mutations.

[0054] As used herein, the phrase “endogenous gene” or “endogenous gene segment” refers 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 (“WT”) organism. In some embodiments, a reference organism is an engineered organism. In some embodiments, a reference organism is a laboratory-bred organism (whether WT or engineered).

[0055] The term “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. To give 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.Attorney Docket No. RPB-03125 (11689WO)

[0056] 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 (“WT”) locus. In some embodiments, an endogenous locus is an engineered locus.

[0057] The phrase “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 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.

[0058] The phrase “operably linked”, as used herein, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. 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 the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “expression control sequence” as used herein refers to 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 poly adenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein 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.Attorney Docket No. RPB-03125 (11689WO)

[0059] The terms “polynucleotide” , and “nucleic acid” are used interchangeably. They refer 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.

[0060] The term “polypeptide” , as used herein, refers to any polymeric chain of amino acids. 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 is engineered in that it is designed and / or produced through action of the hand of man.

[0061] The term “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 “endogenous promoter” refers to a promoter that is naturally associated, e.g., in a wild-type (“WT”) organism, with an endogenous gene.

[0062] The term “replacement” is used herein to refer 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 and / or contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.). In some embodiments, a replaced nucleic acid sequence includes one or moreAttorney Docket No. RPB-03125 (11689WO) of a promoter, an enhancer, a splice donor site, a splice receiver 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 of the replaced nucleic acid sequence. In some embodiments, a replacement nucleic acid sequence is an ortholog 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 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, the 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; the replacement is of an endogenous genomic sequence with a nucleic acid sequence that encodes a protein that has a similar function as a protein 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 gene or 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.

[0063] Variant” as the term is 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, 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.Attorney Docket No. RPB-03125 (11689WO)

[0064] The term “vector”, as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. In some embodiment, 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 operatively linked genes are referred to herein as “expression vectors.”DETAILED DESCRIPTION

[0065] The present disclosure describes mutation of the gene encoding a- 1,3,- galactosyltransferase (GGTA1) in non-human animals (e.g., rodents, e.g., mice or rats), wherein the genome also comprises a humanized FcsRla locus. Such genetically modified non-human animals (e.g., rodents) may be useful for interrogation of polyclonal alpha-gal specific human IgE. The present disclosure recognizes that such a model best mimics the human condition and allows for the use of passive local or systemic sensitization rodent models. Validation data provided herein confirms the absence of the alpha-gal epitope in humanized FcsR I a / Ggtal KO mice and demonstrates a passive cutaneous anaphylaxis response in vivo in these mice.Galactose-a-l,3-galactose (alpha-gal) and alpha-gal syndrome

[0066] Galactose-a-l,3-galactose (alpha-gal) is a unique carbohydrate structure absent in humans but naturally produced on glycolipids and glycoproteins in non-primate mammals, prosimians and new world monkeys. Alpha-gal is produced by the enzyme a-1,3,- galactosyltransferase (GGTA1). The genomes of humans, apes and old world monkeys do not encode an active GGTA1.

[0067] The core structure of the alpha-gal epitope is a terminal disaccharide galactose-a-l,3-galactose. This disaccharide is often followed by N-acetyl glucosamine (GlcNAc) in the third position forming a trisaccharide (Gal-a-l,3-Gal-P-l,4-GlcNAc). In glycoproteins, an alpha-gal epitope can occur as part of a bi-or tri-antennary N-glycan. In glycolipids, an alpha-gal epitope is often part of a pentasaccharide (e.g., ceramide pentahexoside).

[0068] IgG, IgM and IgA antibodies to alpha-gal are present in all nonimmunocompromised humans. Such antibodies are produced as a result of continuous antigenic stimulation by carbohydrate antigens on gastrointestinal bacterial of the normal flora. It is estimated that the antibody response to alpha-gal is as much as 1-5% of totalAttorney Docket No. RPB-03125 (11689WO) circulating IgG and IgM. (See, Galili (2005) Immunol Cell Biol. 83(6):674-686 and Macher and Galili (2008) Biochim Biophys Act., 1780(2):75-88, herein incorporated by reference in their entireties.)

[0069] Alpha-gal syndrome, also known as mammalian meat allergy (MMA), is an allergic reaction to alpha-gal. A link has been described between the development of alphagal syndrome and prior tick bites. Alpha-gal syndrome has been shown to be due to IgE specific to alpha-gal. Following tick bites, primarily from the lone star tick, some individuals produce IgE to alpha-gal and subsequently undergo anaphylaxis upon consumption of mammalian meat or exposure to alpha-gal containing mammal byproducts. Commins, S. P., et al. (2009). The Journal of allergy and clinical immunology, 123(2), 426-433, herein incorporated by reference in its entirety.

[0070] Sensitization to the a-gal epitope is of key importance to cetuximab-induced anaphylaxis (an a-gal-containing monoclonal antibody produced in murine NS0 cells), MMA (primary), which can extend to dairy, gelatin, collagen (secondary), and the rejection of xenotransplants. Alpha-gal IgE isolated from allergic human donor demonstrated to bind trisaccharide with P-1,4 linkage to GlcNAc or a P-1,3- linkage to GlcNAc. Platts- -Mills, et al. (2020). The journal of allergy and clinical immunology. In practice, 8(1), 15-23, herein incorporated by reference in its entirety.

[0071] Alpha-gal is structurally similar to B-group antigen. Anti-alpha-gal antibodies demonstrated to bind B group antigen. B antigen blood group patients may have decreased anti-alpha-gal IgG and some protection from allergic sensitization to alpha-gal and the development of red meat allergy.

[0072] Sensitized individuals may have greater burden of atherosclerotic plaques and / or high risk features (atheroma calcification and necrosis).

[0073] Desensitization via oral immunotherapy (OfT) has been reported in patients with delay ed-type reaction to red meat is reported; successful reintroduction of meat following decrease in alpha-gal serum IgE reported Serum IgE to alpha-gal associated with higher risk of coronary artery disease. See Unal et al. (2017) The Journal of allergy and clinical immunology, 5(2): P502-503 and Pelletier et al. (2018) Ann Allergy Asthma Immunol. 121( 1): 123- 124, herein incorporated by reference in their entireties.

[0074] Rodents express alpha-gal and therefore are tolerized to the alpha-gal epitope. In order to model the allergic response to alpha-gal in vivo in rodents (e.g., mice or rats), rodents were genetically modified to mutate the endogenous gene encoding the GGTA1 enzyme.Attorney Docket No. RPB-03125 (11689WO) a-1, 3, -galactosyltransferase (GGTA1) Knock-Out

[0075] Provided genetically modified rodents (e.g., mice or rats) and cells (e.g., ES cells) described herein do not express a functional endogenous GGTA1 enzyme and / or lack GGTA1 enzymatic activity. In some embodiments, provided genetically modified rodents (e.g., mice or rats) and cells (e.g., ES cells) comprise a GGTA1 knock-out.

[0076] GGTA1 is a member of the galactosyltransferase family of intracellular, membrane-bound enzymes that are involved in the biosynthesis of glycoproteins and glycolipids. GGTA1 protein catalyzes the transfer of galactose from UDP-galactose to N- acetyllactosamine in an alpha(l,3) linkage to form galactose alpha(l,3)-galactose.

[0077] The endogenous mouse GGTA1 locus is present on chromosome 2, genome coordinates 35,400,183-35,463,231, complement strand (GRCm38 / mml0).

[0078] Alternative splicing of the GGTA1 gene results in multiple transcript variants encoding different isoforms, such as, but not limited to, those listed in Table 1 below.Table 1: Genbank Accession Numbers for mouse GGTA1Attorney Docket No. RPB-03125 (11689WO)

[0079] In some embodiments, rodents provided herein do not express a functional GGTA1 enzyme. In some embodiments, provided genetically modified rodents (e.g., mice or rats) comprise a knock-out of the gene encoding GGTA1.

[0080] In some embodiments, GGTA1 mutant rodents animals comprises a deletion of at least part of an endogenous GGTA1 locus, such that the rodent does not express a functional rodent GGTA1 enzyme. In some embodiments, provided genetically modified rodents (e.g., mice) comprise a deletion of at least part of the encoding region of rodent GGTA1.

[0081] In some embodiments, rodents provided herein comprise a deletion of the endogenous genomic sequence encoding GGTA1, wherein the deletion is a deletion of at least 20,000 nt, at least 21,000 nt, at least 22,000 nt, at least 23,000 nt, at least 24,000 nt, at least 25,000 nt, at least 26,000 nt, at least 27,000 nt, at least 28,000 nt, at least 29,000 nt, or at least 30,000 nt.

[0082] In some embodiments, rodents provided herein comprise a deletion of the endogenous genomic sequence encoding GGTA1, wherein the deletion is a deletion of between 5,000 nt to 50,000 nt. In some embodiments, rodents provided herein comprise a deletion of the endogenous genomic sequence encoding GGTA1, wherein the deletion is a deletion of between 10,000 nt to 45,000 nt. In some embodiments, rodents provided herein comprise a deletion of the endogenous genomic sequence encoding GGTA1, wherein the deletion is a deletion of between 15,000 nt to 40,000 nt. In some embodiments, rodents provided herein comprise a deletion of the endogenous genomic sequence encoding GGTA1, wherein the deletion is a deletion of between 20,000 nt to 35,000 nt. In some embodiments, rodents provided herein comprise a deletion of the endogenous genomic sequence encoding GGTA1, wherein the deletion is a deletion of between 20,000 nt to 30,000 nt.

[0083] In some embodiments, rodents provided herein comprise a deletion of the genomic sequence encoding GGTA1, wherein the deletion is within genomic coordinates chr2: 35,399,144 to 35,463,231 (GRCm38 / mml0, - strand).

[0084] In some embodiments, rodents provided herein comprise a deletion of about 20,000 nt, about 21,000 nt, about 22,000 nt, about 23,000 nt, about 24,000 nt, about 25,000Attorney Docket No. RPB-03125 (11689WO) nt, about 26,000 nt, about 27,000 nt, about 28,000 nt, or about 29,000 nt, wherein the deletion is within genomic coordinates chr2: 35,399,144 to 35,427,764 (GRCm38 / mrnl0, - strand).

[0085] In some embodiments, rodents provided herein comprise a deletion of about 20,000 nt, about 21,000 nt, about 22,000 nt, about 23,000 nt, about 24,000 nt, or about 25,000 nt, wherein the deletion is within genomic coordinates chr2: 35,399,144 to 35,427,764 (GRCm38 / mml0, - strand).

[0086] In some embodiments, provided rodents comprise a deletion of 5,000 to 24,247 bp of genomic sequence within genomic coordinates chr2: 35,401,275 to 35,425,521 (GRCm38 / mml0, - strand). In some embodiments, provided rodents comprise a deletion of 10,000 to 24,247 bp of genomic sequence within genomic coordinates chr2: 35,401,275 to 35,425,521 (GRCm38 / mml0, - strand). In some embodiments, provided rodents comprise a deletion of 20,000 to 24,247 bp of genomic sequence within genomic coordinates chr2: 35,401,275 to 35,425,521 (GRCm38 / mml0, - strand). In some embodiments, rodents provided herein comprise a deletion of the genomic sequence corresponding to GRCm38 / mml0:chr2: 35,401,273-35,425,528.

[0087] In some embodiments, provided rodents comprise a deletion of 5,000 to 28,620 bp of genomic sequence within genomic coordinates chr2: 35,399,144 to 35,427,764 (GRCm38 / mml0, - strand). In some embodiments, provided rodents comprise a deletion of 10,000 to 28,620 bp of genomic sequence within genomic coordinates chr2: 35,399,144 to 35,427,764 (GRCm38 / mml0, - strand). In some embodiments, provided rodents comprise a deletion of 20,000 to 28,620 bp of genomic sequence within genomic coordinates chr2: 35,399,144 to 35,427,764 (GRCm38 / mml0, - strand). In some embodiments, rodents provided herein comprise a deletion of the genomic sequence corresponding to GRCm38 / mml0:chr2: 35,399,144-35,427,764.

[0088] In some embodiments, rodents provided herein comprise a deletion of about 20,000 nt, about 21,000 nt, about 22,000 nt, about 23,000 nt, about 24,000 nt, or about 25,000 nt, wherein the deletion is within genomic coordinates chr2: 35,399,144 to 35,425,528 (GRCm38 / mml0, - strand).

[0089] In some embodiments, provided rodents comprise a deletion of 5,000 to 24,247 bp of genomic sequence within genomic coordinates chr2: 35,399,144 to 35,427,764 (GRCm38 / mml0, - strand).

[0090] In some embodiments, provided rodents comprise a deletion of at least 10,000 nt, at least 11,000 nt, at least 12,000 nt, at least 13,000 nt, at least 14,000 nt, at least 15,000 nt, at least 16,000 nt, at least 17,000 nt, at least 18,000 nt, at least 19,000 nt, at least 20,000 ntAttorney Docket No. RPB-03125 (11689WO) within genomic coordinates chr2: 35,399,144 to 35,427,764 (GRCm38 / mmlO, - strand). In some embodiments, provided rodents comprise a deletion of 20,000 to 24,247 bp of genomic sequence within genomic coordinates chr2: 35,399,144 to 35,427,764 (GRCm38 / mml0, - strand).

[0091] In some embodiments, rodents provided herein comprise a deletion of at least a portion of an endogenous genomic GGTA1 sequence, such that the rodent genome comprises a sequence corresponding to SEQ ID NO: 32.

[0092] In some embodiments, the genome of provided rodents comprise a sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to the sequence corresponding to SEQ ID NO: 32.

[0093] In some embodiments, rodents provided herein comprise a deletion of exon 1 of GGTA1. In some embodiments, rodents provided herein comprise a deletion of 2 or more exons of GGTA1. In some embodiments, rodents provided herein comprise a deletion of all or substantially of the exons of GGTA1.

[0094] In some embodiments, provided genetically modified rodents (e.g., mice) comprise a deletion of the entire GGTA1 protein-coding sequence.

[0095] In some embodiments, provided genetically modified rodents (e.g., mice) comprise a deletion of the entire GGTA1 protein-coding sequence and the GGTA1 promoter sequence.

[0096] In some embodiments, provided mice are homozygous for the GGTA1 deletion.

[0097] In some embodiments, provided mice are heterozygous for the GGTA1 deletion.Humanized Fc epsilon receptor 1 alpha

[0098] Provided genetically modified rodents (e.g., mice or rats) and cells (e.g., ES cells) described herein comprise a GGTA1 knock-out as described above, and also include a humanized or human Fc epsilon receptor 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.

[0099] 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.Attorney Docket No. RPB-03125 (11689WO)In some embodiments, the FceRla locus comprises a nucleic acid sequence encoding an FceRla polypeptide comprising a human extracellular domain, a human transmembrane domain and a rodent (e.g., mouse or rat) cytoplasmic domain. In some embodiments, the FceRla locus comprises a nucleic acid sequence encoding an FceRla polypeptide comprising a human extracellular domain, a human transmembrane domain and a human cytoplasmic domain. An exemplary embodiment of an engineered FceRla locus is described in WO2019 / 190990, incorporated herein by reference.

[0100] In some embodiments, the nucleic acid sequence encoding the FceRla polypeptide is positioned at an endogenous rodent (e.g., mouse) FceRla locus. In certain embodiments, the nucleic acid sequence encoding the FceRla polypeptide replaces all or part of an endogenous rodent (e.g., mouse) FceRla gene. For example, in some embodiments, the nucleic acid sequence encoding the extracellular domain of an endogenous FceRla at an endogenous FceRla locus is replaced with a nucleic acid sequence encoding the extracellular domain of a human FceRla such that a rodent (e.g., mouse) comprising such a locus expresses a FceRla 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 FceRla polypeptide comprising a human extracellular domain, a human transmembrane domain, and a human cytoplasmic domain is positioned at an endogenous rodent (e.g., mouse) FceRla locus. In some embodiments, the nucleic acid sequence encoding a FceRla 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) FceRla gene. In some embodiments, the mouse does not express a rodent (e.g., mouse) FceRla, or does not express a functional rodent (e.g., mouse) FceRla. In some embodiments, the FceRla 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).

[0101] 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) FceRItx are replaced by part of human coding exon 1, human coding exon 2, human coding exon 3, human coding exon 4, and human coding exon 5 of the human FceRItx gene. In some embodiments, the FceRItx 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 andAttorney Docket No. RPB-03125 (11689WO) poly A, followed by the mouse 3’UTR and poly A. 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 FceRItx that are believed to interact with IgE, and exon 5 encodes the cytoplasmic and transmembrane domains of the protein (see FIG. 1).

[0102] GenBank accession nos. NC_000001.l l (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.

[0103] 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.Genetically modified mice and mouse ES cells

[0104] In some embodiments, the present disclosure provides genetically modified mice and / or mouse ES cells comprising a human or humanized FcsRla locus that do not express a functional GGTA1 protein. In some embodiments, provided genetically modified mice and / or mouse ES cells comprise a GGTA1 knock-out.

[0105] 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.Attorney Docket No. RPB-03125 (11689WO)

[0106] 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.

[0107] 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

[0108] Genetically modified rodents (e.g., mice or rats) and cells (e.g., ES cells) comprising a GGTA1 knock-out and a humanized or human Fc epsilon receptor 1 alpha (FcsRla) locus described herein find many uses in the art. For example, provided rodents (e.g., mice or rats) may be useful as a model for alpha-gal syndrome and / or for screening agents for treatment and / or prevention thereof.

[0109] In some embodiments, provided herein are methods of testing an agent. In some embodiments, provided methods of testing an agent comprise administering the agent to a rodent (e.g., mouse or rat) described herein.

[0110] In some embodiments, provided herein are methods of testing or determining the ability of an agent to reduce or alleviate an alpha gal-induced allergy response. In some embodiments, the ability of an agent to reduce or alleviate an alpha gal-induced allergy response is determined by measuring mast cell degranulation. In some embodiments, provided methods of testing an agent comprise administering the agent to a rodent (e.g., mouse or rat) described herein. In some embodiments, provided methods comprise assessing and / or measuring passive cutaneous anaphylaxis (PCA) response. In some embodiments,Attorney Docket No. RPB-03125 (11689WO) provided methods comprise assessing and / or measuring passive systemic anaphylaxis (PSA) response.

[0111] In some embodiments, provided methods comprise measuring the therapeutic efficacy of the administered agent (e.g., therapeutic protein) in a rodent (e.g., mouse or rat) as described herein. For example, in some embodiments, provided methods measure the ability of an administered dose of the agent (e.g., therapeutic protein) to reduce or eliminate one or more symptoms in the rodent model. In some embodiments, the rodent model is passive cutaneous anaphylaxis (PCA) model and / or passive systemic anaphylaxis (PSA) model.

[0112] In some embodiments, provided methods comprise measuring the safety and dosing of the administered agent (e.g., therapeutic protein) in the rodent model. In some embodiments, provided methods comprises measuring the extent to which an administered dose of the therapeutic protein produces one or more adverse effects in the non-human 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 of fertility, 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 (PCA) and / or passive systemic anaphylaxis (PSA) models.

[0113] 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.

[0114] In some embodiments, provided mice are useful in a passive cutaneous anaphylaxis (PCA) model of alpha gal-induced allergy response. The PCA model is an in vivo model of local allergen-induced mast cell degranulation mediated by allergen- specific crosslinking of IgE bound to FCER1. The PCA model measures degranulation-induced vascular permeability, and has been used to study hypersensitivity in various settings. SeeAttorney Docket No. RPB-03125 (11689WO)Gilfillan A M and Tkaczyk C. (2006) Nature Reviews Immunology, 6(3):218-30; Orengo JM, et al. (2018) Nature Communications, 9( 1): 1421 ; Zhu D, et al. (2005) Nature Medicine, 11(4):446-9, each of which is herein incorporated by reference in its entirety. However, as human IgE does not bind mouse FCER1. See Dombrowicz D, et al. (1996) Journal of Immunology, 157(4): 1645-51, herein incorporated by reference in its entirety. As such, the standard PCA model cannot be used to test sensitization by human allergen- specific IgE in rodents, such as mice. In some embodiments, to study sensitization with human IgE in the PCA model, a humanized mouse strain is used in which the entire coding sequence of the mouse FcsRla gene was replaced with the corresponding human sequence (designated 7v: / ? / zllu / llu), as described in Example 1 below. This model may be used to evaluate the capacity of human mAbs to block mast cell degranulation in the context of mice sensitized with IgE containing antisera isolated from allergic human donors.

[0115] 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.

[0116] In some embodiments, provided herein are methods of screening agents for the ability to reduce or alleviate an alpha gal-induced allergy response. In some embodiments, provided screening assays comprise contacting or administering a candidate agent of interest to a genetically modified rodent of the present disclosure, wherein the candidate agent is administered prior to sensitization and / or challenge. In some embodiments, provided screening assays comprise contacting or administering a candidate agent of interest to a genetically modified rodent of the present disclosure, wherein the candidate agent is administered after sensitization and / or challenge.

[0117] In some embodiments, provided herein are methods of screening agents for the ability to prevent an alpha gal-induced allergy response. In some embodiments, provided screening assays comprise contacting or administering a candidate agent of interest to a genetically modified rodent of the present disclosure, wherein the candidate agent is administered prior to sensitization and / or challenge.

[0118] In some embodiments, the agent is administered to a rodent (e.g., mouse or rat) provided herein as part of a pharmaceutical composition e.g., a pharmaceutical composition, containing a human 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.Attorney Docket No. RPB-03125 (11689WO)

[0119] 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).

[0120] In various embodiments, rodents (e.g., mice or rats) 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 or rat) 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.

[0121] In some embodiments, rodents (e.g., mice or rats) as described herein provide an improved in vivo system for development and selection of human antibodies or Fc fusion polypeptides. Alternatively and / or additionally, an immune response may be monitored in such mice so as to characterize and select potential human antibodies that may be developed as a therapeutic. In some embodiments, rodents (e.g., mice or rats) as described herein provide an in vivo system for development and / or selection of agents to treat or prevent an alpha gal-induced allergy response.

[0122] 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

[0123] 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.

[0124] 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, homologousAttorney Docket No. RPB-03125 (11689WO) 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), herein incorporated by reference in their entireties.

[0125] 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, which is incorporated by reference herein). Offspring 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.).

[0126] 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.Attorney Docket No. RPB-03125 (11689WO)7,659,442, U.S. Pat. No. 7,294,754, and Kraus et al. (2010, Genesis 48:394-399), herein incorporated by reference in their entireties.

[0127] 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, herein incorporated by reference in their entireties), 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, which are each incorporated herein by reference in their entireties). 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) Nat Protoc. 6(6): doi: 10.1038 / nprot.2011.338 (all of which are each incorporated herein by reference in their entireties).

[0128] In some embodiments, genetically modified founder animals can be bred to additional animals carrying one or more genetic modifications.

[0129] 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 described herein, and such stem cells may be introduced into an embryo to generate genetically modified animals with several genetic modifications.

[0130] Various embodiments of the present disclosure provide genetically modified rodents (e.g., mice) that include a human nucleic acid in substantially all of their cells, as well as genetically modified rodents (e.g., mice) that include a human nucleic acid in some, but not all their cells. In some instances, e.g., targeted recombination, one copy of the human nucleic acid will be integrated into the genome of the genetically modified rodents. In other instances, e.g., random integration, multiple copies, adjacent or distant to one another, of the human nucleic acid may be integrated into the genome of the genetically modified rodents.Attorney Docket No. RPB-03125 (11689WO)Kits

[0131] Provided herein a pack or kit comprising one or more containers filled with at least one rodent (e.g., mouse), rodent cell (e.g., mouse cell), DNA fragment and / or targeting vector as described in the description, examples, and / or figures herein. Kits may be used in any applicable method (e.g., a research method). Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human administration, (b) directions for use, (c) a contract that governs the transfer of materials and / or biological products (e.g., a mouse or mouse cell as described herein) between two or more entities and combinations thereof.Exemplary Embodiments

[0132] Embodiment 1. A rodent whose genome:(a) does not encode a functional a a- 1,3, -galactosyltransferase (GGTA1), and(b) comprises a human or a humanized Fc epsilon receptor 1 alpha (FcsRla) locus, wherein the human or the humanized FcsRla locus comprises a nucleic acid sequence encoding a FcsRla polypeptide comprising a human extracellular domain.

[0133] Embodiment 2. The rodent of embodiment 1, wherein the rodent genome comprises a deletion of at least part of the GGTA1 locus.

[0134] Embodiment 3. The rodent of embodiment 1 or embodiment 2, wherein the rodent genome comprises a deletion of a sequence that is 10,000 bp to 64,087 bp, wherein the deletion is within genomic coordinates chr2: 35,399,144 to 35,463,231 (GRCm38 / mml0,- strand).

[0135] Embodiment 4. The rodent of any one of embodiments 1 to 3, wherein the rodent genome comprises a deletion of a sequence that is 10,000 bp to 24,247 bp, wherein the deletion is within genomic coordinates chr2: 35,401,275 to 35,425,521 (GRCm38 / mml0,- strand).

[0136] Embodiment 5. The rodent of any one of embodiments 1 to 4, wherein the rodent genome comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to the sequence corresponding to SEQ ID NO: 32.

[0137] Embodiment 6. The rodent of any one of embodiments 1 to 4, wherein the rodent genome comprises the sequence corresponding to SEQ ID NO: 32.Attorney Docket No. RPB-03125 (11689WO)

[0138] Embodiment 7. The rodent of any one of embodiments 1 to 4, wherein the rodent genome comprises a deletion of one or more GGTA1 exons.

[0139] Embodiment 8. The rodent of any one of embodiments 1 to 4, wherein the rodent genome comprises a deletion of all of the GGTA1 exons.

[0140] Embodiment 9. The rodent of any one of embodiments 1 to 4, wherein the rodent genome comprises a deletion of the entire GGTA1 encoding region.

[0141] Embodiment 10. The rodent of any one of embodiments 2 to 9, wherein rodent genome is homozygous for the deletion.

[0142] Embodiment 11. The rodent of any one of embodiments 1 to 10, theFcsRla polypeptide further comprises a rodent transmembrane domain.

[0143] Embodiment 12. The rodent of any one of embodiments 1 to 10, wherein the FcsRla polypeptide further comprises a human transmembrane domain.

[0144] Embodiment 13. The rodent of any one of embodiments 1 to 12, wherein the FcsRla polypeptide further comprises a rodent cytoplasmic domain.

[0145] Embodiment 14. The rodent of any one of embodiments 1 to 12, wherein the FcsRla polypeptide further comprises a human cytoplasmic domain.

[0146] Embodiment 15. The rodent of any one of embodiments 1 to 14, wherein the nucleic acid sequence encoding the human or humanized FcsRla polypeptide is positioned at an endogenous rodent FcsRla locus.

[0147] Embodiment 16. The rodent of any one of embodiments 1 to 15, wherein the nucleic acid sequence encoding the human or humanized FcsRla polypeptide replaces all or part of an endogenous rodent FcsRla gene.

[0148] Embodiment 17. The rodent of any one of embodiments 1 to 16, wherein the nucleic acid sequence encoding the human FcsRla extracellular domain replaces an endogenous nucleic acid sequence encoding a rodent FcsRla extracellular domain.

[0149] Embodiment 18. The rodent of any one of embodiments 1 to 17, wherein the rodent does not express a rodent FcsRla.

[0150] Embodiment 19. The rodent of any one of embodiments 1 to 18, wherein the rodent is heterozygous for the human or humanized FcsRla locus.

[0151] Embodiment 20. The rodent of any one of embodiments 1 to 18, wherein the rodent is homozygous for the human or humanized FcsRla locus.

[0152] Embodiment 21. The rodent of any one of embodiments 1 to 20, wherein the rodent is a mouse.Attorney Docket No. RPB-03125 (11689WO)

[0153] Embodiment 22. A cell isolated from a genetically modified rodent of any one of embodiments 1 to 21.

[0154] Embodiment 23. An isolated rodent cell whose genome comprises a a-1 ,3,-galactosyltransl'crasc (GGTA1) knock-out and a human or humanized Fc epsilon receptor 1 alpha (FcsRla) locus.

[0155] Embodiment 24. An isolated rodent cell whose genome comprises a deletion of at least part of an a- 1,3, -galactosyltransferase (GGTA1) locus and a human or humanized Fc epsilon receptor 1 alpha (FcsRla) locus.

[0156] Embodiment 25. The rodent cell of embodiment 23 or 24, wherein the rodent cell genome comprises a deletion of a sequence that is 10,000 bp to 64,087 bp, wherein the deletion is within genomic coordinates chr2: 35,399,144 to 35,463,231 (GRCm38 / mml0, - strand).

[0157] Embodiment 26. The rodent cell of embodiment 23 or 24, wherein the rodent cell genome comprises a deletion of a sequence that is 10,000 bp to 24,247 bp, wherein the deletion is within genomic coordinates chr2: 35,401,275 to 35,425,521 (GRCm38 / mml0, - strand).

[0158] Embodiment 27. The rodent cell of any one of embodiments 23 to 26, wherein the rodent cell genome comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to the sequence corresponding to SEQ ID NO: 32.

[0159] Embodiment 28. The rodent cell of any one of embodiments 23 to 26, wherein the rodent cell genome comprises the sequence corresponding to SEQ ID NO: 32.

[0160] Embodiment 29. The rodent cell of any one of embodiments 24 to 26, wherein the rodent cell genome comprises a deletion of one or more GGTA1 exons.

[0161] Embodiment 30. The rodent cell of any one of embodiments 24 to 26, wherein the rodent cell genome comprises a deletion of all of the GGTA1 exons.

[0162] Embodiment 31. The rodent cell of any one of embodiments 23 to 26, wherein the rodent cell genome comprises a deletion of the entire GGTA1 encoding region.

[0163] Embodiment 32. The rodent cell of any one of embodiments 23 to 31, wherein rodent cell genome is homozygous for the deletion.

[0164] Embodiment 33. The rodent cell of any one of embodiments 23 or to 32, wherein the FcsRla polypeptide further comprises a rodent transmembrane domain.

[0165] Embodiment 34. The rodent cell of any one of embodiments 23 or to 32, wherein the FcsRla polypeptide further comprises a human transmembrane domain.Attorney Docket No. RPB-03125 (11689WO)

[0166] Embodiment 35. The rodent cell of any one of embodiments 23 or to 34, wherein the FcsRla polypeptide further comprises a rodent cytoplasmic domain.

[0167] Embodiment 36. The rodent cell of any one of embodiments 23 to 34, wherein the FcsRla polypeptide further comprises a human cytoplasmic domain.

[0168] Embodiment 37. The rodent cell of any one of embodiments 23 to 36, wherein the nucleic acid sequence encoding the human or humanized FcsRla polypeptide is positioned at an endogenous rodent FcsRla locus.

[0169] Embodiment 38. The rodent cell of any one of embodiments 23 to 37, wherein the nucleic acid sequence encoding the human or humanized FcsRla polypeptide replaces all or part of an endogenous rodent FcsRla gene.

[0170] Embodiment 39. The rodent cell of any one of embodiments 23 to 38, wherein the nucleic acid sequence encoding the human FcsRla extracellular domain replaces an endogenous nucleic acid sequence encoding a rodent FcsRla extracellular domain.

[0171] Embodiment 40. The rodent of any one of embodiments 23 to 39, wherein the rodent cell is heterozygous for the human or humanized FcsRla locus.

[0172] Embodiment 41. The rodent of any one of embodiments 23 to 39, wherein the rodent cell is homozygous for the human or humanized FcsRla locus.

[0173] Embodiment 42. The rodent cell of any one of embodiments 23 to 41, wherein the rodent cell is a mouse cell.

[0174] Embodiment 43. The rodent cell of any one of embodiments 22 to 42, wherein the rodent cell is an ES cell.

[0175] Embodiment 44. A method of making a genetically modified rodent of any one of embodiments 1 to 21.

[0176] Embodiment 45. A method of making a genetically modified rodent, the method comprising: deleting at least a portion of an endogenous a- 1,3, -galactosyltransferase (GGTA1) locus in the genome of a rodent, wherein the rodent genome comprises a humanized Fc epsilon receptor 1 alpha (FcsRla) locus at the endogenous rodent FcsRla locus.

[0177] Embodiment 46. A method of making a genetically modified rodent, the method comprising: inserting a humanized Fc epsilon receptor 1 alpha (FcsRla) locus in an endogenous FcsRla locus of the rodent genome, wherein rodent genome does not encode a functional endogenous a- 1,3, -galactosyltransferase (GGTA1).Attorney Docket No. RPB-03125 (11689WO)

[0178] Embodiment 47. A method of making a genetically modified rodent, the method comprising: generating a rodent ES cell of embodiment 23; and generating a rodent from said ES cell.

[0179] Embodiment 48. A method of making a genetically modified rodent, the method comprising: breeding a rodent comprising a GGTA1 mutation with a rodent comprising humanized FcsRIa locus, selecting for rodents that are homozygous for the GGTA1 mutation and the humanized FcsRIa locus.

[0180] Embodiment 49. The method of embodiment 48, wherein the GGTA1 mutation comprises a deletion of at least a portion of the endogenous GGTA1 locus.

[0181] Embodiment 50. The method of embodiment 48, wherein the GGTA1 mutation comprises a knock-out of the endogenous GGTA1 locus.

[0182] Embodiment 51. A method of making a rodent model of alpha-gal allergy, the method comprising: genetically modifying a rodent such that its genome comprises a humanized FcsRIa locus and does not encode a functional GGTA1.

[0183] Embodiment 52. The method of embodiment 51, comprising deleting at least a portion of the endogenous GGTA1 locus.

[0184] Embodiment 53. The method of embodiment 51, comprising a knock-out of the endogenous GGTA1 locus.

[0185] Embodiment 54. Use of a genetically modified rodent of any one of embodiments 1 to 21 for testing an agent.

[0186] Embodiment 55. The use of embodiment 54, wherein the agent is tested for its ability to prevent and / or treat alpha-gal allergy.

[0187] Embodiment 56. A method of testing an agent, the method comprising administering the agent to a rodent of any one of embodiments 1 to 21.

[0188] Embodiment 57. A method of testing an agent for preventing and / or treating alpha-gal allergy, the method comprising administering the agent to a rodent of any one of embodiments 1 to 21.

[0189] Embodiment 58. The method of embodiment 56 or 57, further comprising measuring one or more pharmacokinetic properties of the administered agent.Attorney Docket No. RPB-03125 (11689WO)

[0190] Embodiment 59. The method of embodiment 58, wherein the one or more pharmacokinetic properties are selected from one or more of area under the plasma concentration versus time (AUC), in vivo recovery (IVR), clearance rate (CL), mean residence time (MRT), agent half-life (tAz), and volume of distribution at steady state (Vss).

[0191] Embodiment 60. The method of any one of embodiments 56 to 59, further comprising measuring therapeutic efficacy of the administered agent.

[0192] Embodiment 61. The method of any one of embodiments 56 to 60, further comprising administering a plurality of doses of the agent to the genetically modified rodent, and determining one or more of:(a) therapeutic efficacy of each dose of the agent;(b) safety of each dose the agent; and(c) tolerability of each dose the agent.

[0193] Embodiment 62. The method of any one of embodiments 56 to 61, further comprising measuring an immune response generated by the genetically modified rodent against the agent.

[0194] Embodiment 63. The method of any one of embodiments 56 to 62, wherein the agent is an antibody or Fc fusion.

[0195] Embodiment 64. The method of any one of embodiments 56 to 63, wherein the agent targets alpha-gal.

[0196] Embodiment 65. The method of any one of embodiments 56 to 64, further comprising measuring mast cell degranulation observed in the genetically modified rodent.

[0197] Embodiment 66. The method of any one of embodiments 56 to 65, further comprising measuring passive cutaneous anaphylaxis (PC A) response in genetically modified rodents administered the agent.

[0198] Embodiment 67. The method of any one of embodiments 56 to 66, further comprising measuring passive systemic anaphylaxis (PSA) in genetically modified rodents administered the agent.

[0199] Embodiment 68. The method of any one of embodiments 56 to 67, wherein the genetically modified rodent is a mouse.Attorney Docket No. RPB-03125 (11689WO)EXAMPLESExample 1: A Mouse Comprising Humanized Fes receptor alpha (FcsRa) for Studying Human IgE Antibody Interactions

[0200] The FceR protein consists of a single ex subunit, a |3 subunit, and two y subunits. The extracellular portion of the ex subunit, FceRItx, comprises two immunoglobulin- like domains and binds IgE with high affinity, even in the absence of the other subunits. Therefore, in the strategy outlined below, FceRItx, the ex subunit of the FceR was humanized.

[0201] The mouse FceRItx locus, located on mouse chromosome 1, was humanized by construction of unique targeting vectors from human and mouse bacterial artificial chromosomes (BAC) DNA using VELOCIGENE® technology (see, e.g., US Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome couple with high-resolution expression analysis. Nat. Biotech. 21(6): 652-659, both incorporated herein by reference). DNA from mouse BAC RP23-332il4 (Invitrogen-Thermo Fisher) was modified by homologous recombination to delete 5.7Kb of mouse genomic DNA encoding the coding region of the mouse FceRItx, and subsequently to insert 6.1Kb of human FceRItx coding sequence and human 3’ untranslated region from BAC CTD-3064hl7 (Invitrogen-Thermo Fisher) (FIG. 1). Thus, part of mouse coding exon 1, coding exon 2, coding exon 3, coding exon 4, and coding exon 5 of the mouse FceRItx were replaced by part of human coding exon 1, coding exon 2, coding exon 3, coding exon 4, and coding exon 5 of the human FceRItx gene. The resulting chimeric FceRItx gene comprised 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. Chimeric gene exons 1 (partial) and 2 encode the signal peptide, exon 3 and 4 encode the two Ig-like domains of FceRItx that are believed to interact with IgE, and exon 5 encodes the cytoplasmic and transmembrane domains of the protein.

[0202] Specifically, to generate humanized FceRItx mice, the large targeting vector used in construction of the chimeric gene comprised the nucleic acid sequences listed in Table 2 below. Self-deleting neomycin cassette was inserted 3’ of the last coding exon.Table 2: Genome Coordinates for the FceRItx Large Targeting Vector DNAAttorney Docket No. RPB-03125 (11689WO)

[0203] The targeted BAC DNA was used to electroporate mouse ES cells comprising a deletion in mouse FceRItx exons to create modified ES cells for generating mice that express humanized FceRItx (FIG. 1). ES cells containing insertions of human FceRItx exon sequences were identified by a quantitative TAQMAN™ assay (see, e.g., Fie and Petropoulos, 1998. Curr. Opin. Biotechnology 9:43-48, incorporated herein by reference). Specific primer sets and probes were designed for detecting insertion of human sequences (gain-of-allele, GOA) and deletion of mouse sequences (loss-of-allele, EOA) (not shown).

[0204] The junctions of the humanized FceRItx allele prior to the selection cassette deletion are presented in Table 3 below.Table 3: Junctions of the Humanized FceRItx allele

[0205] Bold sequences represent human DNA, underlined sequences are restriction enzyme sites or vector sequences, italicized sequences are loxP site sequences.

[0206] Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VEEOCIMOUSE® method (see, e.g., US 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, each of which are incorporated by reference in their entireties). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing aAttorney Docket No. RPB-03125 (11689WO) humanized FceRItx were identified by genotyping using a modification of allele assay (see above) that detects the presence of the unique human FceRItx gene sequences. Heterozygous mice bearing a chimeric FceRItx gene were bred to homozygosity.

[0207] The resultant FceRItx protein expressed by the mice is completely human, and the sequence of the human FceRItx protein expressed by the genetically modified mice is depicted below (SEQ ID NO:4), with Ig-like domains italicized and the signal peptide and transmembrane domain bold and underlined. The mature protein starts at amino acid 26 of the sequence depicted below. For reference, exemplary Genbank Accession Numbers for human and mouse protein and mRNA sequences are listed in Table 4 below.SEQUENCE OF HUMAN FceRItx PROTEIN (SEQ ID NO:4)MAPAMESPTL LCVALLFFAP DGVIAVPQKP KVSLNPPWNR IFKGENVTLT CNGNNFFEVSSTKWFHNGSL SEETNSSLNI VNAKFEDSGE YKCQHQQVNE SEPVYLEVFS DWLLLQASAEVVMEGQPLFL RCHGWRNWDV YKVIYYKDGE ALKYWYENHN ISITNATVED SGTYYCTGKVWQLDYESEPL NITVIKAPRE KYWLQFFIPL LWILFAVDT GLFISTQQQV TFLLKIKRTRKGFRLLNPHP KPNPKNNTable 4: Genbank Accession Numbers for FCER1A

[0208] To validate expression of humanized FceRItx on the surface of splenic basophils from humanized FceRItx mice, wild type (WT) or homozygous humanized FceRItx mice were sacrificed, the spleens were harvested and single cell suspensions were prepared following red blood cell lysis (Sigma). The cells were then stained with a live / dead cell marker, blocked by Fc block, followed by antibody staining with one of two antibody mixes: (1) mix 1: anti-mouse CD49b antibody (PECy7 conjugated, EBioscience clone DX5), antimouse TCRP (APC conjugated, BD clone H57-597), anti-mouse B220 (BUV395 conjugated, BD clone RA3-682) and anti-mouse FceRItx (eFluor 450 conjugated, EBioscience clone MAR-1) or (2) mix 2: anti-mouse CD49b antibody (PECy7 conjugated, EBioscience clone DX5), anti-mouse TCR|3 (APC conjugated, BD clone H57-597), anti-mouse B220 (BUV395 conjugated, BD clone RA3-682) and anti-human FceRItx (eFluor 450 conjugated, EBioscience clone AER-37(CRA1)). The cells were acquired in an LSRFortessa instrumentAttorney Docket No. RPB-03125 (11689WO) and analyzed using Flow Jo software. Basophils were identified as TCR|3- B220- CD49b+ FceRICx+. The FACS plots in FIG. 2 show the TCR|3- B220- population, with the basophil population identified by arrows. This population is positive for mouse FceRItx only in WT mice, and positive for human FceRItx only in humanized FceRItx mice. The graphs show the quantification of human or mouse FCER1CX+ basophils as percent of live cells in the spleens of 5 mice from of each genotype (FIG. 2).

[0209] FceRItx humanized mice were validated in passive cutaneous anaphylaxis (PCA) model. As shown in the schematic of FIG. 3A, on day 1, groups of WT or humanized mice received an intradermal injection with a cocktail of two allergen- specific human IgE antibodies, or an irrelevant IgG antibody (negative control) into the right and left ears, respectively, thus allowing allergen- specific IgE to bind FceR on mast cells. After twenty- four hours, the mice were challenged by intravenous (IV) injection of Ipg of allergen diluted in 0.5% Evan’s blue dye. One hour after allergen challenge, mice were sacrificed, Evan’s blue dye was extracted from ear tissue and spectrophotometrically quantitated using a standard curve. Ears were then dried and weighed. The results show Evan’s blue dye extravasation in the tissue quantified as ng Evan’s blue / mg tissue as a measure of local mast cell degranulation. The data, shown in FIG. 3B, demonstrate that the human allergen- specific IgE can mediate a local anaphylactic response in humanized FceRItx mice, but not in WT mice.

[0210] FceRItx humanized mice were also validated in passive systemic anaphylaxis (PSA) model. As shown in the schematic of FIG. 4A, on day 1, groups of humanized FceRItx mice received an intravenous (IV) injection with a cocktail of two allergen- specific human IgE antibodies, or an irrelevant IgG antibody (negative control), allowing allergen- specific IgE to bind FcsR I -expressing cells systemically. After twenty-four hours, basal core temperature measurements were taken for all the mice, followed by IV injection of Ipg of allergen. Core temperature measurements were then taken for all the mice at 30, 60, 120 and 240 minutes after the allergen challenge, and changes in core temperature at each time point relative to basal temperature were calculated. A decrease in core temperature is a readout for systemic anaphylaxis. The data, shown in FIG. 4B, demonstrate that the human allergenspecific IgE can mediate the systemic anaphylactic response in humanized FceRItx mice, as measured by a significant drop in core temperature 30-60 minutes after challenge.Attorney Docket No. RPB-03125 (11689WO)Example 2: Generation of a-l,3-galactosyltransferase B (GGTA1) Knock-Out Mice

[0211] The mouse a-l,3-galactosyltransferase B (GGTA1) locus, located on mouse chromosome 2, was inactivated by deletion of at least a portion of the endogenous genomic Ggtal sequence. Collapse of both Ggtal alleles was achieved using a combination of four SpCas9 guide RNAs (gRNA), each consisting of invariant tracr RNA, the scaffold for binding to SpCas9 enzyme, and a 20bp guide sequence specific to Ggtal that allows a precise double- stranded cut. Guides direct SpCas9 cleavage upstream of the Ggtal start ATG (guide mGU, cut site lOlObp upstream from the ATG; guide mGU2, cut site 2184bp upstream of the ATG) and close to the stop codon (guide mGD, cut site 295bp upstream of the stop; guide mGD2, cut site 830bp downstream of the stop). Guide sequences are listed on Table 5A below.

[0212] A mixture of each guide complexed with SpCas9 was electroporated into hybrid 129S6 / SvEvTac:C57Bl / 6NTac Fl embryonic stems cells (ESC). In some example embodiments, genetic editing was performed in mouse embryonic stem cells comprising a start to beyond stop humanization of one allele of the mouse FceRla locus. Resulting clonal colonies were screened first via TaqMan for deletion of both copies of Ggtal, using loss-of- allele assays listed in FIG. 5. The deletion sizes were limited to within the region proscribed by retention (ret) TaqMan assays (FIG. 5). Clones with both Ggtal copies deleted were then subjected to Illumina Next Generation Sequencing (NGS) technology to fully characterize the knockout sequence using primers listed on Table 5B below.Table 5A: SpCas9 guidesTable 5B: NGS amplicon primersAttorney Docket No. RPB-03125 (11689WO)

[0213] The resulting mice comprise a 24,247 bp collapse within the mGgtal genomic locus, specifically of the following coordinates: mmlO chr2:35, 401, 275-35, 425, 521. The sequence collapsed / deleted is provided in Table 6 below.Table 6: Genomic sequence collapsed / deleted in example GGTA1-KO mice.Attorney Docket No. RPB-03125 (11689WO)TGGGGTGAACCAGGTGGGGCTGAAAAGTGGGTGCAGGCTTAGATTATAGCCATTAGAGCAAGTCTTCAGTGTT C C AGC AAC AAC AGT T T T T GAAT GGGAGC C T C AGAGGT AAC T AGAAT T C T ACGGAAC AT C T T AGAGC T AC T T C C TCTCTTTCCAATGCCCACATGGATGGGCTTCAGCATCCTTTCAGATCATGAAGCCTCATTAACTGTGCTGGCC TAATTGGCCATGACTAGTTTGTGTGCTTGAGGGATAGGGGGAGGGGAGACACTTGTCGCTGAGTGAGTTACAA ATGTATCCTGTTAGGAAGGATGTGGGCAGATGCCTTTCATTATCTTTACTGCATCAAACATTTTATGGGTATG AGTGTTTTGCCTGCAAGTATGTATATGTACCACTTGTATATGTGGACCCCATGGAGGCCAGAAGAGCATCAGG TCCTGTGAAACCAGAGTTATGGACACCTGTGAGCTGCAAATGTGGATGCTGGGAACTGAATCGAGCAGGTGTT TCATTGAGGTGTTTCAACCACACAGCTGTTTCTCCAGCCCCAGAAGCCATCTCTCATTCCAGATTTAGTTTAT TTAATCTATTTCCCCCTCTTTTTTTCTCCCTGCCTCTACAGGAGAAAATAATGAATGTCAAGGGAAAAGTAAT CCTGTTGATGCTGATTGTCTCAACCGTGGTTGTCGTGTTTTGGGAATATGTCAACAGGTAATTATGAAGCCAG CTAGAAAGGCTGCTTTCATTCCCTGTGACTGGTGCCAGCTGAGTGACCAATCAGTCTGAACATAAGGGACGGA GCCGTGAGCAGGAGTCCAGTCTTCCTGTGTTCCTGAGCCCCAGATGGCCATTAAAACTGTAGACCATCCAAGT CACTTCTGCCTTAGTAATTATCCTCTTTCATGCCGTGCTCCTCAAACCTCGAATTTCTGTAAGCTAGATGGAG AGAGAAAGTACATTAAGCCAAAACCACCATCTCAAGTAATTTGTATAAGCAGATCCCAGAAGATTCAGGCCAG GCAGGGTAGTGCATGTATGGAGTCCTTGTGCTTGCAAGGCAGAGGCAGGAGCATCATACAAATGGAAGACCAA GC T T GT C T T C AT AGT GAC T T C C AGGC C AGC T GT AGC C T T AC AAGGAGAC C C T GAC T C T AAAAAT AAAGGGGC T GGAAAGGT GGC T C AGC AGC T AAGAGC AC T GGC T GC T C T GT C AGAGGGC C T GGGAT T GAC T C C C AGC AC C C AC A TGCCTTGGTGGCTCACAAACTCCTGTGTCCAGTGTCAGGACTCCTTCATTGTTTTCTGGCCTCCCTGGGCACC AGAAAC AC AAAT GGAGC AT AT C C AT C C AC AC AGGC AAAAT AC AC AT AC T C AT AAAC T C GAAAAAT AAAGAAAG GAAAAAAAGAT AAAC AAAT GAT C C AGGT AGGAC T T C C AC AAGAAAC C AGC TC T GAGT C CCGCAGTCC T AAAAT ATTTGCAGGATGAAAGGGACCATCCAAGTTGCTGGGATTTGCTGCAAAAAAATACCAAGTTTTACCAGTGATT CGTTCGTGAGTGGCTGAAAGCCTCTTCCGCCCTCCCTTGGAAAGAAGGGTGAATGAAATGTTTCTAGGTTGTC AAATGCATCTGTGAATTAAAATCTATCAGCTGCCTTTGCTCCTGGAAGCTCTGCTGTTTGAGGCCTCAGGGGG AGAT AT GT C T C C AT C AAAAAGT C C C T GC AGGC T C C T AAGAAGGAGC AC T GCC T GAGC T C AGT T C C AC T T C AGC TGGTCCAGAGTTTTCTCAGAAGAATACATTTTTTTTTTAAAGGTGAAGTGTTTAGTGGCCCGAGAGTTCTTTA AT C T AGAT TGTCAGCCATCTGATTTATCTCTTT C AGAC T C T T GAGT T T T AAAAT C AGAT GAGC C AGC AGAAC T CTTGGGGCTACAAAAAGAAAACTGTATCTTTAGGTCCAGGAGTAGGAGGGGCAACCTTCCAGTCGGAATGAGC AAGATAAGAGTTTCAAAGTGTAGCAAACATGTAACCACTTCTGGGATGTTCTGTTGTTTCATTCTGAATAGCC ACTTTTGAGAAAAGGCCTGACAAGATAGGGTGTGACCAACTTAAAAGGTCTGGAGAGTAGGCCATTTGAATTG AGGAAGC T AGT T C AAGT T AAGAGAT GATTCACTAATTGGATATGGCTACCCTTAGGCTCTTGGGCT GAAGAAC ATCTTGGCTAGACCTTAAAGCCAGACTGCCTGGTTGGTCCTGGACGCCCATTTACTGCCTGTGTGGCCTTGGG CAAGTTACTCAGCTACTCTGTTCTTTAGTTCCCTACTTTTAAAAAATGAGGATAATATCCCAGCATGGTGGAT CAGACCTGCAATCCCCATACTTGAGTGGCTGAGTCTGGAGGATCACTGTGAGTTAAAGCCAACCTGGGTTACA T AGT AAGC T T AT GGT C AGC C T GAAC T AC AGAAT GAGAC T C C GT T T T AAAAAGAAAAAGAAAAAC C C T AAAAAC AAAACTGTAGAGATCACAGGTCAGTGGTAGAGCACTTGCCTAGCTTTGCAAGGAATCCCTGTACCACTGATGA GCAAACAAGAAAATTCCCACTGCCTTCATCATCCTCATCTTGGTCATCTCTGTCATAATAGCCGGGTACCACA GCTTGTGTGGTGGCTTCAACCAGAGTGTCTTAGGCAAAGGTGCCGTCCTTGTCAGTTCCCATGTGAACCTTTC TCCTTGGTTTGTAGACAGCCACCTTCTCCCCAGACCCACATATGATGGAGAATAAGCAACTTGTAATTGTTTC C T T T T C T TAT GAAAAC AC T AAAT C CAT CAT GAGGC C C C AGC C T T AGGC T C CC AT C T AAAC C T AGAT AC T T C C C AGATGCCTACCTCCAAGTGCCACATCTTGGGGACAGGGCTTTAACAGACACATTCACTACACCGTAGCAGTCT TTGCTCTGATGGGCAAAACATGCATCAAGATTGTGGTGTCTAGAAGGTATGTCTCCAACCACTCACTGGGCTT GTCCTCTGGGGCT GGAAC AC T C AT T T AGAT AAC C AGC AAGC T C T GAAC GGAGT T T GAT T AGAC AC CTTTGGTG TTGTCTTATTAGAGCCATGCCTCTGTTTTTTGAGTGGCTCACTAATGAAGACATGGTGAGTAGAGAGCTGGCT CAGTGGTTAAGAGCACTTGCTGCTCTTCAAGAGGACCTGGGTTCAATTCTCAGCACCCTCATTGGGCAGCTCA CAGCTGCCTGTAACTCCAGCTCCAGGGGATGCAGTGACTTCCTCTGGCCTCCATAGGCACTGCACACATCAGC ATCCCCCCCTCACCCCC GC AAC AC AC AC AC AC C GAGGGGGGGT AC GGAGGGAGGGAGGGAGGGAGGGAGAGGG GGGAGGGGGAGAGAGGGAGAGAGAGAGAGGGAGAGAAGAAGAGAGGAGAGGAGAGTTGAAAGCTCAAGAGTAC ATACATACGGGTGTGGCATCTCTGAAAGAGCATTATTATCTTTGTTGCCAGGCATATGTTCTGAGCATCATGG CTGTAGGTGGTCTGGCTACTTTTTAGGCAGTTGTTTGATACTAAGACTCTAGATACATTGTTTTTCTGATTCAAttorney Docket No. RPB-03125 (11689WO)T GAAAT C AC T T GT T T T T C T GC C AGAGT AC AGAAAGGGGAAC AGGT C T C T GGC AC T AT C C AAAC T GGC T AT GC A C T GAC C T GAGGT GGAC C AAGGGC AT T C AC C C T AGAGAGGC AGGT GGC AC T GT GC T C AAAGGGAT T T AAAT AT A ACTGACTATATCCAAGAACTAGTATATAAGTCATTTAAAACTAATACTGCCTTCTGGGCCAAATAGGACTGCA GTGAGATCCTGTTTCAAAAAATAATAATAAGGGCTGGAGAGATGGCTCAGTGGTTAAGAGTACTCCCTGGTCC TTCAGAGGACCCAGGTTTGATTCCCAGCACCCACACCGTGCCTCACAACCATCTGTAACTCTAGTTCAGGGGC TCTGATGCCATCTTCTGGCCTCTGTGGAAAGTGATGCTGGCAAAATACCTATTCCCATAAATTTTTTTAAAGA AAATAATAAATGAATAAGGAGAATATGGTCCAGGAAGGCATTTGATTTCAAGGTTTTTGAAAATCCAGTGCCA TGTTTTAGAGCCAGAAGGAATCTTACAGGTTAGTATAACCATTGAGATAAAGAAGGTGGTGAAGGTGGCCGGA GTTACTGAGCATGTGTGGATACCCAGGCTGTGTCACTCCTGTTCTTAGAATGGTTCCGCAAAGTGGGAATGAC TCTGTGTGTGTGTGTGTTGATGGCAACACCAAGTCTCAAGAATGCTCAATTAAAGTTTATGGTCATGTTTGGG GAGGACTTGAGCCCCTGTTGCCTGCCTGCCTGTGAGCCATTCTTTTACTTACAGCACGCCTGTCCTTGAGCTG GGCCATTTTATGCAGATGACAGAGTGGAGGCCAGCCTTGCTGGGGGCCAGCCTTGCAGGGGAACAGGGTGGGG TGGTCAGTAGTGGTTTATATCTTTGTTCTAAGTAGTATTAGATCTTGAGATCCTCCGCCTCAGGCTCCATGGA AGAGT GC T C C C GT C T T C T GAGT GC T C AAGGGC C AC AT GAAAGC C GAAT AGAGC C C T T C AC C AT AGGGC AT T AC AGTATGGATTGGGTGAGCTCCGTGCATAAGGAGCCTGTGATTTAAGGTGAATGAAGGGAGTTGAGGTAGAGAG GAGAC C GT GAGAGAC T GT GAGAC AAGGAGGC AGAGAC TGCCTGCCCT GGAAAGAGAT T C AT AGAC AT T T GT AA AAC AC AT TCTGTGGGC C AAGT GC CATGCCAGGAT C AGAT C AGGGAGGGC AC AGAAGAGGT GAC T T T T T GAGC T GTGCCTGGAGGATTAGTGCTGCTAAAGTGACAGTTGAGCCTGAATATCATTGGCAGATTCTAGGAGAAGATGG TAAAATTTAGAAGGAAGGCTGTTAAAGACATGATGGCAACCATTAACTACAAAATAGTGCCCCAATGTCAAGT AGTGTTCTTAATGTTGGCTTGTCTTCTCCTCTCTTCTCATAGTTGCCCTCTTTTCCTCATGGTCCCCCCTTTT CTAATTGTCTCCCCCTTTTTCCTCATTGCCTTCCTCTCCTTCTCATTGTCTCCTCCTCCTCCTCCTATGGTAA GATGTGGAGTTACTCCACAGGCTCAGATGTGGGGTTCTGCACCTCATTAGTAGAATGACTTTGAGAAGACTCA TGGATTGCCCCTGCCCTCCAGAGTAAGGCTGGGGACAGAGTGGTGTGAGGATTCCCAAAGCCACTGTGTGTCA ACTGCGTGGAACAGTGCATGGCTAGGCTCACCAAAGCACTACATTAATACATTGATTTAGGAGCTAGGTACAT TAGGGCTTTGTCCTGGAGTATGGATCTTGGGGCAAAGCTGGAGGAGGTAACAGGGCAGGAAGGCAGATGTTTG C T C C C T GGGAGGAGGC TTGATCTCT GGT GT GGGGAGGAAGT GGGGAGAGGAGAT AAGAGC C AAAGAGAC C C T G GT AGAAT C T GAAAC AAC T AT T AAC GAT GGGAT GAGC AAAGT C AGC C AT AGGGT T T AGGC AC AGT GC C T C T GC C CAGCACCAAATTACTCAGTATGGCACTAAGTCCTGTCTTTTTGAGACACTTCTCATTGGTCATTGCTATGCAG AGC AGGGAGGC C T C AAAC T T GT AGC T T C T GC T GC T C AAGGC T GGAAT T AGAGGC AC C AAGC C AC C AC AC AC AT TTTCCAGTAGCCTCTCTGGTCAGCACTGTCACCAGCCATGGCTGCCTTGACCACTGCCCACTTCCTAACTGTT CTCCACACCAACTCTTTATGCTCATTGCTGCTCTCTGGCCACACTAGCTTTCTACCAGTTCTTCCCAGGCAAA TTCCCTAGCCAGGATGTATGTTGCTGTATGTTGCCTTCTCTGTTACATTGTATATTTTTCATGAGCCCCAGCA CTCGGTGTGTAGGACTTGCCTAGCACGTGTAAGACTGATGAGAGCTAGTGCCCTAAAGTAGTTGTAGCTGGCC T AGC C T T C T GGT T AAAGC AAC AC C C AT GGGGGC T GC T C AGAAGAAGGGAT C T GAGC T GAAT GT GGC GGC T AT T TCCTGTGGGGAAGAATCCTCAGCCTGAGGTGGCTGGCCGTGGCGCTTCCACCTTCCCCGCCTTCCTCATTGCC CAGCTTCTGGGACTGTGGTGGAAGAGGACCTTCCTGTCATGTAACAAACAGCTGGGTGACTTTAAAAGAGAGA AAGAGGGAAAAAAATCCCCCAAATAAAAACAAGAATTGAGAGTGTTTGGGTGCCCACTTCTGTTCCTCAGTGA T GC T T GT GGGAAT C C C C T GAGAAC C C AAAC GC T T AAGGAAAAC C AC T GC AGT GAAGC C T T T C T GAGAAT T AAA AGTATATGACGTTTCTATTTCTTATTTGTCCTTAGCCCAGAAGGCTCTTTCTTGTGGATATATCACACAAAGT AAGTGTTCTGAATTCTGTGTATCTATTGGATGTCTGGATCACTTGATTTTTTTTTTTTAGCCCCTAAAGTTGA TTTCCTCTCTT C AAGC CAGCCAATGTAGTGCTCGGGC C AC AGT AAAGGGAGGAGAGAGGC C AGGAC AGGGAGG AGGATTGCTAGGGCCCTGGGGTCAGGGCTGCAACTCTGCTAGTCCCCAAACTGGTCTTTGTAGAATAGTGATG AGTTTTGCTCTCGGTTCTGCTCAGGGGACTCTCCTCAAATATTGTCATGGGGACCATTTTTGGTTGACGTAGG GAAAGAGCCCAGGGAACTGCATGCTGTAGTGTGTACCCTCAGTGCTGCTGTGAGGCACTGAGGGAGGACTTAC GTTCAGTTCCAGTTTCTTATCTTCTGCATGGTGGCCAGGAAGTTCAGAAGAAATAATTTGTAACAGCTTTCAC CACACGTAGCCTTCATTTGGGTGTAATTCATTCCCTGGTGTTACACCTGCCTTGTATCCTTACCTGCTGAGGG GTCTTGGCCACACACT C AAAC C C AC T GAGC CCTCACTTCAGC C GT AAAGAGGAGAAT AGAC GT GAAAC T T GAA TTTGAGATTGCCAAGGTCATGCGAAAATATACACTGGGTAACCAAATGAACCAGTGGCATTTTCTTGGTGTAT GTGTGTGGTATGTGCACGTGTGTGAGTGGGTGTCTATGGTTATGCCTGCCTATGCATGTACCAGTGTGGGCCAAttorney Docket No. RPB-03125 (11689WO)GAGGGCAAGGTTTATTATTCTATTCTTCTGTACCTCAAGTTTGGAGACAAAGCTTCCTACTGAATTTGGGTAT TAATTTTGGCTATACCAGCTGGCCAGCCAACCCAGGACTCCTGGTTTCCCCCCCTACCCCCCACTAAGTGCCA GGGTTCTACACTTGTACCGCTTGCTTTGGATTTTGTCAGAGTGCTGGAGATCTGACCTGGGGTCCTCATGCTT TTGAAGCAAGCACTTTATTCACTGAGCAATCTTCCTAGCCACTAATATTTCTTTTATTAAAATTGTGTGTGTG TCTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGCGTGTGCGTGCGCAGATTGGAGGACAACTTGGTTCTT TCCTTCTACTCTTATGGGTCGAGGAATTGAACTTGGGTCATCAGACTTGGTAGCAAGTACCTTTGTACCCACT GAGATATCTTATCCGCCTTTCTCTATTGCCTATGACTTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTT T T C T T T C C C AAT T AT T AT AAGC AC GT C T T GAAT T C AC T AAC T AT C T C AGAC AT T T C C AGAGGC AAT T GAGGC C TAGAGTGAATTTCTTAGCCAGGTGTGGTGGAGTGAGCCTCTTGTCTCTGCCATAGGCCACATGGGAGGGCGGC TGTGGTAGCCTGAGCTACAAGCCAGGACCCCACCTCCAAACAGGGTCCCTAATGTTGTATGCCTGTTCAGAGT CACCTTGTCACTGCATC C AAC TTTCTCTCAC T AGAGAC AC C T GAAAGT AGAGAT TAT T AAC CATGATTATCAA AAGTCAGGGCCAAGATTGATATATAACTATTTTCTAACTTTTCTGTTGTCAATCTTCAGATTTCCTGAAGCTT TCATTGATTGGGCAATGGGATTTTTTTCTCAGATTAATCTCTATAATACATGCATGTATACAGACACACACAG ACACACACGCATGCAGTCATTCTCGGGAAGGTGCTTTTTCTTATTTTAATATTACCCCTCGTTACAGCCGCTT TATGTTCACCAGGCTCTTGCATATCTGCTGTCTCATTGGTCATTACAGATCCCTTCGTGGAAGGATTATTATT GATTACCCTTTTCAGAGAAGAACGTGGCAGTTTAGACAGTGTGAGTGTATGCCAAAGTCACTCCACTAGCAGG AGGAGATCGTGACCACAGGCTCTCAGGTCTGCAGGGTCTCCACCATTCTGATTTCCCTGCCCCTTATCCTTCA GGGGTCCCAGGGATGAGCAGAGTGCTCAGGGCTGCCCAGAAGGGCGCAGCTGAGGCCCCTCAAGTCCACTCTC TGCCTTTAGCTCAGCTGCCTTTTGCGTGTCCATGTTTCATGAGCTGCATCTTGACGTTCACTTTTTCTAGTGC TACCCGACCCTTAAAGTTCAGGACCGCCTCGATTTCTAGATGTGTTTATATTCTTTTTCATTTCCTAGAATTC CAGAGGTTGGTGAGAACAGATGGCAGAAGGACTGGTGGTTCCCAAGCTGGTTTAAAAATGGGTAAGGGATCAG GATGGGTTCCTAAGTCCCTGAAACCCACAGAGGACCCATGGCCTCCTCCCTCCCTTCTTCTGGCTCACTGGAC TCACTCATGGAGTCTCCTCATTGCTGTTGTTGTTTTTGTTGTTGTTAGCTTCTATTGTTATTGTGAGGGGTGG GGAGTGTGTTTGTGTGTATGACGTGTGTATGATTGCAGCTGTGTGTACACCATAGTACTCATCGGAGGTCAGA AGGCAGCTTTCAGGAGGCAATTCTGCCTTTCCAGTACGGGTTCCAGTGTGTGATCACCAGACTCAGATGCTCA GGC T T T C AGGAC AAGC AGT T T T AC AGGAT GAGC CATCTCACCAGTT C AGAGGAGGC T AAC T T AC AAGT GGAAT TCTGAGTGTCTCCCCGCTTCCCTCCCCCTCCTCCCACTCCCTCCTCCCCCCCTTCCTGTCTGCCTTCCTCCTT CCTCTGCCCTTTCTTCTC C AGT T AAGGGT GAAGT T C AGGC T GAAGT GGAAAT T T C AGAAT AGAC AC AGAAC AG AAATGTCCCTTGGAGTACTGTTCTGAAACATCTCACCGACTTCTGAAATAACTGAGGGTTACAGGGTCACTGG AACCTCAGCCCCTGACCCACATGGTGGCCAGAGAGGCAAATGCTGTACCTTTTATCAGAAGTGTGTAGGGATC AAGGGGTCAGTGCCCTGAGTCCTCCAGTCCACCCAGTGGTGTGAGTGATGCCTTCTTTCCCTTGAGACACGAG T C AT GGAAGC C AC C T GT C C T T AC C AAC T T T GT C C T AC C T T T GT C C AC AGGAC C C AC AGT T AT C AAGAAGAC AA C GT AGAAGGAC GGAGAGAAAAGGGT AGAAAT GGAGAT C GC AT T GAAGAGC CTCAGCTAT GGGAC TGGTTCAAT CCAAAGTAAGGACAGACAGGAGATTGGGGTGGGGGGTGCTGAGTGGGGTTCTGAGGAGATGCTGAGGGGAGTG CTGAGGGGGTGCTGGCAGGAGGGGGTGCTGGCAGGAGAGGGTGCTGGCAGGAGGGGGTGCTGGCAGGAGGGGA TGCTGGCAGGAGGGGGTGCTGGCAGGAGGGGATGCTGGCAGGAGGGGGTGCTGGCAGGAGGGGATGCTGGCAG GAGT GGGT AGAC CTTCCTCAATGGGCTTTGGC T AAGAAAC T AAGAT CTGGGTGCTTT GAAC C AGAC T GAAC AC TGTGGTAATTGCAGCAGGAAATGGCCAGTGGTAGGTTAAACATAAACACTGGGTGTTAAGGACTTTACAGGCC ACATAGGATGCTGCT GAGAAAAT GAC AAGGT C T AGGGT GAGC C AAGAAAAC T C T C T C T AGAT T C AGC T AAAAC ATATCCAT GAGGT AGAAAT TAT GAGC T AGGAGAT C C C AGAGC C AGGAAT C C AC T C C GT AC AC AAGC AGAGAAC GCGTGTCTATCAGAAACTAGGAAATGGTAGCTGGTGTTTTCCAAACTGCTGCTCGCTACCCACTCACGTTCTC AGAAAGCAGCCGAAGTATCAAGCCTAAACAGACTTTAAAAACAAGCAAGGAATGGTAGCGTTGGTGCAGCGGG CAAAGCACTTGCCGTGCTAGTGTGCAAACTGGAGGTCAGACCCCTGGAAGCTGTGTGAAGCCAGATGCAGTGG CCTGCGTGTGTGAGCCCAAGCATAAAGGTGGAAATACAGACAAGAGAATCCCTGGAAGGTCTTAGGTCAGTCA GGCTGCTTTAGGAAGTGGGAACCCCCAAGAAACCCTGCCTCAAACATGGTGGGAAGTGAGAACTGTCACCTGT GGTTGTCCTCTGACCTCCACATGTGGGCTGTGGAATCTGCATGCCCACACACTACATACACGTCACACACACA CCTACACATAGCATTGAAGTATAAGAGATGATCAAAGTGCATACCCTAAGGATGAGTGTTTGTAAGTAGAGTT TTGTTTCTGTGCATATGTGTATTATTTGCATATAATATAAACTATTTCTTCACTTGATCACAACCAAACTTGT AGTTCTTTCAAATTAAAGCTTTAAAAGATACTGTGTTAGGCACTGTGTTGGGGACACTATGATACGTTTAAGAAttorney Docket No. RPB-03125 (11689WO)AGTTCCTCATCTCAGAACTGGGGTTTCTTAGCTCAGTAGCGGAGTATGTAGTTAGCATGCATGCGACCCTGGG T T GAT C C C AAGGAC T AAT AT C GAAAGAAGAC AGGAAAGAAGGGAGGAGAC GGGAT GGGC AGGAAGT GGGGAGT GGGTAGAGGGGTGGGGTGTGTGTACGTCATAGTCCTAGAGGGCAGTAGATGTTAAGGTCATGTGATCAGCTGA CGGGAAAGGTGTGATCAGAGCTGCCCAGGGCGACTGTCAGCCCTGGGAGACTTGTGATTGATGGTTCTTCTAG GAAATGGACATTAGGAACTGCCACAGGCTTAGGACCTGAGAGGTGGCACTGTGTCGTGTATGTGTTAAGGAGT T T C T GGGT GGAAAGC CTCAGCTGCTCAT GAGC T T GGC T GAAGAT GAGC T GAAGAC AGAAGGT GAGGT GT C AT C AGGCTACAGGTAGCAGGGAGATCTGTAGTCACAGATGAGGTCATTGGGGCAGAGGAGTAGGACCGGGTGTGTT CTGTCTCAGCCCT GGAGGAGC AGGC AGAGAAAGAGAC C T C T C AAGGGGT GAGGC AC AGC T GT AGAGGAAGGAG AGAACAGTGCCTCGTTCTTTTTGACGGAGCTCAGGCTCTGGAGAAACTGGAGATGTTCTGGCAAGTCCACTGA GTTCTCAGGAGATTCATGCCAGTGTGCAAGACTGAGAATTCCATCCTGCCCGTATCTTACCATAGGCTGGGCA TGGAGTAGCTGCCTGGGACACTATGAGTGTAAAATGTTGGGAGCACTGCAGCTTTGCTAAGCCTCTTAGATGG GGAT GAGGT C C GGGGC C T AGAC AGC T AGAGAT GGCAGCTATGGGGATGATGTC T AAGAGC T AAAC AGC T AGAG ATGGTGCCTATGGAGGTAAGACACCCCCGCCCCCCCACAAACCCAGGGCTAGACAGCTAGAGATGGCTGCTGT AGATATCTCAGCTCCGCCCCAACCTCTTCTTTCTCATTTCCCATCTTCTTAAATACCTCAGTCCTAGCCTGGG AGAAGTTTTCCGTTGTCCACCAACAGTAAAATTCTATTATCAATTCTCCCTCTCAGATTCTTTCCACAGCCTC TCTCCCTTCCATAGCCACCTCCGCTGTTTGTGCTGACATTACCTCTCTATTGGGATTGTACAGCTTGTGTGAT GTGTGTTCCACATCTTTCCTCAGTCTACTGTGTGCTTCATAAGAATTTTAAACATCGATTCATTTATTTGTGT GTGGCAAGGCCCGCATGTATAAGTCAGAGGACAACCTGTGGGAGTTGGTTCTCTCCTTCCACTGTGTGTGTTC CTGGGATCAAACTCTGGCTGTCAGCTCCCACGAAAGTGCCTTTGCCTGCCGAGCCACCTGTCCAGCCTAATAC ACAGAATTGTAATACTTCCTCTAGCTAACCCGTGGATACCACTAATGTACCTGTGCATGAAGACATACAGTTG GGCATGAACTCCTAGTAACCTAGAGACCCGGATGCTCTATTGCAGGAGGTGGTCATTTTCCTGAAGTTTTTGC CCCCTATGGTGGACCTGGCCATACAGAGGTCACACAGTTTGAGTGCCCAGTGCAGAAGGAAGGGCATTTGAGT GTCACATAGCACCGTTTCCTCCCGCTCTTTAGAACCTTATCATGGCGGTCTTGGCTCAGGCAGTTGATAGTAT CACAGTGGCTTCTTTGAGGGGATGTGCTTGTGGGTTTTGATGACTGAGCTTGCTTCAGGCACAGTTTCCCCCT AGAAACTGGAGTCCCCTCATCCTGCCTCCCACCTGCTCCCACCTGCACTTCCAGGCTGTTGTCTGGCTTCTCC GTCACGCGCCACTGACATGCTGAAATTGCCAGTGGAACTGAACAATTAATGACCGGGAAATAGAATGTTTCAA TTTTTAATTTTGCTTTAATTTTTAATAGCGCTGTTTTGTCTTCCTGCTCAAAGGAGGTTCTCACTTTCAAATA GCCAAGGTGGACACTGGGTTTCCACTCAGTTTCAAGCCAGTAGGGCCCGAGAGGCCACAGAGCTGAGGAGTGG CATGGCAGGGTTGAGCACCCACTATACTGAAGGTTCTCAGGGGAAGAACTGGGTGCCATTTTGTAACTTTAAC TTTTTCAAATCCTATTTTTAATGATAGTTCTTAAACGCTTTAACCTTCCTTGTAACCCACCACCCACCAGAGG TAATGGAAAAGAAAGGATATGGGGGAAGTAGACCTATATTACAAAGGTTCTTTGGAACAACTCCTGTCTGTGT TGTTTGGAAATCAGCAGTTCAGTTCACAGGTTAGCAGGCAGCGAGTGCCAGCTCCATCCACTCGCAAACACCT CACATATACACCAGCAGTCCAGTTTGGTGGTCGGGTTAGCAACAGTGGTTACATGACCTAGCAGAGAGAGCCA GGC T C AGC C T C AGC T C GGGT C AGC AGGAGGGAC T AAT AAGAAC AC C AAGAGAAAGT C T C T GC T C T GC C T C T C T CAGGGAAGTGAAGATCAGGAAAGATAAGAGTCACAAGTGTTGCACAGGTAACTGTACCAGCAAGCCAAGTTCT GTCTCCGTCACTCCATGGAGTCCTATTTATACCCTCCAAACATCACGTGTCCTCCATATACTTTGCCTCAGCA C T T GC AT C T AAT C AGC T AAGT C C T T GAAAGC AGC AGC AAAC T AC AGC AC ACC AC C AGAAT T T C T T T T GGT GC A TTTCTCTCTATGGCATCCCGACAAATGCACCTCAACTATGCAGTGTATAGCGGACCAATACAAAGAATTTTTC ATCATGAGTCCTTTCACATTCTTACTTTAGTAGAACATCCTCTCTCCTCTGTCTGCTTCAGTGAAACGTTCCT TCAAAAGTCTGCCTTAGTCTTTCACCTGTGTGCACCTTGACGAAGTGTTCCTTTACATGTTTGCCCCAGCAAA ACACCATCTAACTGACTTTCCAAAGAACCCTTAGGTTTCCACTCCACCATTTCTCAGTGCTTAAACTTCAGTT TGGCTCTTTGTGAAAATTAAGAGGCCAGTGGCTCAGCCCCCTTCCCCTTCCGGAGCCTCTCTTGCCTTGACAG TGAGTGCTGGCAATGGCTGGTGTGGGGCTAAGTGCAGGAGGGAAAGGCCTGTTCTTAGCTCCTGGTATACACG AC AC AT T AAAT AC AGGC C T AGT C T T T T C AT T AAC C AC AGT AT AAC AT C AT C T GC T C AT AT AGAAC AAT GAGC A AC AC T GT AGT T C AC GC AC AGC AAC AAC AAGAC AGAC T GAGGAGGT T GGC T GGGT T AGAGC TACT GAGAC T T C C TTCCTTCCTT GAC AGC AAAGT C AAC AGC C C AGC AGAT GAGGC T C T GAC AT AGGAGGGAGAT AAC AT T C AC AAA CGCTGCTTTTGAGAAAGAGAATCTGCATATGATTATTTATTTTCTCCTTTTTGTTTTGAAATACCATCACTCA AT AAAGC C AGC C T GGGAAGGGAGT AAAT GT T C T AC C AGGGT AAAGC T C T GC AGGT GAAC AC C C C T C AGAGC T G CCTCCTGTGAGCTCCTAATACCAGTGCCACACTGGCCTATCACAGTTCAGTGCTGACGAAATGCTGCAGTAGCAttorney Docket No. RPB-03125 (11689WO)TACTGAGTGGACAAAGGCAAATCTGGGGTTTAGCCAGACTTTATCAGCTGAGCACGTGTCCATCTAAGGAGCA ATGAAAACTGGGGCTCAAAGCACTAGACTCCACAGAAAGACTCTGGGTTCAAGTCACAGCTTTCCTCCTTAGT AGCTGTGTGACTTTAGGCAGATAAGGGCCAGACTGTCCATACTGCAGACAACAGCACTGGGAACCAGTGAGGT TTAGCTTTAAATAACAGGAAAATGCCACAAGGCCAAGCAAGCTTGCACAACTTGTATTTCACCCATCCAGGAC CTACTATCTTTGTACTTCACTCTATTCCAAGAGTTGGAGGTACCCTATGCATTTGTGCCTGGCCCTTGCCAAG ACTCCACCCCTTCTGTAATTCCTGTCTTTCATGCAGGCAAGATTCAGTGACAGTCACTGGCCTCCCTTCCTTG GCCAGTCTCTCACCACACCTCAGTGTAATGCTTCTGACTCGGTGTTGCATGCTTCTTCTCACCAGGAACCGCC CGGATGTTTTGACAGTGACCCCGTGGAAGGCGCCGATTGTGTGGGAAGGCACTTATGACACAGCTCTGCTGGA AAAGTACTACGCCACACAGAAACTCACTGTGGGGCTGACAGTGTTTGCTGTGGGAAAGTAAGCACCACTGACA AACTCACCCTTGATGATTTGTTCTTGTTCTAGCATCAAAGGATTTGTGTGGGGCTCCAGGGCCCCACAAAGGC T GGAAT T T GAC AGT AGAC TTCCCCCTTCTTTCTTATAATGGCT GAGAAAAAAC AAT GAT AGT AGGT GAT GAGG TATTTCTCTGCCAGTGAATGAGCCAATCCAAGCCAGAGTAGATTGTATTAAATACAGGTTTATTGGGAAGCTG CTCTCAGGTGAGGTCACTGACCCCAAGGACTGAGGCCAGAGATGTCACCGTGGGGAGTATGGTGGGGAAGGGC AGAGAGGTGGGAGGGGAGGGAGGGAGAGGCAAAGAAAGAGAGAGAGAAAGGGCAGAAGGAAGGGAGGGAGGAC TGGGGGAAAGAGAGCGCAAATGTGTGCATGTTAAGATTCTGTCTGGACTCCACGCCCACAGTTACCTGGCAAC GGCCAGGTATGCTCCTACTCATAGTTACCTGGGAACAGCCAGGTAGGCCTGGCCCACAATAAAACGGGCTGCT TGCCCCTTCTCTCTCTCTCTTACTCTTGCCTCTTGCTCCCCCTCTCTCCCCATTCCCTTCCCTCCTCTATCAC CATGGTCATGGCCGGCCTCTACTTCTTTACTCTCTCCTTCTCTCTGCCTTTCTCTGCCTCTAGTACTCTTTAA CTCCCCTCCTCAGGCCCTGAATAAATGCTATTTTATACTATACCGCCATGTGGCTGGTCCCTCAGGGGGAAAG GACACCTTGGCATGGGTCCATTAAGGCACCCCCCTCCCCCCATACCTCACCACACCCCATAGAACATATTCTT ATATTTCTTTATCTTTTTATAATCACAAGCAAAGTGTCTGGATTACAAAGGCAAGAACCTCTGGGGGGAAGGC TGGAAAGTTCAGGCATGGGGGTAAGGTATGTCAGGTAGGGACTGAGGGATGCTGGGAAAACCTGGAGGCCAGG TCTGCTTTGCTATGTAAAATAGGCACCTTGGTCCCTTGTTCCTGGGTTCCAAACCAAACAGGAGGTAAGTCCA CCAATTCCCAC C AC AC AAGGGAGGGAGAC TCTCTTCAATGCCACT GGT AC CC AGAAC C AAGAC CATTCCTGCC TTCAGAACCTTTCTAGAAAGGCCCCAAAAGCCAGCTGTTGACTGGCTGGGATGTAAGTGCTCCTCCTTTCTTA TAAAGCCCAGGGCCACGGACAGGGTGTGGATGTTTGGGTGTGTCAGAAGACAATTTAGGGGTGTTGGCTCTCT CCTTCCACTATATGGGTTCTGGGGATCCACCTCAATGCTCAGGTCTGGCACCCATACCTACTGAAAAGTCTTT CTGGCTTGTTTCTTTTTCTGTCCCCAGGCCCTTGGTGTTGGTACTTCTTCTGATTTAAACACACACACACACA C AC AC AC AC AC AC AC AC AC AC AC AC AC AC AC AT C AAC AGGAAGT T GGCTCGCCTGC C AT AGAC AAGAGGAGT A GAAGGAGC AGGT T GAC CTTCCATT GAGC T T T C AC AGGAC AGGAC C T AAC AGAAGAT GCTGGGGTGATATCCCC GGATCCCCTCCCAGGGGCCAGCAGTCCTCCTTGGCTTTGGGAAGTCCTGACATTGCTCAGTGTGCTCATTCCC TGAGCCTAATGCCATCAAGTGAAAAGCAGTGTCTATTGACATTTCACTAGAGGCATTTGTATTGTTTAGGGAT T GGAGT C AGGGC T C AAC T C AGC AAAGC C T C T GGGC AC T GAAGC C AC AAGAC AGC C AC AT T GC AC AT T GAT T T G ATCCTAGTGTTTCCCTAAAAGAACAGAAAGTTACCCACTGTCCCACAATGCTATCAATCATCCAATCACAACT C T GC AGAAAAAC AGAAGC C AGAAC AC C AC AAGAGC CCTGTGATCCCTCCCACCTCCT GGGGGAAAGC T GT GC T CCCAGATAGACAAGTCTGCCACAGTGAATGCCAGAGAAAGTGCATCTGGACCTTAGGGATTGGTAGTTGCCTG TCTTCAGGCTCCATGCCTGGAGCCACCTGTAGCACTTTAGCCTCTTAAGCTGACTGTTAGACACTGCGGTGAC ACTGCCCTCCCTGCCCTGCTCCTACGGATTCTTTCCCAAGCTTCAGGGCACACTGGGTTTCCTTAGAGGTCCA AGTACAGAGGTGTGTCCTGACCTCTATCCTTGTGGTCCTGTCAGAGGCTCTCCTGGACCATTCAGACCTATTT GCCCATCTAAAGAGTCACACCTACAAGCCTTTGAGCTCCCTGATGCCAAAAATAAAAAGTGCAATTTGTTTCA AC T C C AC T GGAAAAAC C C AAGC AT T GAGAAAAAC AAAAC AGC AAC AAAAGAAAC AAAAC C AAAGC C AC C T T C A TTACATTCAGAACATTGTGATTGGCTACTTGTGTGCTCAGCAAACTGACTGTGGTCCATGTCTGTGTAGGGTT TGCACAAGGACCCTCTGACGTCACCCTCATCCTGCAGTCTCCTTGGTATTTCTTTTTTTTTTTTTAAAGATTT ATTATTTATTATATGTAAGTACACTGTAGCTGTCTTCAGACACTCCAGAAGAGGGCGTCAGATCTCGTTACGG ATGGTTGTGAGCCACCATGTGGTTGCTGGGATTTGAACTCTGGACCTTCAGAAGAGCAGTCGGGTGCTCTTAC CTGCTGAGCCATCTCACCAGCCCCTCCTTGGTATTTCTTGGGAAGACAGTCTATAGCTTGAGCTTGTAAAATG ATCAATATGGTGTCAT T AGAT T GAGC AAAT AT T C AAAT T GAC AAGAGAT AT T T AT AAAAAT GAGAAAAAT AATACTGATACTATAAAATGTTGGAAAATATGTGAAGTAATAGGAACCATTTTTATTGCTAGTTGGGAATACAAAA TGGTGCAGCCACTGCAGAAGAAATTGTGGAAGTTGCTTACACAACAAAACATGCTCTGATCATAATATAATCTAttorney Docket No. RPB-03125 (11689WO)TCTGGTCAGATTCCTTGGTGTTCCCCCACATGAGTTGAGACGTGTCTTCACAACAACCTATGTAGATATTTTT AGAAAT GT T C T T T GT AAC TGC T AAAAC T T GGGAGC AAAC AAGT C AAAT GGAAAGAT GAAC GGAT C AAT T C AGA CAATAGGATAATATTTCCTGCT GAAAAGT GAGC T GT C GAGC C AC AGAGAAGAAGGAGGAGGAAGT T T T T GT C A ATTGTTACTTAGTGCAGAACACAAGCTGGAAAGGTTCCAGTGCTGACCAGTTTTGTGTCAGCTTGACACAGGC T AGAGT C AT C T GAAAGGAAGGAGC C T C C AT GGAGAAAC TGCCTCCAT C AAAT GC AGC C T C AGGC AAGC C T GT A GGACACATTCTTAGTGACTGATGGGGGAGGGACCATCCCACTGTCTGTGGTCCCATTCCAGGGCTGGTGGGCC T GGGT T C TAT TAGAAAGC AGGC T GAGT AAGC CAT GAT GAGC AAGC CAGT AAGC AGAGC C CAT C TAT GGC C T C T GCATCAGCTCCTGCCTCCAGCCCTGCTTGAGTTCCTGTCCTGACTTTCTTCTAGGATGAAAAGTGATGTGGAA GTGGAAACCAAGTACCTCTTTCCTTCCCAACTTGCTTCTGGTCTTGTGTTCTGTCAGAGCAATCTGTCCCCAA AT AGGAC AGAT CTCTACTGTCCACTTTCTGATTCTGATTCT GGAAGAGC C AAGC TAT GGT GT C AGGGAAAAGA ATGGTGCCTGAAAGCAGCTGGGGTGAGAGAAAGGCAGAAAAAGAGGAGCCTGGGAGAGTGAGTTCTAATGCAG TCAGACTCTTGTGATATAGCACTGGTGGGTTTTGCTGATGCCATGCTTTTGCACACTTCCCAACATGAACCAT AGTATAACATAGAATAACCTTATGTTAATGTGAGTGCCAACTGTCACACACATGCCACCAGGAGCACTGGTGG CCCTAGTAGGGAAGTCTATTCACATACCAGGTAGGTACATGGGTACATTCAACACTTTCAGCTCAGTTTTGTT CAGAATTCAAAATTATTCTAAAGCATGCTAATTTTTTTTTTACATTATATCTGTTTTTTATTTTATGTGCAAT GGTGTCCTGCCTGCCTGAGGACGTTGGTTCTCTGGAACTGGAGCTACAGACAGTTGTGAGCTGTCATGTGGGT GC AT GAAAT AGGAT T C T GGGAAT T GAAC CCAGGTCCTCT GGAAGAGC AAC TATTACTCTTAATGCT GAGC CAT CTCTCCAACCCCAAAGAATGCTATTTTTAAAAAGATAAAATTGTTAAAATATAAGCAAACTATAGTGTGGTCT GTAGCTCAGGTGGTTGGCTGCATAGTGGGTATGACACTCTAGGTTTGATCCCAGGCACCTTAAAAATAGCAGA TGTCCTGCTGGTAATCCTAGCAAGACTATGAGAAGTTCAAGGTCATCCGAGACCGCATAGTAAGTTTGAGGAT AGCCTGGTATACATGACCCACTGTCTCCCCCCCCCCCCAATAAATAAATAAATAAATAAATAAATAAATAAAT AAATAAATAAATAAGATGCAATCTGAGATTGTTCTAGTATATGAATGGAATCATAACATGGTTTTTGTCTAGA TGGTCCACTGGTGTTTGGGGTAGCCCGTGAGAGGTTTGTAGAATGGACATGAGTAAGGAATTAGATGTCCATC CTATGAACATGCATCCTCTGTGTCACAGAGGAGGCTGACAGTGCTAGTGTTTGGGGTTAACCTAACCGAGAAG GTAAGGTTGAGTTTTGAATGCTCAGAGGATTCTGGGAAGAATTTTCTTTGTGTTTGGCTTAGCAGATACACTG GCCTCTTCTGGATATT C AAGAGC TAGCTCCTTCTCT GAC AGC C AGC T T C T C AAT CAGAGAAC AGAGC C T T AGC AT GAAC C T T AC T GC AAC GC AGAGT AGT T GAGAAC AC C GAGC TCTCAGTGTGGCAGGCATC GAAGAGC AT GC GG TCGGGGCTGTGCATCCCCAGTTTGCTTAACAAAGCTGGCAGTGAGATAAGTCATGCCACTTTCCCCAAGGACA CAATGACCAGCTAGTGTCGAGTGGTATGTGGAGAAGCCATCCCCTCCTAACATACAATACAGATCATCTACTG TAATGTTAAGTATGGTATTACATGTATATATGTACCCATATATAAGTGTGATAGCCCGTGGTGGTTCAATGTA GCCCTCTCTATTTCAGGTACATTGAGCATTACTTAGAAGACTTTCTGGAGTCTGCTGACATGTACTTCATGGT TGGCCATCGGGTCATATTTTACGTCATGATAGACGACACCTCCCGGATGCCTGTCGTGCACCTGAACCCTCTA C AT T C C T T AC AAGT C T T T GAGAT C AGGT C T GAGAAGAGGT GGC AGGAT AT C AGC AT GATGCGCAT GAAGAC C A TTGGGGAGCACATCCTGGCCCACATCCAGCACGAGGTCGACTTCCTCTTCTGCATGGACGTGGATCAAGTCTT TCAAGACAACTTCGGGGTGGAAACTCTGGGCCAGCTGGTAGCACAGCTCCAGGCCTGGTGGTACAAGGCCAGT C C C GAGAAGT T C AC C T AT GAGAGGC GGGAAC TGTCGGCCGCGTACATTCCATTC GGAGAGGGGGAT T T T T AC T AC C AC GC GGC C AT T T T T GGAGGAAC GC C T AC T C AC AT T C T C AAC C T C AC C AGGGAGT GC T T T AAGGGGAT C C T C C AGGAC AAGAAAC AT GAC AT AGAAGC CCAGTGGCATGAT GAGAGC C AC C TC AAC AAAT AC TTCCTTTT C AAC AAACCCACTAAAATCCTATCTCCAGAGTATTGCTGGGACTATCAGATAGGCCTGCCTTCAGATATTAAAAGTG TCAAGGTAGCTTGGCAGACAAAAGAGTATAATTTGGTTAGAAATAATGTCTGACTTCAAATTGTGATGGAAAC TTGACACTATTACTCTGGCTAATTCCTCAAACAAGTAGCAACACTTGATTTCAACTTTTAAAAGAAACAATCA AAAC C AAAAC C C AC T AC C AT GGC AAAC AGAT GATTTCTCCT GAC AC C T T GAGC C T GT AAT AT GT GAGAAAGAG TCTATGGCAAGTAATCAGGTATAAATTCTCAATGATTTCTTATATATTCTGGGTCTTGGGAAAACTTGATTCT AGAAAT C AAAAT T AAT T T GAC AAAGGAAAAGC AGAT GC C GGAAAC TTCTTCCCAGTCTGTCATACAATTCACC ACTGGCCAGGTGCTGAGAGAAGCATTAGGGAACAGTGTGGGTTGTGTCAGAGTTGGACGGCTCCATCCCTTTG GCTTCATTATCTTCCTCCTCAT GGAGAT T C T AAAGC AAC C C AGAGAGGC T T T GC AGC C AGAGAC C T T T AAT AA GGATGCCAATGTGACCATCAGTCTGTAAAAGCTGATGGCTCCAGGAGCCGCTGGCAGTCCAGGCCCCACTAGG CTATTGTTTCTGTCCTGGGCATAAAGGAGGCAGAGAGTGCCAATAGGTACTTTGGTGGCACATGTTCAGAGTC C AGGAAAAAT GGC AGGGT GAC C AC T TAGAGGGAC AT AGGAC TTGGGGTTGGTGATT GAAC T GAGT T AC AAAC AAttorney Docket No. RPB-03125 (11689WO)

[0214] Ggta^'; Feer la™'- and Ggta^'; Fcerl ahu / humice were generated from embryonic stem cells using the VelociGene® method (Valenzuela 2003 Nat Biotech PMID: 12730667; Poueymirou 2007 Nat Biotech PMID: 17187059, each of which are incorporated by reference in their entirety) and by breeding.Example 3: Evaluation of GGTA1 Expression in WT and GGTA1 knock-out mice

[0215] To validate loss of expression of GGTA1 in GGTAlKOmice, flow cytometry was used to evaluate expression of alpha-gal in splenocytes. The example describes validation of the lack of alpha-gal expression in Fcerlahu / huIGGFA. KO mice via flow cytometry.

[0216] Fcerlahu / humice (n=3, 15 weeks old, female) and Fcerlahu / hu; GGTA1 KO mice (n=3, 13 weeks old, male) were sacrificed and spleens harvested into RPMI media. Individual cell suspensions were generated by manual dissociation in a 70 pM filter plate containing RPMI media. The cells were then collected, centrifuged to remove media and the resulting pellet was resuspended in IX red blood cell lysing buffer to eliminate red blood cells. After incubation for 3 minutes at room temperature, IX Dulbecco's phosphate buffered saline (DPBS) was added to deactivate the red blood cell lysis buffer, cells were centrifuged and resuspended in DPBS for a wash step. The cells were centrifuged again, supernatants discarded, and the pellet resuspended in DPBS. Aliquots of 1710thof each sample were plated, centrifuged, supernatants discarded, and cells resuspended in LIVE / DEAD® Fixable Blue Dead Cell Stain diluted 1:1000 in IX DPBS to determine cell viability. Cells were incubated for 10 minutes at room temperature while protected from light. After one wash in IX DPBS, cells were incubated in a solution of MACS buffer containing 10 g / mL of purified rat anti-mouse CD16 / CD32 Fc Block (Tongo Biosciences), for 15 minutes at 4°C. After IX wash in MACS buffer, cells were incubated in lOOnM M86 anti- alpha-gal hu-IgGl mAb (Abeam, Ab00532-10.0) or isotype control for 20 min at 4°C while protected from light. Following IX wash in MACS buffer, cells were incubated in anti-human IgG-APC (Jackson, 109-136-170) as a secondary antibody, at 4°C while protected from light. Cells were again washed in MACS buffer and then stained at room temperature in a flow antibody mix as indicated in FIG. 6A (CDl lb-BV605, Ey6C-FITC, Ey6G-BUV395, F4 / 80-BV711, TCRb-AF700, CD8a-PeCy7, B220-BV510, CD19-BUV737, and CD3-BV786 purchasedAttorney Docket No. RPB-03125 (11689WO) from BD Biosciences and CDl lc-APC-eF780, CD45-PerCP-Cy5.5, CD4-BV421 from Biolegend). After antibody incubation, the cells were washed twice in MACS buffer, resuspended in BD CytoFix diluted 1:4 in DPBS and then incubated at 4°C while protected from light. The cells were subsequently washed, filtered and resuspended in MACS buffer. Sample data were acquired on a Fortessa X-20 cell analyzer. Data analysis was performed using FlowJo Software.

[0217] Alpha-gal was expressed in splenocytes of GGTA1WTmice, but not in GGTA1KOmice, mice stained with isotype control or splenocytes stained with primary or secondary antibody only (FIG. 6B).

[0218] Flow cytometry was also used to evaluate expression of alpha-gal in different immune cells of GGTA1WTmice and GGTA1KOmice. Neutrophils, macrophages, monocytes, B cells, and T cells were isolated by flow cytometry. The gating strategy was defined as depicted in FIG. 7A and validated using fluorescence minus one (FMO) controls.

[0219] As depicted in FIG. 7B, alpha-gal is expressed on most immune cells in GGTA1WTmice: B cells, neutrophils, macrophages and monocytes; low or no expression of GGTA1 was observed on T cells of GGTA1WTmice. Alpha-gal was not detected in any of the immune cell subtypes for GGTA1KOmice (FIG. 7B).Example 4: Evaluation of alpha-gal induced allergic response in GGTA1-KO mice with humanized FcsRa

[0220] GGTA1 knock-out / humanized FceRIcx (GGTA1KO; FceRIcxhu / hu) rodents were validated in passive cutaneous anaphylaxis (PCA) model. The PCA model assesses type 1 hypersensitivity to allergens (such as alpha-gal), and provides a readout of local mast cell activation-induced vascular permeability in ear tissue (Gilfillan A M and Tkaczyk C. (2006) Nature Reviews Immunology, 6(3):218-30; Orengo JM, et al. (2018) Nature Communications, 9(1): 1421; Zhu D, et al. (2005) Nature Medicine, l l(4):446-9, herein incorporated by reference in their entireties). Here, the PCA model was validated for response to alpha-gal in the context of GGTA1 knock-out / FCERlA-humanized mice, using the alpha-gal allergen and human alpha-gal-specific IgE.

[0221] Fcerlahu / humice (female, aged 15-17 weeks, n=5) and Fcerlahu / hu; GGTA1 KO mice (female, aged 12-14 weeks, n=3-4) were placed into 3 experimental groups each as indicated in FIG. 8. On day 1, a sample of IgE-rich plasma of an alpha-gal allergic individual (alpha-gal syndrome, AGS) or a non-alpha-gal allergic individual (negative control) were injected intradermal (ID) into the right and left mice ears, respectively, allowing IgE to bindAttorney Docket No. RPB-03125 (11689WO)FCERIA on mast cells. Twenty-four hours after local administration of allergen- specific IgE, mice were challenged by IV injection of 1-20 pg of a first example alpha-gal allergen (galactose-alphal,3-galactose-beta 1,4-N-acetyl glucosamine-BSA, NGP0334, Dextra Labs “BSA-alpha-gal trisaccharide”) diluted in PBS containing 0.5% Evans Blue Dye. 1 hour after allergen challenge, mice were sacrificed, and mast cell degranulation was separately quantified for the left and right ear via measurement of Evans Blue dye extravasation into the tissue. Specifically, ears were excised and placed into formamide for incubation at 50°C for approximately 72 hours. Following extraction of Evans Blue dye, ear tissue was removed from formamide, blotted to remove excess liquid, and weighed. Aliquots of each formamide extract were transferred to a 96-well plate in duplicate. The optical density (OD) of the resulting supernatants was measured at 620 nm. The OD620 was converted to Evans blue dye concentration using a standard curve. Results are presented as ng Evans Blue Dye / mg ear tissue, shown in FIG. 9.

[0222] These results demonstrate that the human alpha-gal- specific IgE can mediate a local anaphylactic response in GGTA1 -knock-out mice with humanized FceRItx, but not in GGTA1 -expressing (GGTA1WT) mice with humanized FceRItx.

[0223] Using this same PCA model, the effects of increasing doses of the first example alpha-gal allergen (BSA-alpha-gal trisaccharide, NGP0334, Dextra Labs) were assessed in Fcerlahu / hu; GGTA1-KO mice (female, aged 12-14 weeks, n=4-5) sensitized with plasma from three different AGS donors. On day 1, a sample of IgE-rich plasma of an AGS individual or a non-alpha-gal allergic individual (negative control) was injected intradermally (ID) into the right and left mice ears, respectively. Concentrations of alpha-gal- specific and total IgE in the example donor samples is provided in Table 7 below.

[0224] Table 7: Example Donor Plasma Samples

[0225] Twenty-four hours after local administration of allergen- specific IgE, mice were challenged by IV injection with 10 pg, 25 pg, or 50 pg of the first example alpha-gal allergen, BSA-alpha-gal trisaccharide (NGP0334, Dextra Labs) diluted in PBS containing 0.5% Evans Blue Dye. 1 hour after allergen challenge, mice were sacrificed, and mast cellAttorney Docket No. RPB-03125 (11689WO) degranulation was separately quantified for the left and right ear via measurement of Evans Blue dye extravasation as described above. Results are presented as ng Evans Blue Dye / mg ear tissue, shown in FIG. 10. A dose-dependent increase of mast cell degranulation was observed with challenge of increasing doses of BSA-alpha-gal trisaccharide in samples sensitized using plasma from Donor 1 and Donor 2 and a consistent mast cell degranulation response was observed across all challenge concentrations for Donor 3. No alpha-gal induced mast cell degranulation response was observed in samples sensitized with non- AGS plasma under any of the tested doses.

[0226] GGTA1 knock-out / humanized FceRIcx (GGTA1KO; FceRIcxhu / hu) rodents were further validated using a PCA model for response to a second, different example alpha-gal allergen, bovine thyroglobulin (InBio Catalog AGAL-1, lot 47399), which is a protein antigen that naturally contains alpha-gal. Fcerlahu / hu; GGTA1 KO mice (n=5) were placed into 3 experimental groups each as indicated in FIG. 11. On day 1, a sample of IgE-rich plasma of an alpha-gal allergic individual (AGS donor) or a non-alpha-gal allergic individual (no AGS donor) were injected intradermal (ID) into the right and left mice ears, respectively, allowing IgE to bind FCERIA on mast cells. Twenty-four hours after local administration of allergen- specific IgE, mice were challenged by IV injection of 25-100 pg of bovine thyroglobulin diluted in PBS containing 0.5% Evan’s Blue Dye. Results are presented as ng Evans Blue Dye / mg ear tissue, shown in FIG. 12. A dose-dependent increase of mast cell degranulation was observed with challenge of increasing doses of bovine thyroglobulin in samples sensitized with plasma from an AGS Donor. No alpha-gal induced mast cell degranulation response was observed in samples sensitized with non- AGS plasma under any of the tested doses. Thus, Fcerlahll / hu; GGTA1 KO mice exhibited an alpha-gal allergic response to multiple different example alpha-gal containing antigens over a range of doses.

[0227] Taken together, the present examples demonstrate successful generation of a mouse model for alpha-gal allergic response.INCORPORATION BY REFERENCE

[0228] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0229] Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in aAttorney Docket No. RPB-03125 (11689WO) public database, such as those maintained by The Institute for Genomic Research (TIGR) on the world wide web at tigr.org and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.EQUIVALENTS

[0230] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

Attorney Docket No. RPB-03125 (11689WO)CLAIMS1. A rodent whose genome:(a) does not encode a functional a a- 1 ,3, -galactosyl transferase (GGTA1), and(b) comprises a human or a humanized Fc epsilon receptor 1 alpha (FcsRla) locus, wherein the human or the humanized FcsR I a locus comprises a nucleic acid sequence encoding a FcsR la polypeptide comprising a human extracellular domain.

2. The rodent of claim 1 , wherein the rodent genome comprises a deletion of at least part of the GGTA1 locus.

3. The rodent of claim 1 or claim 2, wherein the rodent comprises a deletion of a sequence that is 10,000 bp to 64,087 bp, wherein the deletion is within genomic coordinates chr2: 35,399,144 to 35,463,231 (GRCm38 / mml0, - strand).4 The rodent of any one of claims 1 to 3, wherein the rodent comprises a deletion of a sequence that is 10,000 bp to 24,247 bp, wherein the deletion is within genomic coordinates chr2: 35,401,275 to 35,425,521 (GRCm38 / mml0, - strand).

5. The rodent of any one of claims 1 to 4, wherein the rodent genome comprises a deletion of the entire GGTA1 encoding region.

6. The rodent of any one of claims 2 to 5, wherein the rodent genome is homozygous for the deletion.

7. The rodent of any one of claims 1 to 6, wherein the FcsR la polypeptide further comprises a rodent or human transmembrane domain.

8. The rodent of any one of claims 1 to 7, wherein the FcsRla polypeptide further comprises a rodent or human cytoplasmic domain.

9. The rodent of any one of claims 1 to 8, wherein the nucleic acid sequence encoding the human or humanized FcsRla polypeptide is positioned at an endogenous rodent FcsRla locus.

10. The rodent of any one of claims 1 to 9, wherein the nucleic acid sequence encoding the human or humanized FcsRla polypeptide replaces all or part of an endogenous rodent FcsRla gene.Attorney Docket No. RPB-03125 (11689WO)11. The rodent of any one of claims 1 to 10, wherein the nucleic acid sequence encoding the human FcsR I a extracellular domain replaces an endogenous nucleic acid sequence encoding a rodent FcsR la extracellular domain.

12. The rodent of any one of claims 1 to 11, wherein the rodent does not express a rodent FcsR la.

13. The rodent of any one of claims 1 to 12, wherein the rodent is heterozygous for the human or humanized FcsR la locus.

14. The rodent of any one of claims 1 to 13, wherein the rodent is homozygous for the human or humanized FcsRla locus.

15. The rodent of any one of claims 1 to 14, wherein the rodent is a mouse.

16. An isolated rodent cell whose genome comprises a a- 1,3, -galactosyltransferase (GGTA1) knock-out and a human or humanized Fc epsilon receptor 1 alpha (FcsRla) locus.

17. The isolated rodent cell of claim 16, wherein the cell is an ES cell.

18. A method of making a genetically modified rodent of any one of claims 1 to 15.

19. A method of making a genetically modified rodent, the method comprising: deleting at least a portion of an endogenous a- 1,3, -galactosyltransferase (GGTA1) locus in the genome of a rodent, wherein the rodent genome comprises a humanized Fc epsilon receptor 1 alpha (FcsRla) locus at an endogenous rodent FcsRla locus; or inserting a humanized FcsRla locus in an endogenous FcsRla locus of the rodent genome, wherein rodent genome does not encode a functional endogenous GGTA1.

20. A method of making a genetically modified rodent, the method comprising: generating a rodent ES cell of claim 17; and generating a rodent from said ES cell.

21. A method of making a genetically modified rodent, the method comprising: breeding rodent comprising an a- 1,3, -galactosyltransferase (GGTA1) knock-out with a rodent comprising a humanized FcsRla locus, and selecting for rodents that are homozygous for the GGTA1 knock-out and the humanized FcsRla locus.Attorney Docket No. RPB-03125 (11689WO)22. Use of a genetically modified rodent of any one of claims 1 to 15 for testing an agent for its ability to prevent and / or treat alpha-gal allergy.

23. A method of testing an agent for preventing and / or treating alpha-gal allergy, the method comprising administering the agent to the genetically modified rodent of any one of claims 1 to 15.

24. The method of claim 23, further comprising measuring one or more pharmacokinetic properties of the administered agent.

25. The method of claim 24, wherein the one or more pharmacokinetic properties are selected from one or more of area under the plasma concentration versus time (AUC), in vivo recovery (IVR), clearance rate (CL), mean residence time (MRT), agent half-life (P / 2), and volume of distribution at steady state (Vss).

26. The method of any one of claims 23 to 25, further comprising measuring therapeutic efficacy of the administered agent.

27. The method of any one of claims 23 to 26, further comprising administering a plurality of doses of the agent and determining one or more of:(a) therapeutic efficacy of each dose of the agent;(b) safety of each dose the agent; and(c) tolerability of each dose the agent.

28. The method of any one of claims 23 to 27, further comprising:(a) measuring an immune response generated by the genetically modified rodent against the agent;(b) measuring mast cell degranulation observed in the genetically modified rodent,(c) measuring passive cutaneous anaphylaxis (PCA) response in the genetically modified rodents administered the agent; and / or(d) measuring passive systemic anaphylaxis (PSA) in genetically modified rodents administered the agent.

29. The method of any one of claims 23 to 28, wherein the agent is an antibody or Fc fusion.

30. The method of any one of claims 23 to 29, wherein the agent targets alpha-gal.

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