WO2022188817A1

WO2022188817A1 - Genetically modified non-human animal with human or chimeric gcgr genes

Info

Publication number: WO2022188817A1
Application number: PCT/CN2022/079987
Authority: WO
Inventors: Rufeng ZHANG; Chengzhang SHANG; Yanhui NIE
Original assignee: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
Priority date: 2021-03-09
Filing date: 2022-03-09
Publication date: 2022-09-15
Also published as: CN114561394A

Abstract

Genetically modified non-human animals that express a human or chimeric (e.g., humanized) GCGR, and methods of use thereof.

Description

GENETICALLY MODIFIED NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC GCGR GENES

CLAIM OF PRIORITY

This application claims the benefit of Chinese Patent Application App. No. CN202110254959.8, filed on March 9, 2021 and Chinese Patent Application App. No. CN202111410563.4, filed on November 25, 2021. The entire contents of the foregoing applications are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) glucagon receptor, and methods of use thereof.

BACKGROUND

Glucagon receptor (GCGR) has been considered an important drug target in the treatment of type 2 diabetes mellitus (T2DM) due to its effect on pancreatic alpha-cells. However, additional and novel effects for glucagon, such as modulation of satiety, thermogenesis, energy expenditure, and control of lipid metabolism have more recently been garnering scientific attention.

The traditional drug research for diabetes and discovery of additional effects for GCGR typically use gene knockout mouse models. Because of the extensive involvement of GCGR in the occurrence of metabolic diseases (e.g., diabetes) , and the huge application value of targeting this signaling pathway, there is a need to develop non-human animal models related to humanized GCGR signaling pathway. The animal model can make preclinical trials more efficient and minimize development failures.

SUMMARY

This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) glucagon receptor (GCGR) and methods of use thereof. The animal model can express human GCGR or chimeric GCGR (e.g., humanized GCGR) protein in its body. It can be used in the studies on the function of GCGR gene, and can be used in the screening and evaluation of anti-human GCGR antibodies. In addition, the animal models prepared by the methods described herein can be used in drug screening, pharmacodynamics studies, treatments for diabetes for human GCGR target sites; they can also be used to facilitate the development and design of new drugs, and save time and cost. In summary, this disclosure provides a powerful tool for studying the function of GCGR proteins, and a platform for screening hypoglycemic drugs.

In one aspect, the disclosure is related to a genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric GCGR (glucagon receptor) . In some embodiments, the sequence encoding the human or chimeric GCGR is operably linked to an endogenous regulatory element at the endogenous GCGR gene locus in the at least one chromosome. In some embodiments, the sequence encoding a human or chimeric GCGR comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human GCGR (NP_000151.1 (SEQ ID NO: 2) ) . In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat. In some embodiments, the animal is a mouse. In some embodiments, the animal does not express endogenous GCGR or expresses a decreased level of endogenous GCGR. In some embodiments, the animal has one or more cells expressing human or chimeric GCGR. In some embodiments, the expressed human or chimeric GCGR can interact with human glucagon, thereby promoting glycogen hydrolysis and/or gluconeogenesis. In some embodiments, the expressed human or chimeric GCGR can interact with endogenous glucagon, thereby promoting glycogen hydrolysis and/or gluconeogenesis.

In one aspect, the disclosure is related to a genetically-modified, non-human animal, in some embodiments, the genome of the animal comprises a replacement of a sequence encoding a region of endogenous GCGR with a sequence encoding a corresponding region of human GCGR at an endogenous GCGR gene locus. In some embodiments, the sequence encoding the corresponding region of human GCGR is operably linked to an endogenous regulatory element at the endogenous GCGR locus, and one or more cells of the animal expresses a human or chimeric GCGR. In some embodiments, the sequence encoding the corresponding region of human GCGR is immediately after endogenous 5'-UTR. In some embodiments, the sequence encoding a region of endogenous GCGR comprises the full-length coding sequence of endogenous GCGR (e.g., a nucleic acid sequence encoding amino acids 1-485 of SEQ ID NO: 1) . In some embodiments, the sequence encoding a region of endogenous GCGR comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14, or a part thereof, of the endogenous GCGR gene. In some embodiments, the replaced sequence starts with the start codon and ends with the stop codon of the endogenous mouse GCGR gene. In some embodiments, the animal is heterozygous with respect to the replacement at the endogenous GCGR gene locus. In some embodiments, the animal is homozygous with respect to the replacement at the endogenous GCGR gene locus.

In one aspect, the disclosure is related to a method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous GCGR gene locus, a sequence encoding a region of endogenous GCGR with a sequence encoding a corresponding region of human GCGR. In some embodiments, the sequence encoding the corresponding region of human GCGR comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14, or a part thereof, of a human GCGR gene. In some embodiments, the sequence encoding the corresponding region of human GCGR comprises a portion of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and a portion of exon 14, of a human GCGR gene. In some embodiments, the sequence encoding the corresponding region of human GCGR encodes amino acids 1-477 of SEQ ID NO: 2. In some embodiments, the sequence encoding a region of endogenous GCGR comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14, or a part thereof, of the endogenous GCGR gene. In some embodiments, the animal is a mouse, and the sequence encoding a region of endogenous GCGR starts within exon 2 and ends within exon 14 of the endogenous mouse GCGR gene.

In one aspect, the disclosure is related to a method of making a genetically-modified animal cell that expresses a human or chimeric GCGR, the method comprising: replacing at an endogenous GCGR gene locus, a nucleotide sequence encoding a region of endogenous GCGR with a nucleotide sequence encoding a corresponding region of human GCGR, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the human or chimeric GCGR, in some embodiments, the animal cell expresses the human or chimeric GCGR. In some embodiments, the animal is a mouse. In some embodiments, the nucleotide sequence encoding the human or chimeric GCGR is operably linked to an endogenous GCGR regulatory region, e.g., promoter.

In some embodiments, the animal further comprises a sequence encoding an additional human or chimeric protein. In some embodiments, the additional human or chimeric protein is programmed cell death protein 1 (PD-1) , Toil-like receptor (TLR) , CD40, tumor necrosis factor receptor superfamily member 9 (4-1BB) , glucagon-like peptide-1 receptor (GLP1R) , programmed cell death ligand 1 (PD-L1) , IL4, IL6, B7 Homolog 3 (B7-H3) , T-Cell Immunoreceptor with Ig and ITIM Domains (TIGIT) , or CD28.

In one aspect, the disclosure is related to a method of determining effectiveness of a GCGR modulator for treating a metabolic disorder (e.g., diabetes) , comprising: a) administering the GCGR modulator to an animal as described herein; b) optionally, administering glucose to the animal; and c) determining blood glucose level of the animal.

In one aspect, the disclosure is related to a method of determining effectiveness of a GCGR modulator for reducing blood glucose level, comprising: a) administering the GCGR modulator to an animal as described herein; b) optionally, administering glucose to the animal; and c) determining blood glucose level of the animal.

In some embodiments, the methods described herein further comprises: comparing the blood glucose level of the animal with blood glucose level of a reference animal, in some embodiments, the reference animal is not administered with the GCGR modulator.

In one aspect, the disclosure is related to a method of determining effectiveness of a GCGR modulator for increasing glucagon (e.g., free glucagon) level, increasing GLP-1, and/or decreasing insulin level, comprising: a) administering the GCGR modulator to an animal as described herein; and b) determining glucagon (e.g., free glucagon) level, GLP-1 level, and/or insulin level in the serum of the animal. In some embodiments, the methods described herein further comprises: comparing the glucagon (e.g., free glucagon) level and/or insulin level in the serum of the animal with glucagon (e.g., free glucagon) level and/or insulin level in the serum of a reference animal, in some embodiments, the reference animal is not administered with the GCGR modulator.

In some embodiments, the GCGR modulator is an anti-GCGR antibody or antigen-binding fragment thereof. In some embodiments, the anti-GCGR antibody or antigen-binding fragment thereof is an anti-human GCGR antibody. In some embodiments, the GCGR modulator is a drug (e.g., a small-molecule drug) targeting GCGR. In some embodiments, the animal is induced to make a diet-induced obesity (DIO) model.

In one aspect, the disclosure is related to a protein comprising an amino acid sequence, in some embodiments, the amino acid sequence is one of the following: (a) an amino acid sequence set forth in SEQ ID NO: 1 or 2; (b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 1 or 2; (c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1 or 2; (d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and (e) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 1 or 2.

In one aspect, the disclosure is related to a nucleic acid comprising a nucleotide sequence, in some embodiments, the nucleotide sequence is one of the following: (a) a sequence that encodes the protein as described herein; (b) SEQ ID NO: 3, 4, 5, 6, 7, 8, 32, or 33; (c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 32, or 33; (d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 32, or 33; and (e) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 32, or 33.

In some embodiments, the animal does not express endogenous GCGR.

In some embodiments, the replaced sequence starts within exon 2 and ends within exon 14 of the endogenous mouse GCGR gene.

In one aspect, the disclosure is related to a cell comprising the protein and/or the nucleic acid as described herein. In one aspect, the disclosure is related to an animal comprising the protein and/or the nucleic acid as described herein.

In one aspect, the disclosure relates to a genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric GCGR. In some embodiments, the sequence encoding the human or chimeric GCGR is operably linked to an endogenous regulatory element at the endogenous GCGR gene locus in the at least one chromosome.

In some embodiments, the sequence encoding a human or chimeric GCGR comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human GCGR (NP_000151.1 (SEQ ID NO: 2) . In some embodiments, the sequence encoding a human or chimeric GCGR comprises a sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 1-477 of SEQ ID NO: 2.

In one aspect, the disclosure relates to methods of determining effectiveness of an anti-GCGR antibody and an additional therapeutic agent for the treatment of diabetes. The methods involve administering the anti-GCGR antibody and the additional therapeutic agent to the animal as described herein; optionally administering glucose to the animal; and determining blood glucose level of the animal.

In some embodiments, the animal further comprises a sequence encoding a human or chimeric programmed cell death protein 1 (PD-1) , Toil-like receptor (TLR) , CD40, tumor necrosis factor receptor superfamily member 9 (4-1BB) , glucagon-like peptide-1 receptor (GLP1R) , programmed cell death ligand 1 (PD-L1) , IL, 4, IL6, B7 Homolog 3 (B7-H3) , T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , or CD28. In some embodiments, the additional therapeutic agent is an antibody or antigen-binding fragment thereof that specifically binds to PD-1, TLR, , CD40, 4-1BB, GLP1R, PD-L1, IL4, IL6, B7-H3, TIGIT, or CD28.

In one aspect, the disclosure relates to methods of determining effectiveness of an anti-GCGR antibody (e.g., an anti-human GCGR antibody) for the treatment of diabetes.

In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal's endogenous GCGR gene, wherein the disruption of the endogenous GCGR gene comprises deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14 or part thereof of the endogenous GCGR gene.

In some embodiments, the disruption of the endogenous GCGR gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14 of the endogenous GCGR gene.

In some embodiments, the disruption of the endogenous GCGR gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, and/or intron 13 of the endogenous GCGR gene.

In some embodiments, wherein the deletion can comprise deleting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or more nucleotides.

In some embodiments, the disruption of the endogenous GCGR gene comprises the deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nucleotides of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14.

The disclosure also relates to a cell including the targeting vector as described herein.

The disclosure also relates to non-human mammal generated through the methods as described herein.

In some embodiments, the genome thereof contains human gene (s) .

In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

In some embodiments, the non-human mammal expresses a protein encoded by a humanized GCGR gene.

The disclosure also relates to an offspring of the non-human mammal.

The disclosure also relates to a cell (e.g., stem cell or embryonic stem cell) or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or an animal model (e.g., diabetes or obesity animal model) thereof.

The disclosure further relates to the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or an animal model (e.g., diabetes or obesity animal model) derived or induced from the non-human mammal or an offspring thereof.

In another aspect, the disclosure relates to a tissue derived from the non-human mammal or an offspring thereof.

The disclosure further relates to GCGR genomic DNA sequences of a humanized mouse, a DNA sequence obtained by a reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence; a construct expressing the amino acid sequence thereof; a cell comprising the construct thereof; a tissue comprising the cell thereof.

The disclosure further relates to the use of the non-human mammal or an offspring thereof, the animal model generated through the method as described herein in the development of a product related to diabetes, the manufacture of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.

The disclosure also relates to the use of the non-human mammal or an offspring thereof, the animal model generated through the method as described herein in the production and utilization of an animal experimental disease model of diabetes, the study on regulation of blood glucose, or the development of a new diagnostic strategy and/or a therapeutic strategy.

The disclosure further relates to the use of the non-human mammal or an offspring thereof, the animal model generated through the methods as described herein, in the screening, verifying, evaluating or studying the GCGR gene function; anti-human GCGR antibodies; drugs for human GCGR targeting sites; the drugs or efficacies for human GCGR targeting sites; and the drugs for diabetes and hypoglycemic drugs.

In one aspect, the disclosure is related to a method of determining effectiveness (e.g., side effects) of a GCGR modulator for decreasing triglyceride (TG) , comprising: a) administering the GCGR modulator to an animal as described herein; and b) determining TG level in the serum of the animal. In some embodiments, the method further comprises: comparing the TG level in the serum of the animal with TG level in the serum of a reference animal, wherein the reference animal is not administered with the GCGR modulator. In some embodiments, the disclosure is related to a method of determining effectiveness (e.g., side effects) of a GCGR modulator for increasing total cholesterol (TC) , high density lipoprotein-cholesterol (HDL-C) and/or low density lipoprotein-cholesterol (LDL-C) , comprising: a) administering the GCGR modulator to an animal as described herein; and b) determining TC, HDL-C, and/or LDL-C levels in the serum of the animal. In some embodiments, the method further comprises: comparing the TC, HDL-C, and/or LDL-C levels in the serum of the animal with TC, HDL-C, and/or LDL-C levels in the serum of a reference animal, wherein the reference animal is not administered with the GCGR modulator.

As used herein, the term “nucleotide” refers to native or modified ribonucleotide sequences or deoxyribonucleotide sequences (e.g., DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, or tRNA)

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing mouse and human GCGR gene loci.

FIG. 2 is a schematic diagram showing humanized GCGR gene locus.

FIG. 3 is a schematic diagram showing a GCGR gene targeting strategy.

FIG. 4 shows Southern Blot results of cells after recombination using the 5'Probe, 3' Probe, and Neo Probe. WT is a wild-type control. ES-01, ES-02, ES-03, and ES-04 are clone numbers.

FIG. 5 is a schematic diagram showing the FRT recombination process in GCGR gene humanized mice.

FIG. 6A shows PCR identification results ofF1 generation mice by primers WT-F1 and WT-R1. M is a marker. PC is a positive control. WT is a wild-type control. H ₂O is a water control.

FIG. 6B shows PCR identification results of F1 generation mice by primers WT-F1 and Mut-R1. M is a marker. PC is a positive control. WT is a wild-type control. H ₂O is a water control.

FIG. 6C shows PCR identification results of F1 generation mice by primers Frt-F and Frt--R.M is a marker. PC is a positive control. WT is a wild-type control. H ₂O is a water control.

FIG. 6D shows PCR identification results of F1 generation mice by primers Flp-F1 and Flp-R1. M is a marker. PC is a positive control. WT is a wild-type control. H ₂O is a water control.

FIG. 7 shows mRNA transcription results of humanized GCGR gene in F2 generation of GCGR gene humanized mice. “+/+” represents wild-type mouse, and “H/H” represents GCGR gene humanized homozygous mouse.

FIG. 8 is an experimental scheme of in vivo efficacy verification using GCGR gene humanized mice. “RBG” stands for random blood glucose. “OGTT” stands for oral glucose tolerance test.

FIG. 9A shows the RBG level of C57BL/6 mice treated with human IgG4 (G1) , GCGR gene humanized mice treated with human IgG4 (G2) , and GCGR gene humanized mice treated with Crotedumab (G3) in the in vivo efficacy verification experiment.

FIG. 9B shows the body weight of C57BL/6 mice treated with human IgG4 (G1) , GCGR gene humanized mice treated with human IgG4 (G2) , and GCGR gene humanized mice treated with Crotedumab (G3) in the in vivo efficacy verification experiment.

FIG. 9C shows the OGTT test results of C57BL/6 mice treated with human IgG4 (G1) , GCGR gene humanized mice treated with human IgG4 (G2) , and GCGR gene humanized mice treated with Crotedumab (G3) on day 4 of the in vivo efficacy verification experiment.

FIG. 9D shows the glucose AUC of C57BL/6 mice treated with human IgG4 (G1) , GCGR gene humanized mice treated with human IgG4 (G2) , and GCGR gene humanized mice treated with Crotedumab (G3) on day 4 of the in vivo efficacy verification experiment.

FIG. 9E shows the serum insulin level of C57BL/6 mice treated with human IgG4 (G1) , GCGR gene humanized mice treated with human IgG4 (G2) , and GCGR gene humanized mice treated with Crotedumab (G3) on day 7 of the in vivo efficacy verification experiment.

FIG. 9F shows the serum glucagon level of C57BL/6 mice treated with human IgG4 (G1) , GCGR gene humanized mice treated with human IgG4 (G2) , and GCGR gene humanized mice treated with Crotedumab (G3) on day 7 of the in vivo efficacy verification experiment.

FIGS. 10A-10D show the serum level of TG (triglyceride) , TC (total cholesterol) , HDL-C (high density lipoprotein-cholesterol) , and LDL-C (low density lipoprotein-cholesterol) , respectively, in C57BL/6 mice treated with human IgG4 (G1) , GCGR gene humanized mice treated with human IgG4 (G2) , and GCGR gene humanized mice treated with Crotedumab (G3) on day 11 of the in vivo efficacy verification experiment.

FIG. 11 shows Western Blot results of GCGR gene humanized homozygous mouse (H/H) and wild-type C57BL/6 mouse (+/+) . β-actin is an internal reference.

FIGS. 12A-12B show flow cytometry detection results of leukocyte subtypes and T cell subtypes, respectively, in the spleen of GCGR gene humanized homozygous mouse (H/H) and wild-type C57BL/6 mouse (+/+) .

FIGS. 13A-13B show flow cytometry detection results of leukocyte subtypes and T cell subtypes, respectively, in the lymph nodes of GCGR gene humanized homozygous mouse (H/H) and wild-type C57BL/6 mouse (+/+) .

FIGS. 14A-14B show flow cytometry detection results of leukocyte subtypes and T cell subtypes, respectively, in the peripheral blood of GCGR gene humanized homozygous mouse (H/H) and wild-type C57BL/6 mouse (+/+) .

FIGS. 15A-15B show body weight and RBG level, respectively, in a diet-induced obesity (DIO) model of GCGR gene humanized mice for efficacy validation.

FIGS. 16A-16B show the blood glucose-time curve and the area under the curve, respectively, in the OGTT test on day 4 of the pharmacodynamic validation experiment.

FIGS. 16C-16E show serum level changes of insulin, glucagon, and GLP-1, respectively, on day 14 and day 28 of the pharmacodynamic validation experiment.

FIGS. 17A-17F show the detection results of triglyceride (TG) , total cholesterol (TC) , high-density lipoprotein cholesterol (HDL-C) , low-density lipoprotein cholesterol (LDL-C) , alanine aminotransferase (ALT) and aspartate aminotransferase (AST) , respectively, on day 28 of the pharmacodynamic validation experiment.

FIGS. 18A-18B show stained sections at different magnifications (100× and 200×) for glucagon and insulin in the pancreas of G1 and G4 group mice, respectively.

FIGS. 19A-19C show islet alpha cell area, islet beta cell area, and the count of islet number per pancreas area, respectively, in the pancreas of G1-G4 group mice.

FIG. 20 shows the alignment between human GCGR amino acid sequence (NP_000151.1; SEQ ID NO: 2) and mouse GCGR amino acid sequence (NP_032127.2; SEQ ID NO: 1) .

FIG. 21 shows the alignment between human GCGR amino acid sequence (NP_000151.1; SEQ ID NO: 2) and rat GCGR amino acid sequence (NP_742089.1; SEQ ID NO: 34) .

DETAILED DESCRIPTION

This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) GCGR, and methods of use thereof.

GCGR plays a central role in the regulation of blood glucose levels and glucose homeostasis. It regulates the rate of hepatic glucose production by promoting glycogen hydrolysis and gluconeogenesis, and plays an important role in mediating the responses to fasting. Ligand (e.g., glucagon) binding causes a conformation change of GCGR that triggers signaling via guanine nucleotide-binding proteins (G proteins) and modulates the activity of down-stream effectors, such as adenylate cyclase. GCGR can promote activation of adenylate cyclase. Besides, GCGR plays a role in signaling via a phosphatidylinositol-calcium second messenger system. GCGR is a member of the glucagon receptor family that also includes GLP-1, GLP-2, secretin, GHRH and GIP receptors. GCGR regulates blood glucose levels and is predominantly expressed in the pancreas, liver and kidneys. Thus, anti-GCGR antibodies can be potentially used as diabetes therapies.

Experimental animal models are an indispensable research tool for studying the effects of these antibodies (e.g., GCGR antibodies) . Common experimental animals include mice, rats, guinea pigs, hamsters, rabbits, dogs, monkeys, pigs, fish and so on. However, there are many differences between human and animal genes and protein sequences, and many human proteins cannot bind to the animal's homologous proteins to produce biological activity, leading to that the results of many clinical trials do not match the results obtained from animal experiments. A large number of clinical studies are in urgent need of better animal models. With the continuous development and maturation of genetic engineering technologies, the use of human cells or genes to replace or substitute an animal's endogenous similar cells or genes to establish a biological system or disease model closer to human, and establish the humanized experimental animal models (humanized animal model) has provided an important tool for new clinical approaches or means. In this context, the genetically engineered animal model, that is, the use of genetic manipulation techniques, the use of human normal or mutant genes to replace animal homologous genes, can be used to establish the genetically modified animal models that are closer to human gene systems. The humanized animal models have various important applications. For example, due to the presence of human or humanized genes, the animals can express or express in part of the proteins with human functions, so as to greatly reduce the differences in clinical trials between humans and animals, and provide the possibility of drug screening at animal levels.

Unless otherwise specified, the practice of the methods described herein can take advantage of the techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology. These techniques are explained in detail in the following literature, for examples: Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) ; DNA Cloning, Volumes I and II (D.N. Glovered., 1985) ; Oligonucleotide Synthesis (M.J. Gaited., 1984) ;Mullisetal U.S. Pat. No. 4,683, 195; Nucleic Acid Hybridization (B.D. Hames&S.J. Higginseds. 1984) ; Transcription And Translation (B.D. Hames&S.J. Higginseds. 1984) ;Culture Of Animal Cell (R.I. Freshney, Alan R. Liss, Inc., 1987) ; Immobilized Cells And Enzymes (IRL Press, 1986) ; B. Perbal, A Practical Guide To Molecular Cloning (1984) , the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York) , specifically, Vols. 154 and 155 (Wuetal. eds. ) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed. ) ; Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Caloseds., 1987, Cold Spring Harbor Laboratory) ; Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987) ; Hand book Of Experimental Immunology, Volumes V (D.M. Weir and C.C. Blackwell, eds., 1986) ; and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986) ; each of which is incorporated herein by reference in its entirety.

Glucagon and GCGR

Glucagon is a 29-amino acid polypeptide secreted by the α-cell of the islet of Langerhans. Initially synthesized as a larger precursor “proglucagon, ” it is cleaved by a specific enzyme, proconvertase to the active molecule “glucagon. ” Glucagon is also produced by the central nervous system on which its actions may include the regulation of glucose production. Glucagon secretion is stimulated by hypoglycaemia, arginine, gastric inhibitor polypeptide, gastrin and potassium chloride and inhibited by hyperglycaemia, insulin, zinc, GLP-1 (glucagon-like peptide 1) and somatostatin. Glucagon also mediates an increase in intracellular calcium in a phospholipase-C-dependent manner and it activates AMPK and JNK. Binding sites for glucagon have been identified in liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreatic islet β-cells.

Glucagon receptor is a 62 kDa protein that is activated by glucagon and is a member of the class B G-protein coupled family of receptors, coupled to G alpha i, G _s and to a lesser extent G alpha q. Stimulation of the receptor results in the activation of adenylate cyclase and phospholipase C and in increased levels of the secondary messengers intracellular cAMP and calcium. In humans, the glucagon receptor is encoded by the GCGR gene. Glucagon receptors are mainly expressed in liver and in kidney with lesser amounts found in heart, adipose tissue, spleen, thymus, adrenal glands, pancreas, cerebral cortex, and gastrointestinal tract.

Upon binding with the signaling molecule glucagon, GCGR initiates a signal transduction pathway that begins with the activation of adenylate cyclase, which in turn produces cyclic AMP (cAMP) . Protein kinase A, whose activation is dependent on the increased levels of cAMP, is responsible for the ensuing cellular response in the form of

protein kinase

1 and 2. The ligand-bound glucagon receptor can also initiate a concurrent signaling pathway that is independent of cAMP by activating phospholipase C. Phospholipase C produces DAG and IP3 from PIP2, a phospholipid phospholipase C cleaves off of the plasma membrane. Ca ²⁺ stores inside the cell release Ca ²⁺ when its calcium channels are bound by IP ₃.

A detailed description of GCGR and its function can be found, e.g., in Vuguin, P.M., et al. "Novel insight into glucagon receptor action: lessons from knockout and transgenic mouse models. " Diabetes, Obesity and Metabolism 13 (2011) : 144-150; A1-Massadi, O., et al. "Glucagon control on food intake and energy balance. " International Journal of Molecular Sciences 20.16 (2019) : 3905; Okamoto, H., et al. "Glucagon receptor inhibition normalizes blood glucose in severe insulin-resistant mice. " PNAS 114.10 (2017) : 2753-2758; each of which is incorporated by reference in its entirety.

In human genomes, GCGR gene (Gene ID: 2642) locus has fourteen exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and exon 14 (FIG. 1) . The GCGR protein also has four extracellular regions, seven transmembrane regions, and four cytoplasmic regions, and the signal peptide is located in the N-terminal extracellular region of GCGR. The nucleotide sequence for human GCGR mRNA is NM_000160.5, and the amino acid sequence for human GCGR is NP_000151.1 (SEQ ID NO: 2) . The location for each exon and each region in human GCGR nucleotide sequence and amino acid sequence is listed below:

Table 1

The human GCGR gene (Gene ID: 2642) is located in Chromosome 17 of the human genome, which is located from 81,804,150 to 81,814,008 of NC_000017.11.5’ UTR is from 81804150 to 81804249, and from 81808842 to 81808979. exon 1 is from 81,804,150 to 81,804,249, intron 1 is from 81,804,250 to 81,808,841, exon 2 is from 81,808,842 to 81809019, intron 2 is from 81,809,079 to 81,809,781, exon 3 is from 81,809,782 to 81,809,884, intron 3 is from 81,809,885 to 81,810,824, exon 4 is from 81,810,825 to 81,810,932, intron 4 is from 81,810,933 to 81,811,009, exon 5 is from 81,811,010 to 81,811,131, intron 5 is from 81,811,132 to 81,811,221, exon 6 is from 81,811,222 to 81,811,328, intron 6 is from 81,811,329 to 81,811,403, exon 7 is from 81,811,404 to 81,811,560, intron7 is from 81,811,561 to 81,811,650, exon 8 is from 81,811,651 to 81,811,810, intron 8 is from 81,811,811 to 81,811,885, exon 9 is from 81,811,886 to 81,811,946, intron 9 is from 81,811,947 to 81,812,182, exon 10 is from 81,812,183 to 81,812,252, intron 10 is from 81,812,253 to 81,812,576, exon 11 is from 81,812,577 to 81,812,665, intron 11 is from 81,812,666 to 81,812,806, exon 12 is from 81,812,807 to 81,812,945, intron 12 is from 81,812,946 to 81,813,015, exon 13 is from 81,813,016 to 81,813,057, intron 13 is from 81,813,058 to 81,813,473, exon 14 is from 81,813,474 to 81,814,008. The 3’ UTR is from 81813690 to 81814008, based on transcript NM_000160.5. All relevant information for human GCGR locus can be found in the NCBI website with Gene ID: 2642, which is incorporated by reference herein in its entirety.

In mice, GCGR gene locus has fourteen exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and exon 14 (FIG. 1) . The mouse GCGR protein also has four extracellular regions, seven transmembrane regions, and four cytoplasmic regions, and the signal peptide is located in the N-terminal extracellular region of GCGR. The nucleotide sequence for mouse GCGR mRNA is NM_008101.2, the amino acid sequence for mouse GCGR is NP_032127.2 (SEQ ID NO: 1) . The location for each exon and each region in the mouse GCGR nucleotide sequence and amino acid sequence is listed below:

Table 2

The mouse GCGR gene (Gene ID: 14527) is located in Chromosome 11 of the mouse genome, which is located from 120420011 to 120429815 of NC_000077.7.5’UTR is from 120,530,699 to 120,530,833, and from 120425506 to 120425582. exon 1 is from 120,530,699 to 120,530,833, intron 1 is from 120,530,834 to 120,534,679, exon 2 is from 120,534,680 to 120,534,819, intron 2 is from 120,534,820 to 120,534,915, exon 3 is from 120,534,916 to 120,535,018, intron 3 is from 120,535,019 to 120,536,059, exon 4 is from 120,536,060 to 120,536,167, intron 4 is from 120,536,168 to 120,536,244, exon 5 is from 120,536,245 to 120,536,366, intron 5 is from 120,536,367 to 120,536,461, exon 6 is from 120,536,462 to 120,536,568, intron 6 is from 120,536,569 to 120,536,656, exon 7 is from 120,536,657 to 120,536,813, intron 7 is from 120,536,814 to 120,536,894, exon 8 is from 120,536,895 to 120,537,054, intron 8 is from 120,537,055 to 120,537,128, exon 9 is from 120,537,129 to 120,537,189, intron 9 is from 120,537,190 to 120,537,318, exon 10 is from 120,537,319 to 120,537,388, intron10 is from 120,537,389 to 120,537,389, exon 11 is from 120,537,703 to 120,537,791, intron 11 is from 120,537,792 to 120,537,911, exon 12 is from 120,537,912 to 120,538,050, intron 12 is from 120,538,051 to 120,538,116, exon 13 is from 120,538,117 to 120,538,158, intron 13 is from 120,538,159 to 120,538,460, exon 14 is from 120,538,461 to 120,538,984. The 3’ UTR is from 120429524 to 120429810, based on transcript NM_ NM_008101.2. All relevant information for mouse GCGR locus can be found in the NCBI website with Gene ID: 14527, which is incorporated by reference herein in its entirety.

FIG. 20 shows the alignment between human GCGR amino acid sequence (NP_000151.1; SEQ ID NO: 2) and mouse GCGR amino acid sequence (NP_032127.2; SEQ ID NO: 1) . Thus, the corresponding amino acid residue or region between human and mouse GCGR can be found in FIG. 20.

GCGR genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for GCGR in Rattusnorvegicus (rat) is 24953, the gene ID for GCGR in Macaca mulatta (Rhesus monkey) is 714542, the gene ID for GCGR in Canis lupus familiaris (dog) is 483368, and the gene ID for GCGR in Pan troglodytes (chimpanzee) is 468357. The relevant information for these genes (e.g., intron sequences, exon sequences, amino acid residues of these proteins) can be found, e.g., in NCBI database, which is incorporated by reference herein in its entirety. FIG. 21 shows the alignment between human GCGR amino acid sequence (NP_000151.1; SEQ ID NO: 2) and a rodent GCGR amino acid sequence (NP_742089.1; SEQ ID NO: 34) . Thus, the corresponding amino acid residue or region between human and rodent GCGR can be found in FIG. 21.

The present disclosure provides human or chimeric (e.g., humanized) GCGR nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, signal peptide, extracellular regions, transmembrane regions, and/or cytoplasmic regions are replaced by the corresponding human sequence. In some embodiments, a “region” or “portion” of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, signal peptide, extracellular regions, transmembrane regions, and/or cytoplasmic regions are replaced by the corresponding human sequence. The term “region” or “portion” can refer to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or 1400 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, or 480 amino acid residues. In some embodiments, the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, signal peptide, extracellular regions, transmembrane regions, or cytoplasmic regions. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14 (e.g., a portion of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and a portion of exon 14) are replaced by a region, a portion, or the entire sequence of the human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14 (e.g., a portion of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and a portion of exon 14) sequence.

In some embodiments, the present disclosure is related to a genetically-modified, non-human animal whose genome comprises a chimeric (e.g., humanized) GCGR nucleotide sequence. In some embodiments, the chimeric (e.g., humanized) GCGR nucleotide sequence encodes a GCGR protein comprising one or more (e.g., 1, 2, 3, or 4) extracellular regions, one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) transmembrane regions, one or more (e.g., 1, 2, 3, or 4) cytoplasmic regions, and/or a signal peptide. In some embodiments, the signal peptide described herein is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-25 of SEQ ID NO: 2. In some embodiments, the signal peptide described herein is at least 80%, 85%, 90%, 95%, or 100%identical to amino acids 1-26 of SEQ ID NO: 1. In some embodiments, the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO:3, 4, 5, 6, 7, 8, 32, or 33.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a human or humanized GCGR protein. In some embodiments, the humanized GCGR protein comprises one or more (e.g., 1, 2, 3, or 4) human cytoplasmic regions. In some embodiments, the humanized GCGR protein comprises one or more (e.g., 1, 2, 3, or 4) endogenous cytoplasmic regions. In some embodiments, the humanized GCGR protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) human transmembrane regions. In some embodiments, the humanized GCGR protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) endogenous transmembrane regions. In some embodiments, the humanized GCGR protein comprises one or more (e.g., 1, 2, 3, or 4) human extracellular regions. In some embodiments, the humanized GCGR protein comprises one or more (e.g., 1, 2, 3, or 4) endogenous extracellular regions. In some embodiments, the humanized GCGR protein comprises a human or humanized signal peptide (e.g., amino acids 1-25 of SEQ ID NO: 2) .

In some embodiments, the genetically-modified non-human animal described herein comprises a human or humanized GCGR gene. In some embodiments, the humanized GCGR gene comprises 14 exons. In some embodiments, the humanized GCGR gene comprises humanized exon 1, humanized exon 2, humanized exon 3, humanized exon 4, humanized exon 5, humanized exon 6, humanized exon 7, humanized exon 8, humanized exon 9, humanized exon 10, humanized exon 11, humanized exon 12, humanized exon 13, and/or humanized exon 14. In some embodiments, the humanized GCGR gene comprises humanized intron 1, humanized intron 2, humanized intron 3, humanized intron 4, humanized intron 5, humanized intron 6, humanized intron 7, humanized intron 8, humanized intron 9, humanized intron 10, humanized intron 11, humanized intron 12, and/or humanized intron 13. In some embodiments, the humanized GCGR gene comprises human or humanized 5' UTR. In some embodiments, the humanized GCGR gene comprises human or humanized 3' UTR. In some embodiments, the humanized GCGR gene comprises endogenous 5' UTR. In some embodiments, the humanized GCGR gene comprises endogenous 3' UTR.

In some embodiments, the present disclosure also provides a chimeric (e.g., humanized) GCGR nucleotide sequence and/or amino acid sequences, wherein in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%of the sequence are identical to or derived from mouse GCGR mRNA sequence (e.g., NM_008101.2) , mouse GCGR amino acid sequence (e.g., SEQ ID NO: 1) , or a portion thereof (e.g., exon 1, a portion of exon 2, and a portion of exon 14) ; and in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%of the sequence are identical to or derived from human GCGR mRNA sequence (e.g., NM_000160.5) , human GCGR amino acid sequence (e.g., SEQ ID NO: 2) , or a portion thereof (e.g., a portion of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and a portion of exon 14) .

In some embodiments, the sequence encoding amino acids 1-485 of mouse GCGR (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human GCGR (e.g., amino acids 1-477 of human GCGR (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding amino acids 27-485 of mouse GCGR (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human GCGR (e.g., amino acids 26-477 of human GCGR (SEQ ID NO: 2) ) .

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse GCGR promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from part of or the entire mouse GCGR nucleotide sequence (e.g., a portion of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and a portion of exon 14 of NM_008101.2) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as part of or the entire mouse GCGR nucleotide sequence (e.g., exon 1, a portion of exon 2, and a portion of exon 14 of NM_008101.2) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from part of or the entire human GCGR nucleotide sequence (e.g., exon 1, a portion of exon 2, and a portion of exon 14 of NM_000160.5) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as part of or the entire human GCGR nucleotide sequence (e.g., a portion of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and a portion of exon 14 of NM_000160.5) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire mouse GCGR amino acid sequence (e.g., NP_032127.2 (SEQ ID NO: 1) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire mouse GCGR amino acid sequence (e.g., NP_032127.2 (SEQ ID NO: 1) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from part of or the entire human GCGR amino acid sequence (e.g., NP_000151.1 (SEQ ID NO: 2) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as part of or the entire human GCGR amino acid sequence (e.g., NP_000151.1 (SEQ ID NO: 2) ) .

The present disclosure also provides a humanized GCGR mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) an amino acid sequence shown in SEQ ID NO: 1 or 2;

b) an amino acid sequence having a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 1 or 2;

c) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 1 or 2 under a low stringency condition or a strict stringency condition;

d) an amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 1 or 2;

e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

f) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 or 2.

The present disclosure also relates to a GCGR nucleic acid (e.g., DNA or RNA) sequence, wherein the nucleic acid sequence can be selected from the group consisting of:

a) a nucleic acid sequence as shown in SEQ ID NO: 5 or 8, or a nucleic acid sequence encoding a homologous GCGR amino acid sequence of a humanized mouse GCGR;

b) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 5 or 8 under a low stringency condition or a strict stringency condition;

c) a nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleotide sequence as shown in SEQ ID NO: 5 or 8;

d) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 1 or 2;

e) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 1 or 2;

f) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

g) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 or 2.

The present disclosure further relates to a GCGR genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 5 or 8.

The disclosure also provides an amino acid sequence that has a homology of at least 90%with, or at least 90%identical to the sequence shown in SEQ ID NO: 1 or 2, and has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 1 or 2 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 1 or 2 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

The disclosure also provides a nucleotide sequence that has a homology of at least 90%, or at least 90%identical to the sequence shown in SEQ ID NO: 5 or 8, and encodes a polypeptide that has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 5 or 8 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 5 or 8 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein. In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percentage of residues conserved with similar physicochemical properties (percent homology) , e.g. leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . The homology percentage, in many cases, is higher than the identity percentage.

Cells, tissues, and animals (e.g., mouse) are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express human or chimeric (e.g., humanized) GCGR from an endogenous non-human GCGR locus.

Genetically modified animals

As used herein, the term “genetically-modified non-human animal” refers to a non-human animal having exogenous DNA in at least one chromosome of the animal's genome. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%of cells of the genetically-modified non-human animal have the exogenous DNA in its genome. The cell having exogenous DNA can be various kinds of cells, e.g., hepatocytes, lymphocytes, monocytes, macrophages, endothelial cells, epithelial cells, CD34+thymocytes, neurons or tumor cells. In some embodiments, the cell is an islet alpha cell or an islet beta cell of pancreas. In some embodiments, genetically-modified non-human animals are provided that comprise a modified endogenous GCGR locus that comprises an exogenous sequence (e.g., a human sequence) , e.g., a replacement of one or more non-human sequences with one or more human sequences. The animals are generally able to pass the modification to progeny, i.e., through germline transmission.

As used herein, the term “chimeric gene” or “chimeric nucleic acid” refers to a gene or a nucleic acid, wherein two or more portions of the gene or the nucleic acid are from different species, or at least one of the sequences of the gene or the nucleic acid does not correspond to the wild-type nucleic acid in the animal. In some embodiments, the chimeric gene or chimeric nucleic acid has at least one portion of the sequence that is derived from two or more different sources, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) protein of two or more different species. In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized gene or humanized nucleic acid.

As used herein, the term “chimeric protein” or “chimeric polypeptide” refers to a protein or a polypeptide, wherein two or more portions of the protein or the polypeptide are from different species, or at least one of the sequences of the protein or the polypeptide does not correspond to wild-type amino acid sequence in the animal. In some embodiments, the chimeric protein or the chimeric polypeptide has at least one portion of the sequence that is derived from two or more different sources, e.g., same (or homologous) proteins of different species. In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized protein or a humanized polypeptide.

As used herein, the term “humanized protein” or “humanized polypeptide” refers to a protein or a polypeptide, wherein at least a portion of the protein or the polypeptide is from the human protein or human polypeptide. In some embodiments, the humanized protein or polypeptide is a human protein or polypeptide.

As used herein, the term “humanized nucleic acid” refers to a nucleic acid, wherein at least a portion of the nucleic acid is from the human. In some embodiments, the entire nucleic acid of the humanized nucleic acid is from human. In some embodiments, the humanized nucleic acid is a humanized exon. A humanized exon can be e.g., a human exon or a chimeric exon.

In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized GCGR gene or a humanized GCGR nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human GCGR gene, at least one or more portions of the gene or the nucleic acid is from a non-human GCGR gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an GCGR protein. The encoded GCGR protein is functional or has at least one activity of the human GCGR protein or the non-human GCGR protein, e.g., regulating blood glucose levels, regulating glucose homeostasis, regulating the rate of hepatic glucose production, promoting glycogen hydrolysis, promoting gluconeogenesis, mediating response to fasting, promoting activation of adenylate cyclase, and/or signaling via a phosphatidylinositol-calcium second messenger system.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized GCGR protein or a humanized GCGR polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a human GCGR protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human GCGR protein. The humanized GCGR protein or the humanized GCGR polypeptide is functional or has at least one activity of the human GCGR protein or the non-human GCGR protein.

The genetically modified non-human animal can be various animals, e.g., a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo) , deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey) . For the non-human animals where suitable genetically modifiable embryonic stem (ES) cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo. These methods are known in the art, and are described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003, which is incorporated by reference herein in its entirety.

In one aspect, the animal is a mammal, e.g., of the superfamily Dipodoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In some embodiments, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters) , Cricetidae (e.g., hamster, New World rats and mice, voles) , Muridae (true mice and rats, gerbils, spiny mice, crested rats) , Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice) , Platacanthomyidae (e.g., spiny dormice) , and Spalacidae (e.g., mole rates, bamboo rats, and zokors) . In some embodiments, the genetically modified rodent is selected from a true mouse or rat (family Muridae) , a gerbil, a spiny mouse, and a crested rat. In some embodiments, the non-human animal is a mouse.

In some embodiments, the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/1 0Cr, and C57BL/Ola. In some embodiments, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2. These mice are described, e.g., in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999) ; Auerbach et al., Establishment and Chimera Analysis of 129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000) , both of which are incorporated herein by reference in the entirety. In some embodiments, the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a mix of the 129 strains, or a mix of the BL/6 strains. In some embodiments, the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129) . In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse having a BALB/c, A, A/He, A/J, A/WySN, AKR, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL (C57BL/1 0Cr and C57BL/Ola) , C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, or CBA/H background.

In some embodiments, the animal is a rat. The rat can be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

The animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the humanized GCGR animal is made. For example, suitable mice for maintaining a xenograft (e.g., a human cancer or tumor) , can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part. Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin) , physical means (e.g., irradiating the animal) , and/or genetic modification (e.g., knocking out one or more genes) . Non-limiting examples of such mice include, e.g., NOD mice, SCID mice, NOD/SCID mice, IL2Rγknockout mice, NOD/SCID/γcnull mice (Ito, M. et al., NOD/SCID/γcnull mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100 (9) : 3175-3182, 2002) , nude mice, and Rag 1 and/or Rag2 knockout mice. These mice can optionally be irradiated, or otherwise treated to destroy one or more immune cell type. Thus, in various embodiments, a genetically modified mouse is provided that can include a humanization of at least a portion of an endogenous non-human GCGR locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part. In some embodiments, modification is, e.g., selected from the group consisting of a modification that results in NOD mice, SCID mice, NOD/SCID mice, IL-2Rγ knockout mice, NOD/SCID/γc ^null mice, nude mice, Ragl and/or Rag2 knockout mice, NOD-Prkdc ^scid IL-2rγ ^null mice, NOD-Rag 1 ^-/--IL2rg ^-/- (NRG) mice, Rag 2 ^-/--IL2rg ^-/- (RG) mice, and a combination thereof. These genetically modified animals are described, e.g., in US20150106961, which is incorporated herein by reference in its entirety. In some embodiments, the mouse can include a replacement of all or part of mature GCGR coding sequence with human mature GCGR coding sequence.

Genetically modified non-human animals that comprise a modification of an endogenous non-human GCGR locus. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature GCGR protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the mature GCGR protein sequence) . Although genetically modified cells are also provided that can comprise the modifications described herein (e.g., ES cells, somatic cells) , in many embodiments, the genetically modified non-human animals comprise the modification of the endogenous GCGR locus in the germline of the animal.

Genetically modified animals can express a human GCGR and/or a chimeric (e.g., humanized) GCGR from endogenous mouse loci, wherein the endogenous mouse GCGR gene has been replaced with a human GCGR gene and/or a nucleotide sequence that encodes a region of human GCGR sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70&, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the human GCGR sequence. In various embodiments, an endogenous non-human GCGR locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature GCGR protein.

In some embodiments, the genetically modified mice express the human GCGR and/or chimeric GCGR (e.g., humanized GCGR) from endogenous loci that are under control of mouse promoters and/or mouse regulatory elements. The replacement (s) at the endogenous mouse loci provide non-human animals that express human GCGR or chimeric GCGR (e.g., humanized GCGR) in appropriate cell types and in a manner that does not result in the potential pathologies observed in some other transgenic mice known in the art. The human GCGR or the chimeric GCGR (e.g., humanized GCGR) expressed in animal can maintain one or more functions (e.g., regulating blood glucose level) of the wild-type mouse or human GCGR in the animal. For example, human or non-human GCGR ligands (e.g., glucagon) can bind to the expressed GCGR, which further promotes glycogen hydrolysis and/or gluconeogenesis. Furthermore, in some embodiments, the animal does not express endogenous GCGR. In some embodiments, the animal expresses a decreased level of endogenous GCGR as compared to a wild-type animal. As used herein, the term “endogenous GCGR” refers to GCGR protein that is expressed from an endogenous GCGR nucleotide sequence of the non-human animal (e.g., mouse) before any genetic modification.

The genome of the animal can comprise a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human GCGR (NP_000151.1) (SEQ ID NO: 2) .

The genome of the genetically modified animal can comprise a replacement at an endogenous GCGR gene locus of a sequence encoding a region of endogenous GCGR with a sequence encoding a corresponding region of human GCGR. In some embodiments, the sequence that is replaced is any sequence within the endogenous GCGR gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, 5’-UTR’3’-UTR, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, and/or intron 13, etc. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous GCGR gene. In some embodiments, the sequence that is replaced is exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, or a portion thereof, of an endogenous mouse GCGR gene locus.

In some embodiments, the non-human animal can have, at an endogenous GCGR gene locus, a nucleotide sequence encoding a chimeric human/non-human GCGR polypeptide, wherein a human portion of the chimeric human/non-human GCGR polypeptide comprises a portion of human GCGR, and wherein the animal expresses a functional GCGR on a surface of a cell of the animal. The human portion of the chimeric human/non-human GCGR polypeptide can comprise an amino acid sequence encoded by exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, or a portion thereof, of human GCGR. In some embodiments, the human portion of the chimeric human/non-human GCGR polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 1-477, or 26-477 of SEQ ID NO: 2. In some embodiments, the human portion of the chimeric human/non-human GCGR polypeptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) human GCGR transmembrane regions. In some embodiments, the human portion of the chimeric human/non-human GCGR polypeptide comprises one or more (e.g., 1, 2, 3, or 4) human GCGR cytoplasmic regions. In some embodiments, the human portion of the chimeric human/non-human GCGR polypeptide comprises one or more (e.g., 1, 2, 3, or 4) human GCGR extracellular regions.

In some embodiments, the non-human portion of the chimeric human/non-human GCGR polypeptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) transmembrane regions, one or more (e.g., 1, 2, 3, or 4) cytoplasmic regions, and/or one or more (e.g., 1, 2, 3, or 4) cytoplasmic regions of an endogenous non-human GCGR polypeptide.

Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous GCGR locus, or homozygous with respect to the replacement at the endogenous GCGR locus.

In some embodiments, the humanized GCGR locus lacks a human GCGR 5'-UTR. In some embodiment, the humanized GCGR locus comprises an endogenous (e.g., mouse) 5'-UTR. In some embodiments, the humanization comprises an endogenous (e.g., mouse) 3'-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human GCGR genes appear to be similarly regulated based on the similarity of their 5'-flanking sequence. As shown in the present disclosure, humanized GCGR mice that comprise a replacement at an endogenous mouse GCGR locus, which retain mouse regulatory elements but comprise a humanization of GCGR encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized GCGR are grossly normal.

The present disclosure further relates to a non-human mammal generated through the method mentioned above. In some embodiments, the genome thereof contains human gene (s) .

In some embodiments, the non-human mammal is a rodent, and preferably, the non-human mammal is a mouse.

In addition, the present disclosure also relates to a non-human mammal model for diabetes and/or obesity, characterized in that the non-human mammal model is obtained through the methods as described herein. In some embodiments, the non-human mammal is a rodent (e.g., a mouse) .

The present disclosure further relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or an animal model (e.g., diabetes or obesity animal model) induced from the non-human mammal or an offspring thereof; the tissue (e.g., pancreas or kidney) , organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the animal model.

The present disclosure also provides non-human mammals produced by any of the methods described herein. In some embodiments, a non-human mammal is provided; and the genetically modified animal contains the DNA encoding human or humanized GCGR in the genome of the animal.

In some embodiments, the non-human mammal comprises the genetic construct as described herein (e.g., gene construct as shown in FIGS. 2, 3, and 5) . In some embodiments, a non-human mammal expressing human or humanized GCGR is provided. In some embodiments, the tissue-specific expression of human or humanized GCGR protein is provided.

In some embodiments, the expression of human or humanized GCGR in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance. In some embodiments, the specific inducer is selected from Tet-Off System/Tet-On System, or Tamoxifen System.

Non-human mammals can be any non-human animal known in the art and which can be used in the methods as described herein. Preferred non-human mammals are mammals, (e.g., rodents) . In some embodiments, the non-human mammal is a mouse.

Genetic, molecular and behavioral analyses for the non-human mammals described above can performed. The present disclosure also relates to the progeny produced by the non-human mammal provided by the present disclosure mated with the same or other genotypes.

The present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof. A model based on cell culture can be prepared, for example, by the following methods. Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cells can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques. The integration of genetic constructs containing DNA sequences encoding human GCGR protein can be detected by a variety of methods.

There are many analytical methods that can be used to detect exogenous DNA, including methods at the level of nucleic acid (including the mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies) . In addition, the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art. Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human or humanized GCGR protein.

Vectors

The present disclosure relates to a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of a region to be altered (5' arm) , which is selected from the GCGR gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3' end of the region to be altered (3' arm) , which is selected from the GCGR gene genomic DNAs in the length of 100 to 10,000 nucleotides.

In some embodiments, a) the DNA fragment homologous to the 5' end of a conversion region to be altered (5' arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000077.7; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000077.7.

In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be more than about 3 kb, about 3.5 kb, about 4 kb, about 4.5 kb, about 5 kb, about 5.5 kb, or about 6 kb.

In some embodiments, the region to be altered is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14 of GCGR gene (e.g., exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and exon 14 of mouse GCGR gene) .

The targeting vector can further include one or more selectable markers, e.g., positive or negative selectable markers. In some embodiments, the positive selectable marker is a Neo gene or Neo cassette. In some embodiments, the negative selectable marker is a DTA gene.

In some embodiments, the sequence of the 5' arm is shown in SEQ ID NO: 3; and the sequence of the 3' arm is shown in SEQ ID NO: 4.

In some embodiments, the sequence is derived from human (e.g., 81809019-81813689 of NC_000017.11) . For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human GCGR gene, preferably exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14 of the human GCGR gene. In some embodiments, the nucleotide sequence of the humanized GCGR encodes the entire or the part of human GCGR protein with the NCBI accession number NP_000151.1 (SEQ ID NO: 2) .

The disclosure also relates to a cell comprising the targeting vectors as described above.

In addition, the present disclosure further relates to a non-human mammalian cell, having any one of the foregoing targeting vectors, and one or more in vitro transcripts of the construct as described herein. In some embodiments, the cell includes Cas9 mRNA or an in vitro transcript thereof.

In some embodiments, the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.

In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic stem cell.

Methods of making genetically modified animals

Genetically modified animals can be made by several techniques that are known in the art, including, e.g., nonhomologous end-joining (NHEJ) , homologous recombination (HR) , zinc finger nucleases (ZFNs) , transcription activator-like effector-based nucleases (TALEN) , and the clustered regularly interspaced short palindromic repeats (CRISPR) -Cas system. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these genome editing techniques are known in the art, and is described, e.g., in Yin et al., "Delivery technologies for genome editing, " Nature Reviews Drug Discovery 16.6 (2017) : 387-399, which is incorporated by reference in its entirety. Many other methods are also provided and can be used in genome editing, e.g., micro-injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.

Thus, in some embodiments, the disclosure provides replacing in at least one cell of the animal, at an endogenous GCGR gene locus, a sequence encoding a region of an endogenous GCGR with a sequence encoding a corresponding region of human or chimeric GCGR. In some embodiments, the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.

FIG. 3 shows a humanization strategy for a mouse GCGR locus. In FIG. 3, the targeting strategy involves a vector comprising the 5' end homologous arm, human GCGR gene fragment, 3' homologous arm. The process can involve replacing endogenous GCGR sequence with human sequence by homologous recombination. In some embodiments, the cleavage at the upstream and the downstream of the target site (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double strands break, and the homologous recombination is used to replace endogenous GCGR sequence with human GCGR sequence.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous GCGR locus (or site) , a nucleic acid encoding a sequence encoding a region of endogenous GCGR with a sequence encoding a corresponding region of human GCGR. The sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14 of a human GCGR gene. In some embodiments, the sequence includes a region of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and a region of exon 14 of a human GCGR gene (e.g., nucleic acids 278-1711 of NM_000160.5) . In some embodiments, the endogenous GCGR locus is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14 of mouse GCGR. In some embodiments, the sequence includes a region of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and a region of exon 14 of mouse GCGR gene (e.g., nucleic acids 185-1642 of NM_008101.2) .

In some embodiments, the methods of modifying a GCGR locus of a mouse to express a chimeric human/mouse GCGR peptide can include the steps of replacing at the endogenous mouse GCGR locus a nucleotide sequence encoding a mouse GCGR with a nucleotide sequence encoding a human GCGR, thereby generating a sequence encoding a chimeric human/mouse GCGR.

In some embodiments, the nucleotide sequences as described herein do not overlap with each other (e.g., the first nucleotide sequence, the second nucleotide sequence, and/or the third nucleotide sequence do not overlap) . In some embodiments, the amino acid sequences as described herein do not overlap with each other.

The present disclosure further provides a method for establishing a GCGR gene humanized animal model, involving the following steps:

(a) providing the cell (e.g. a fertilized egg cell) based on the methods described herein;

(b) culturing the cell in a liquid culture medium;

(c) transplanting the cultured cell to the fallopian tube or uterus of the recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;

(d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c) .

In some embodiments, the non-human mammal in the foregoing method is a mouse (e.g., a C57BL/6 mouse) .

In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or false pregnancy) .

In some embodiments, the fertilized eggs for the methods described above are C57BL/6 fertilized eggs. Other fertilized eggs that can also be used in the methods as described herein include, but are not limited to, FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs and DBA/2 fertilized eggs.

Fertilized eggs can come from any non-human animal, e.g., any non-human animal as described herein. In some embodiments, the fertilized egg cells are derived from rodents. The genetic construct can be introduced into a fertilized egg by microinjection of DNA. For example, by way of culturing a fertilized egg after microinjection, a cultured fertilized egg can be transferred to a false pregnant non-human animal, which then gives birth of a non-human mammal, so as to generate the non-human mammal mentioned in the methods described above.

Methods of using genetically modified animals

Replacement of non-human genes in a non-human animal with homologous or orthologous human genes or human sequences, at the endogenous non-human locus and under control of endogenous promoters and/or regulatory elements, can result in a non-human animal with qualities and characteristics that may be substantially different from a typical knockout-plus-transgene animal. In the typical knockout-plus-transgene animal, an endogenous locus is removed or damaged and a fully human transgene is inserted into the animal's genome and presumably integrates at random into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of the human gene and/or protein assay and/or functional assay. Inclusion in the human transgene of upstream and/or downstream human sequences are apparently presumed to be sufficient to provide suitable support for expression and/or regulation of the transgene.

In some cases, the transgene with human regulatory elements expresses in a manner that is unphysiological or otherwise unsatisfactory, and can be actually detrimental to the animal. The disclosure demonstrates that a replacement with human sequence at an endogenous locus under control of endogenous regulatory elements provides a physiologically appropriate expression pattern and level that results in a useful humanized animal whose physiology with respect to the replaced gene are meaningful and appropriate in the context of the humanized animal's physiology.

Genetically modified animals that express human or humanized GCGR protein, e.g., in a physiologically appropriate manner, provide a variety of uses that include, but are not limited to, developing therapeutics for human diseases and disorders, and assessing the toxicity and/or the efficacy of these human therapeutics in the animal models.

In various aspects, genetically modified animals are provided that express human or humanized GCGR, which are useful for testing agents that can decrease or block the interaction between GCGR and GCGR ligands (e.g., glucagon) or the interaction between GCGR and anti-human GCGR antibodies, testing whether an agent can increase or decrease blood glucose level, and/or determining whether an agent is an GCGR agonist or antagonist. The genetically modified animals can be, e.g., an animal model of a human disease, e.g., the disease is induced genetically (a knock-in or knockout) . In some embodiments, the anti-GCGR antibody blocks or inhibits the GCGR-related signaling pathway (e.g., glycogen hydrolysis) .

In some embodiments, the genetically-modified animals can be used for determining effectiveness of a GCGR modulator (e.g., an anti-GCGR antibody) for treating metabolic disorders (e.g., diabetes) and/or reducing blood glucose level. The methods involve administering the anti-GCGR antibody (e.g., an anti-human GCGR antibody) to the animal as described herein; optionally administering a pre-determined amount of glucose to the animal; and determining blood glucose level of the animal. The method can further include comparing the blood glucose level of the animal with blood glucose level of a reference animal. In some embodiments, the reference animal is not administered with the anti-GCGR antibody, or administered with an isotype control (e.g., human IgG4) . In some embodiments, the reference animal is not genetically modified (e.g., a wild-type animal) . In some embodiments, the reference animal includes an endogenous GCGR gene locus. In some embodiments, the reference animal has the same background as the animal used for determining effectiveness of an anti-GCGR antibody for the treatment of diabetes. In some embodiments, the diabetes is type I or type II diabetes.

In some embodiments, the genetically-modified animals can be used for determining effectiveness of a GCGR modulator (e.g., an anti-GCGR antibody) , optionally in combination with one or more additional therapeutic agents (e.g. a second therapeutic agent) , for lowering blood glucose or reducing at least one symptom in a patient suffering from a disease or condition characterized by high blood glucose levels, such as diabetes mellitus.

The GCGR modulator described herein may function to block the interaction between glucagon and GCGR, thereby inhibiting the glucose elevating effects of glucagon. The use of glucagon receptor antagonists, such as the antibodies described herein, may be an effective means of achieving normal levels of glucose, thereby ameliorating, or preventing one or more symptoms of, or long term complications associated with, for example, diabetes. The use of GCGR antagonists, such as the antibodies described herein, may also be an effective means of achieving normal levels of glucose in non-diabetic patients, who experience hyperglycemia as a result of conditions or disorders not related to diabetes, such as perioperative hyperglycemia (hyperglycemia observed in patients just prior to surgery, or after surgery) . In certain embodiments, methods of lowering blood glucose levels or ketone levels in diabetic ketoacidosis are envisioned using the antibodies described herein. In certain embodiments, methods of treating patients to achieve a reduction in body weight, or to prevent weight gain, or to maintain a normal body weight, are also envisioned using the antibodies described herein.

In some embodiments, the genetically-modified animals described herein are useful for determining effectiveness of a GCGR modulator (e.g., an anti-GCGR antibody) for ameliorating conditions such as, for example, impaired glucose tolerance, obesity, or for treating diabetic conditions, or for preventing or reducing the severity of any one or more of the long-term complications associated with diabetes, such as nephropathy, neuropathy, retinopathy, cataracts, stroke, atherosclerosis, impaired wound healing and other complications associated with diabetes, known to those skilled in the art.

Other conditions or disorders treatable by the GCGR modulator include diabetic ketoacidosis, hyperglycemia (including perioperative hyperglycemia, hyperglycemia in the intensive care unit patient, and hyperosmolar hyperglycemia syndrome) , hyperinsulinemia, the metabolic syndrome, insulin resistance syndrome, impaired fasting glucose, or hyperglycemia associated with hypercholesterolemia, hypertriglyceridemia, hyperlipidemia, and general dyslipidemias. In certain embodiments, the disclosure provides for an isolated antibody or antigen-binding fragment thereof specific for GCGR, as described herein, for use in lowering blood glucose or ketone levels, or for treating a patient having a disease or condition associated with, or characterized in part by high blood glucose or ketone levels, wherein the condition or disease is selected from diabetes, impaired glucose tolerance, obesity, nephropathy, neuropathy, retinopathy, cataracts, stroke, atherosclerosis, impaired wound healing, diabetic ketoacidosis, hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia, hyperglycemia in the intensive care unit patient, hyperinsulinemia, the metabolic syndrome, insulin resistance syndrome and impaired fasting glucose. In certain embodiments, use of the isolated antibody or antigen-binding fragment is contemplated for preparation of a medicament for lowering blood glucose or ketone levels, or for treating a patient having a disease or condition associated with, or characterized in part by high blood glucose or ketone levels, or for ameliorating at least one symptom of such disease or condition, wherein the condition or disease is selected from any of the above-noted diseases or conditions. The antibodies may also be useful for treating patients with inoperable glucagonoma (pancreatic endocrine tumor with or without necrolytic migratory erythema and hyperglycemia) .

In some embodiments, embodiments, the second therapeutic agent may be an agent that helps to counteract or reduce any possible side effect (s) associated with the anti-GCGR antibody or antigen-binding fragment thereof, if such side effect (s) should occur. For example, in the event that any of the anti-GCGR antibodies increases lipid or cholesterol levels, it may be beneficial to administer a second agent that is effective to lower lipid or cholesterol levels. The second therapeutic agent may be a small molecule drug, a protein/polypeptide, an antibody, a nucleic acid molecule, such as an anti-sense molecule, or a siRNA. The second therapeutic agent may be synthetic or naturally derived. Additional possibilities of the second agent can be found, e.g., in WO2012071372A2, which is incorporated herein by reference in its entirety.

In one embodiment, the GCGR modulators (e.g., anti-GCGR antibodies) described herein may be used in combination with one or more of the following type 2 diabetes treatments: biguanide (metformin) , sulfonylureas (e.g., glyburide, glipizide) , peroxisome pro! iferator-activated receptor (PPAR) gamma agonists (e.g., pioglitazone, rosiglitazone) , and alpha glucosidase inhibitors (e.g., acarbose, voglibose) . Additional treatments include injectable treatments such as

(glucagon-like peptide 1) , and

(pramlintide) .

The present disclosure also provides methods of determining toxicity of a GCGR modulator (e.g., anti-GCGR antibodies) . The methods involve administering the antibody to the animal as described herein. The animal is then evaluated for its weight change, red blood cell count, hematocrit, and/or hemoglobin. In some embodiments, the antibody can decrease the red blood cells (RBC) , hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%. In some embodiments, the animals can have a weight that is at least 5%, 10%, 20%, 30%, or 40%smaller than the weight of the control group (e.g., average weight of the animals that are not treated with the antibody) .

The present disclosure also relates to the use of the animal model generated through the methods as described herein in the development of a product related to an immunization processes of human cells, the manufacturing of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.

In some embodiments, the disclosure provides the use of the animal model generated through the methods as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.

The disclosure also relates to the use of the animal model generated through the methods as described herein in the screening, verifying, evaluating or studying the GCGR gene function, human GCGR antibodies, drugs for human GCGR targeting sites, the drugs or efficacies for human GCGR targeting sites, the drugs for diabetes and hypoglycemic drugs.

In some embodiments, the genetically-modified non-human animals described herein can be used to generated animal models for preparation and screening of drugs for the treatment of metabolic diseases and related conditions, e.g., lowering blood glucose and/or improving blood glucose tolerance. In some embodiments, the metabolic diseases and related conditions refers to diseases caused by metabolic problems, including metabolic disorders and hypermetabolism, diabetes, diabetic ketoacidosis, hyperglycemia-hyperosmotic syndrome, hypoglycemia, gout, protein-energy malnutrition disease, vitamin A deficiency, scurvy, vitamin D deficiency, and osteoporosis. In some embodiments, the non-human animals are particularly suitable for diabetes and obesity researches, including but not limited to, type 1 diabetes, type 2 diabetes, hyperglycemia, impaired fasting glucose, impaired glucose tolerance, obesity, dyslipidemia, diabetic ketoacidosis, hyperglycemia hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia, insulin resistance syndrome and/or metabolic syndrome.

In some embodiments, the GCGR modulator is selected from CAR-T and small-molecular drugs. In some embodiments, the GCGR modulator is an anti-GCGR antibody or antigen-binding fragment thereof.

In some embodiments, the genetically-modified non-human animals can be detected to assess the individual animal's body weight, fat mass, glucose metabolism, activation pathways, neuroprotective activity or metabolic changes, including changes in food consumption or water consumption. Further, levels of insulin, glucagon, RBG, OGTT, IPGTT, TG, TC, HDL-C, LDL-C can be measured.

Genetically modified animal model with two or more human or chimeric genes

The present disclosure further relates to methods for generating genetically modified animal model with two or more human or chimeric genes. The animal can comprise a human or chimeric GCGR gene and a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein can be PD-1, TLR, CD40, 4-1BB, GLP1R, PD-L1, IL4, IL6, B7-H3, TIGIT, or CD28.

The methods of generating genetically modified animal model with two or more human or chimeric genes (e.g., humanized genes) can include the following steps:

(a) using the methods of introducing human GCGR gene or chimeric GCGR gene as described herein to obtain a genetically modified non-human animal;

(b) mating the genetically modified non-human animal with another genetically modified non-human animal, and then screening the progeny to obtain a genetically modified non-human animal with two or more human or chimeric genes.

In some embodiments, in step (b) of the method, the genetically modified animal can be mated with a genetically modified non-human animal with human or chimeric PD-1, TLR, or Leptin. In some embodiments, the GCGR humanization is directly performed on a genetically modified animal having a human or chimeric PD-1, TLR, or Leptin gene.

As these proteins may involve different mechanisms, a combination therapy that targets two or more of these proteins thereof may be a more effective treatment. In fact, many related clinical trials are in progress and have shown a good effect. The genetically modified animal model with two or more human or humanized genes can be used for determining effectiveness of a combination therapy that targets two or more of these proteins, e.g., an anti-GCGR antibody and an additional therapeutic agent for the treatment of diabetes. The methods include administering the anti-GCGR antibody and the additional therapeutic agent to the animal; optionally administering glucose to the animal; and determining blood glucose of the animal after the combined treatment. In some embodiments, the additional therapeutic agent is an antibody or antigen-binding fragment thereof that specifically binds to PD-1, TLR, Leptin, CD40, 4-1BB, GLP1R, PD-L1, IL4, IL6, B7-H3, TIGIT, or CD28.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

The following materials were used in the following examples.

C57BL/6 mice and Flp transgenic mice were purchased from the China Food and Drugs Research Institute National Rodent Experimental Animal Center.

Human IgG4 was purchased from Crown Bioscience (Cat#: C0004-3) .

GCGR Antibody (human, mouse) (mGCGR) was purchased from MyBioSource (Cat#: MBS7048237) .

GCGR Rabbit pAb (human) (hGCGR) was purchased from ABclonal (Cat#: A10617) .

Brilliant Violet 510 ^TM anti-mouse CD45 Antibody was purchased from BioLegend (Cat#: No. 103138) .

PE/Cy ^TM 7 Mouse anti-mouse NK1.1 antibody was purchased from BioLegend (Cat#: 552878) .

FITC anti-Mouse CD19 antibody was purchased from BioLegend (Cat#: 115506) .

PerCP/Cy5.5 anti-mouse TCRβ chain antibody was purchased from BioLegend (Cat#: 109228) .

PE anti-mouse CD8a Antibody was purchased from BioLegend (Cat#: 100708) .

Brilliant Violet 421 ^TM anti-mouse CD4 Antibody was purchased from BioLegend (Cat#: 100438) .

PE anti-mouse/human CD 11 b antibody was purchased from BioLegend (Cat#: 101208) .

PerCP anti-mouse Ly-6G/Ly-6C Antibody was purchased from BioLegend (Cat#: 108426) .

Brilliant Violet 605 ^TM anti-mouse CD 11 c antibody was purchased from BioLegend (Cat#: 117334) .

APC anti-mouse/rat Foxp3 antibody was purchased from eBioscience (Cat#: 17-5773-82) .

FITC anti-mouse F4/80 Antibody was purchased from BioLegend (Cat#: 123108) .

Ultra-Sensitive Mouse Insulin ELISA Kit was purchased from Crystal Chem (Cat#: 90080) .

Mouse Glucagon ELISA Kit was purchased from Crystal Chem (Cat#: 81518) .

Mouse GLP-1 ELISA Kit was purchased from Crystal Chem (Cat#: 81508) .

EXAMPLE 1: Preparation of humanized mice with GCGR gene

In this example, a non-human animal (e.g., a mouse) was modified to include a nucleotide sequence encoding human GCGR proteins, and the obtained genetically-modified non-human animal can express human or humanized GCGR protein in vivo. The mouse GCGR gene (NCBI Gene ID: 14527, Primary source: MGI: 99572, UniProt ID: Q61606) is located at 120420011 to 120429815 of chromosome 11 (NC_000077.7) , and the human GCGR gene (NCBI Gene ID: 2642, Primary source: HGNC: 4192, UniProt ID: P47871) is located at 81804078 to 81814008 of chromosome 17 (NC 000017.11) . The mouse GCGR transcript is NM_008101.2, and the corresponding protein sequence NP_032127.2 is set forth in SEQ ID NO: 1. The human GCGR transcript is NM_000160.5, and the corresponding protein sequence NP_000151.1 is set forth in SEQ ID NO: 2. Mouse and human GCGR gene loci are shown in FIG. 1.

All or part of nucleotide sequences encoding human GCGR protein can be introduced into the mouse endogenous GCGR locus, so that the mouse expresses human or humanized GCGR protein. Specifically, mouse cells can be modified by various gene-editing techniques to replace specific mouse GCGR gene sequences with human GCGR gene sequences (e.g., genomic DNA sequence, cDNA sequence or CDS sequence) at the endogenous mouse GCGR locus. For example, a sequence starting from the start codon ATG to the stop codon TGA of the mouse GCGR gene can be replaced with the corresponding human DNA sequence, to obtain a humanized GCGR gene locus as shown in FIG. 2, thereby humanizing mouse GCGR gene.

As shown in the schematic diagram of the targeting strategy in FIG. 3, the targeting vector contains homologous arm sequences upstream and downstream of the mouse GCGR gene, and an “A Fragment” containing DNA sequences of human GCGR gene. Specifically, sequence of the upstream homologous arm (5′ homologous arm, SEQ ID NO: 3) is identical to nucleotide sequence of 120420601-120425582 of NCBI accession number NC_000077.7, and sequence of the downstream homologous arm (3′ homologous arm, SEQ ID NO: 4) is identical to nucleotide sequence of 120430149-120434935 of NCBI accession number NC_000077.7. The A Fragment contains a human genomic DNA sequence from GCGR genes (SEQ ID NO: 5) , which is identical to nucleotide sequence of 81809019-81813689 of NCBI accession number NC_000017.11.

The targeting vector also includes an antibiotic resistance gene for positive clone screening (neomycin phosphotransferase gene, or Neo) , and two Frt recombination sites flanking the antibiotic resistance gene, that formed a Neo cassette (within the A Fragment) . The connection between the 5' end of the Neo cassette and the mouse sequence was designed as: 5'-TGTGGCCAACCTCTAGGGTGGTCCCTTTGGCTGTTGCTGTGATGCCTGCCTAGTGGA GTGTGGA GATAGGGT

AAGCTTGATATCGAATTCCGAAGTTCCTATTCTCTAGAAAGTATAGG-3' (SEQ ID NO: 6) , wherein the last “T” in sequence “ GATAGGGT” is the last nucleotide of the mouse sequence, and the first “A” in sequence

is the first nucleotide of the Neo cassette. The connection between the 3' end of the Neo cassette and the mouse sequence was designed as: 5'-TCTAGAAAGTATAGGAACTTCATCAGTCAGGTACATAATTAGGTG GATCC

GGGGAGACTCAACCAGCTACCTCTGTTCCAGTTGCTGGTAGGAAACCTGGGGGAGG-3' (SEQ ID NO: 7) , wherein the last “C” in sequence “GATCC” is the last nucleotide of the Neo cassette, and the first “G” in sequence

is the first nucleotide of the mouse sequence. In addition, a coding gene with a negative selectable marker (a gene encoding diphtheria toxin A subunit (DTA) ) was also constructed downstream of the 3′ homologous arm of the targeting vector. The connection between the mouse and human sequences was designed as: 5'-GATGTGGGGCGTGGCTACCCAGAGGC

CCCTGCCAGCCACAGCGACCCCT-3' (SEQ ID NO: 32) , wherein the “C” in sequence “GAGGC” is the last nucleotide of the mouse sequence, and the “A” in sequence

is the first nucleotide of the human sequence. The connection between the human and mouse sequences was designed as: 5'-TAGATTGGCTGAGAGCCC CTTCTGA

CTGGAGCCTAGCCAGGCTGCGTTCA GAAAGGGCC-3' (SEQ ID NO: 33) , wherein the “A” in sequence “CTTCTGA” is the last nucleotide of the human sequence, and the first “A” in sequence

is the first nucleotide of the mouse sequence. The mRNA sequence of the engineered mouse GCGR after humanization and its encoded protein sequence are shown in SEQ ID NO: 8 and SEQ ID NO: 2, respectively.

The targeting vector was constructed, e.g., by restriction enzyme digestion and ligation. The constructed targeting vector sequences were preliminarily confirmed by restriction enzyme digestion, and then verified by sequencing. Embryonic stem cells of C57BL/6 mice were transfected with the correct targeting vector by electroporation. The positive selectable marker genes were used to screen the cells, and the integration of exogenous genes was confirmed by PCR (PCR primers are shown in Table 3) and Southern Blot. Specifically, after mouse embryonic stem cells were transfected with targeting vectors, the clones identified as positive by PCR were then verified by Southern Blot (cell DNA was digested with BglII and HindIII, respectively, and hybridized with three probes) to screen out correct positive clone cells. The restriction enzymes, probes, and the size of target fragments are shown in Table 4. The Southern Blot detection results are shown in FIG. 4. The results indicate that the 4 PCR-positive embryonic stem cells (ES-01, ES-02, ES-03, and ES-04) were all positive clones without random insertions.

Table 3. PCR primer sequences and target fragment sizes

Table 4. Enzymes and probes used in Southem Blot

Restriction enzyme	Probe	wild-type fragment size	Recombinant fragment size
Bglll
	5′Probe	12.8kb	16.1kb
Hindlll
	3′Probe	17.1kb	8.3 kb
Hindlll	Neo Probe	--	8.3kb

The following primers were used for probe synthesis in Southern Blot assays:

5'Probe:

5'Probe-F: 5'-CAGCTCCCTGTCAGGATTTCTGGTG-3' (SEQ ID NO: 13) ,

5'Probe-R: 5'-ATGTCTGTGTCCTCTCCTCCACCTC-3' (SEQ ID NO: 14) ;

3'Probe:

3'Probe-F: 5'-CTTCCCGCAGAGGAAGGAACAAACT-3' (SEQ ID NO: 15) ,

3'Probe-R: 5'-CGCTGAGCTTCCGGATAGATGGTTT-3' (SEQ ID NO: 16) ;

Neo Probe:

Neo Probe-F: 5'-GGATCGGCCATTGAACAAGA-3' (SEQ ID NO: 17) ,

Neo Probe-R: 5'-CAGAAGAACTCGTCAAGAAG-3' (SEQ ID NO: 18) .

The positive clones that had been screened (black mice) were introduced into isolated blastocysts (white mice) , and the resulted chimeric blastocysts were transferred to a culture medium for short-term culture and then transplanted to the fallopian tubes of the recipient mother (white mice) to produce the F0 chimeric mice (black and white) . The F2 generation homozygous mice were obtained by backcrossing the F0 generation chimeric mice with wild-type mice to obtain the F1 generation mice, and then breeding the F1 generation heterozygous mice with each other. The positive mice were also bred with the Flp transgenic mice to remove the positive selectable marker genes (schematic diagram shown in FIG. 5) , and then the humanized homozygous mice with a humanized GCGR gene were obtained by breeding the heterozygous mice with each other.

The genotype of the humanized heterozygous and homozygous mice can be verified by PCR using primers shown in the table below. Specifically, one wild-type C57BL/6 mouse and one GCGR gene humanized heterozygous mouse (F1 generation) prepared by the methods described herein were selected. Mouse Tail blood was collected for PCR verification using primers shown in the table below, to test whether the wild-type C57BL/6 mouse and the humanized GCGR heterozygous mouse had correct gene sequences. As shown in FIGS. 6A-6D, mice numbered F1-01, F1-02, F1-03, and F1-04 were identified as positive clones, indicating that genetically engineered mice with a humanized GCGR gene can be constructed using the methods described herein.

Table 5. PCR primer sequences and target fragment sizes

Transcription of humanized GCGR mRNA in F2 generation GCGR gene humanized mice was detected by RT-PCR. Specifically, one 8-week-old wild-type C57BL/6 mouse and one GCGR gene humanized homozygous mouse prepared by the methods described herein were selected. Liver tissues were collected after euthanasia, and the primer sequences shown in the table below were designed to detect the mRNA transcription level in hepatocytes of the C57BL/6 mouse and GCGR gene humanized homozygous mouse. As shown in FIG. 7, only mouse GCGR mRNA transcription was detected in the hepatocytes of C57BL/6 mice (+/+) , whereas only human GCGR mRNA transcription was detected in the hepatocytes of GCGR gene humanized homozygous mouse (H/H) .

Table 6. PCR primer sequences and target fragment sizes

Expression of GCGR proteins in mice was detected by Western Blot. Specifically, one 9-week-old female wild-type C57BL/6 mouse (+/+) and one GCGR gene humanized homozygous mouse (H/H) prepared by the method described herein were selected. After euthanasia, kidney tissues were collected and detected by Western Blot using an anti-human GCGR antibody (hGCGR) or an anti-mouse GCGR antibody (mGCGR) that can cross-react with both human and mouse GCGR. As shown in FIG. 11, in the wild-type C57BL/6 mouse, only expression of mouse GCGR was detected, whereas expression of human GCGR was not detected. By contrast, expression of both human and mouse GCGR proteins was detected in the GCGR gene humanized homozygous mouse, which is likely due to the cross-reactivity of the anti-mouse GCGR antibody to both human and mouse GCGR proteins.

The immunophenotyPing of leukocyte subtyPes and T cell subtypes in mice was further detected by flow cytometry. Four 7-week-old female wild-type C57BL/6 mice and four GCGR gene humanized homozygous mice prepared by the methods described herein were selected. After euthanasia, spleen, lymph nodes, and peripheral blood were collected and stained using: Brilliant Violet 510 ^TM anti-mouse CD45 Antibody (mCD45; an anti-mouse CD45 antibody) , PerCP anti-mouse Ly-6G/Ly-6C Antibody (mGr-1; an anti-mouse Gr-1 antibody) , Brilliant Violet 421 ^TM anti-mouse CD4 Antibody (mCD4; an anti-mouse CD4 antibody) , FITC anti-mouse F4/80 Antibody (mF4/80; an anti-mouse F4/80 antibody) , PE anti-mouse CD8a Antibody (mCD8; an anti-mouse CD8 antibody) , PE/Cy ^TM 7 Mouse anti-mouse NK1.1 antibody (mNK1.1; an anti-mouse NK1.1 antibody) , APC anti-mouse/rat Foxp3 antibody (mFoxp3; an anti-mouse Foxp3 antibody) , FITC anti-Mouse CD19 antibody (mCD19; an anti-mouse CD19 antibody) , PerCP/Cy5.5 anti-mouse TCRβ chain antibody (mTCRβ; an anti-mouse Mouse TCR β antibody) , Brilliant Violet 605 ^TM anti-mouse CD11c antibody (mCD11c; an anti-mouse CD11c antibody) , or PE anti-mouse/human CD11b antibody (mCD11b; an anti-mouse/human CD11b antibody) , respectively. The stained cells were subject to flow cytometry detection.

T cells were characterized by mCD45+mTCRβ+; B cells were characterized by mCD45+mCD19+; NK cells were characterized by mCD45+mTCRβ-mNK1.1+; dendritic cells were characterized by mCD45+mTCRβ-mCD11c+; granulocytes were characterized by mCD45+mGr-1 +; monocytes were characterized by mCD45+mGr-1-mCD11b+mF4/80+; macrophages were characterized by mCD45+ mGr-1-mCD11b+mF4/80+; helper T cells (CD4+T cells) were characterized by mCD45+mCD4+; killer T cells (CD8+ T cells) were characterized by mCD45+mCD8+; and regulatory T cells were characterized by mCD45+mCD4+mFoxp3+.

The detection results of spleen, lymph nodes and peripheral blood are shown in FIGS. 12A-12B, FIGS. 13A-13B, and FIGS. 14A-14B, respectively. The results showed that the percentages of leukocyte subtypes in GCGR gene humanized homozygous mice (H/H) , including T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages, were basically the same as those in wild-type C57BL/6 mice (+/+) . The percentages of t cell subtypes, including CD4+ T cells, CD8+ T cells, and regulatory T cells (Treg) , were basically the same as those in wild-type C57BL/6 mice. The results indicate that humanization of GCGR gene in wild-type mice did not affect the overall development, differentiation or distribution of leukocyte subtypes and T cell subtypes in the spleen, lymph nodes and peripheral blood in mice.

EXAMPLE 2. In vivo efficacy verification

Humanized GCGR mice prepared by the method described herein can be used to evaluate the efficacy of modulators (e.g., antibodies) targeting human GCGR. In this example, the humanized GCGR mice prepared in Example 1 were used to evaluate the efficacy of the anti-human GCGR antibody Crotedumab (for the sequence of Crotedumab, see GenomeNet database, ID number: D 11230) . Detailed experiments are discussed as follows.

1. Experimental method

Six C57BL/6 male mice and twelve humanized GCGR male mice (homozygous) were selected. The humanized GCGR mice were randomly grouped and administered with human IgG4 (G2) or Crotedumab (G3) , as shown in the table below. The six C57BL/6 mice were administered with human IgG4 (G1) . The day of grouping was recorded as D0 (day 0) . An experimental scheme is shown in FIG. 8. The random blood glucose (RBG) on D0, D3, D6, D8 and D 11 were measured and the mice were weighed. Euthanasia was performed when a single mouse lost more than 20%of its body weight. The specific treatment, dose level, administration route and frequency are shown in the table below.

Table 7.

On D1 and D8, 10 mg/kg Crotedumab was administered intraperitoneally (i.p. ) to the treatment group mice (G3) , and the same dose level of isotype control (human IgG4) was administered to the control group mice (G1 and G2) . The detection scheme is described as follows. On D3, RBG was measured, followed by fasting treatment. On D4, oral glucose tolerance test (OGTT) was performed. Specifically, after the fasting blood glucose was measured, the mice were intraperitoneally injected with 2 g/kg D-glucose. Blood glucose levels were measured at 15 minutes, 30 minutes, 60 minutes, and 120 minutes after injection. Glucose solution was prepared at 20% (w/v) in saline, and the dose level was 10 ul/g of body weight. On D6, RBG was measured, followed by fasting treatment. On D7, fasting blood glucose was measured. Inner canthus blood was also collected, and centrifuged to obtain serum from supernatant. Glucagon and insulin levels in serum were determined by ELISA. On D11, RBG was measured, and lipid levels from blood serum (obtained by centrifuging blood) were determined. This lipid tests mainly examined the blood biochemical indexes of TG (triglyceride) , TC (total cholesterol) , LDL-C (low density lipoprotein-cholesterol) and HDL-C (high density lipoprotein-cholesterol) in mice.

2. Experimental result

The body weights of the G1-G3 group mice are shown in FIG. 9B. The results showed that the body weights of the humanized GCGR mice in control group G2 and treatment group G3 were not significantly different from that of the wild-type mice (G1) . The results indicate that humanization of GCGR gene and GCGR antibody drug treatment had no effect on the body weight of mice. According to the RBG levels shown in the table below and FIG. 9A, the GCGR gene humanized mice used in group G2 had no significant difference in blood glucose levels as compared with wild-type mice (G1) , indicating that the blood glucose homeostasis of mice after GCGR humanization was not affected. However, after the anti-human GCGR antibody was administered in G3 group mice, the blood glucose levels on D3, D7 and D11 were significantly lower than those of the isotype control group mice (G1 and G2) , indicating that the GCGR humanized mice had a significant response to the GCGR antibody.

Table 8. RBG levels and P value

The results of the OGTT test on D4 are shown in the table below and FIG. 9C. After intraperitoneal injection of 2 g/kg glucose, the blood glucose concentration of the three groups reached the peak in 15 minutes. The increase in blood glucose at each time point after glucose administration in the G3 group mice was significantly lower than that in the G1 and G2 group mice. The area under the glucose tolerance curve (AUC) was also statistically analyzed. As shown in FIG. 9D, the G3 group mice showed a significantly lower glucose AUC than the G1 and G2 group mice. The results indicate that the glucose tolerance of GCGR gene humanized mice was significantly enhanced after GCGR antibody drug treatment.

Table 9. OGTT blood glucose test results on D4

Studies have shown that GCGR antibodies can block the binding of endogenous glucagon to its receptor GCGR after binding to GCGR on the cell membrane surface. As shown in the table below and FIGS. 9E-9F, after the G3 group mice were treated with the anti-human GCGR antibody, the serum glucagon level was significantly higher than that of the G2 group mice, indicating that the anti-human GCGR antibody can block the binding of GCGR to glucagon. The insulin level in the G3 group mice was lower than that in the G2 group mice, which may be caused by the binding of Crotedumab to human GCGR, which inhibited the increase of blood glucose level.

Table 10. Insulin and glucagon test results on D7

Group	Insulin (ng/mL)	Glucagon (pg/mL)
G1	8.3±3.6	ND
G2	7.8±4.1	2.76
G3	3.2±0.7	227.96±182.1

On D11, mouse blood was collected for blood biochemical testing, and the results are shown in the table below and FIGS. 10A-10D. After the G3 group mice were treated with Crotedumab, the serum level of triglyceride (TG) significantly reduced, while the serum levels of total cholesterol (TC) , HDL-C and LDL-C increased.

Table 11. Blood biochemical test results on D11

Group	TG (mmol/L)	TC (mmol/L)	HDL-C (mmol/L)	LDL-C (mmol/L)
G1	2.69±0.45	2.83±0.29	1.69±0.18	0.26±0.05
G2	2.04±0.31	2.7±0.39	1.59±0.27	0.27±0.1
G3	1.5±0.43	3.36±0.31	1.97±0.18	0.41±0.06

The above results showed that after humanization of GCGR, the basic characteristics of mice such as body weight and blood glucose homeostasis did not change significantly. After treatment with the anti-human GCGR antibody, the blood glucose level was significantly reduced and the glucose tolerance was enhanced, indicating that the human GCGR protein in mice can recognize the anti-human GCGR antibody Crotedumab and bind to it, thereby blocking the downstream signaling pathway. After treatment with the anti-human GCGR antibody Crotedumab, the serum level of free glucagon in GCGR gene humanized mice increased significantly, which also demonstrated that the human GCGR in mice can bind to the anti-human GCGR antibody, and block the binding and function of glucagon and GCGR.

EXAMPLE 3. Pharmacodynamic validation of diet-induced obesity model using GCGR gene humanized mice

Forty 7-8-week-old male GCGR gene humanized homozygous mice prepared herein were induced by a high-fat diet for 12 weeks to prepare a diet-induced obesity (DIO) model. From the 13th week, all mice were placed into a control group (G1) and three treatment groups (G2, G3, and G4) according to body weight and RBG. Treatment started on the day of grouping (day 0) . The control group mice were administered with an isotype control (human IgG4) at a dose level of 30 mg/kg. Anti-human GCGR antibody Ab1 (obtained by immunizing mice using methods described in Janeway's Immunobiology (9th Edition) ) was administered to mice in treatment groups G2, G3, and G4 at dose levels of 3, 10, and 30 mg/kg, respectively. The antibodies were subcutaneously injected once a week, for a total of four injections. Non-fasting blood glucose levels on

days

3, 7, 10, 14, 18, 21, and 28 after administration were measured and body weight was recorded. Fasting treatment was performed in the afternoon of day 3. The fasting blood glucose level was measured on day 4, and then the OGTT test was performed. Specifically, intraperitoneal glucose tolerance test (IPGTT) was performed as follows. Glucose solution (100 mg/ml) was injected intraperitoneally at a dose level of 10 ul/g, and the blood glucose level was measured at 15 minutes, 30 minutes, 60 minutes and 120 minutes after injection. On day 14, inner canthus blood was also collected to determine the serum insulin, glucagon and glucagon-like peptide-1 (GLP-1) levels by ELISA. On day 28, blood was collected to detect triglyceride (TG) , total cholesterol (TC) , high-density lipoprotein cholesterol (HDL-C) , low-density lipoprotein cholesterol (LDL-C) , alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. After euthanizing the mice, pancreas tissues were isolated for formalin fixation, and immuno-histochemical staining was performed to detect insulin and glucagon.

FIG. 15A shows the body weight changes of the mice in each group; FIG. 15B shows the RBG levels of the mice in each group. The results showed that the body weight and RBG value of the treatment group mice (G2, G3, and G4) decreased as compared to those of the control group mice (G1) . The results indicate that the anti-human GCGR antibody Ab1 can reduce body weight and RBG level in DIO mice.

FIGS. 16A-16B show the blood glucose-time curve and the area under the curve in the glucose tolerance test, respectively. The table below shows the detailed detection results.

Table 12. IPGTT blood glucose test results

The results indicate that anti-human GCGR antibody can facilitate to improve the regulation ability of blood glucose in DIO mice and reduce blood sugar fluctuation.

The levels of insulin, glucagon and GLP-1 in serum were determined by ELISA. The biomarkers and corresponding kits used were as follows: mouse insulin (Ultra-Sensitive Mouse Insulin ELISA Kit) , mouse glucagon (Mouse Glucagon ELISA Kit) , and mouse GLP-1 (Mouse GLP-1 ELISA Kit) . Serum level changes of insulin, glucagon and GLP-1 are shown in FIG. 16C, FIG. 16D, and FIG. 16E, respectively. The table below shows the specific detection results.

Table 13. Insulin, glucagon, and GLP-1 test results

The data show that the anti-human GCGR antibody can reduce blood glucose levels in mice in a dose-dependent manner, that is, the higher the dose level, the better the hypoglycemic effect was. Moreover, after treatment with the anti-human GCGR antibody, the serum level of free glucagon of GCGR gene humanized mice increased as compared to that of control group mice, demonstrating that the human GCGR protein in mice can bind to the anti-human GCGR antibody Ab1 and block the binding of glucagon to GCGR. In addition, the anti-human GCGR antibody also facilitated reduction of blood lipid levels in DIO mice.

FIGS. 17A-17F and the table below show the detection results of TG, TC, HDL-C, LDL-C, ALT and AST.

Table 14. TG, TC, HDL-C, LDL-C, ALT and AST test results

The results showed that the serum TG, ALT and AST levels of the G2, G3 and G4 group mice were similar to those of the G1 group mice. Compared with control group mice (G1) , HDL- C and LDL-C levels in high-dose treatment group mice (G4) increased significantly. Level of TC also increased with the increase of antibody dose level.

FIGS. 18A-18B show stained sections at different magnifications (100× and 200×) for glucagon and insulin in the pancreas of G1 and G4 group mice, respectively. FIGS. 19A-19C show islet alpha cell area, islet beta cell area, and the count of islet number per pancreas area, respectively. As shown in FIG. 19A, the increase in alpha cell area was dose-dependent. However, the anti-human GCGR antibody had no effect on beta cell area or the islet number per pancreatic area in GCGR gene humanized mice.

The above experimental results indicate that the GCGR gene humanized mice prepared herein can be used as a model for evaluating the efficacy ofhypoglycemic drugs.

EXAMPLE 4. Generation of double-or multi-gene humanized mice

The GCGR gene humanized mice generated using the methods described herein can also be used to generate double-or multi-gene humanized mouse models. For example, in Example 1, the embryonic stem (ES) cells for blastocyst microinjection can be selected from mice comprising other genetic modifications such as modified (e.g., human or humanized) PD-1, TLR, and/or Leptin genes. Alternatively, embryonic stem cells from humanized GCGR mice described herein can be isolated, and gene recombination targeting technology can be used to obtain double-gene or multi-gene-modified mouse models of GCGR and other gene modifications. In addition, it is also possible to breed the homozygous or heterozygous GCGR gene humanized mice obtained by the methods described herein with other genetically modified homozygous or heterozygous mice, and the offspring can be screened. According to Mendel's law, it is possible to generate double-gene or multi-gene modified heterozygous mice comprising modified (e.g., human or humanized) GCGR gene and other genetic modifications. Then the heterozygous mice can be bred with each other to obtain homozygous double-gene or multi-gene modified mice. These double-gene or multi-gene modified mice can be used for in vivo validation of gene regulators targeting human GCGR and other genes.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric GCGR (glucagon receptor) .
The animal of claim 1, wherein the sequence encoding the human or chimeric GCGR is operably linked to an endogenous regulatory element at the endogenous GCGR gene locus in the at least one chromosome.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric GCGR comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human GCGR (NP_000151.1 (SEQ ID NO: 2) ) .
The animal of any one of claims 1-3, wherein the animal is a mammal, e.g., a monkey, a rodent, a mouse, or a rat.
The animal of any one of claims 1-4, wherein the animal is a mouse.
The animal of any one of claims 1-5, wherein the animal does not express endogenous GCGR or expresses a decreased level of endogenous GCGR.
The animal of any one of claims 1-6, wherein the animal has one or more cells expressing human or chimeric GCGR.
The animal of claim 7, wherein the expressed human or chimeric GCGR can interact with human glucagon, thereby promoting glycogen hydrolysis and/or gluconeogenesis.
The animal of claim 7, wherein the expressed human or chimeric GCGR can interact with endogenous glucagon, thereby promoting glycogen hydrolysis and/or gluconeogenesis.
A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous GCGR with a sequence encoding a corresponding region of human GCGR at an endogenous GCGR gene locus.
The animal of claim 10, wherein the sequence encoding the corresponding region of human GCGR is operably linked to an endogenous regulatory element at the endogenous GCGR locus, and one or more cells of the animal expresses a human or chimeric GCGR.
The animal of claim 10 or 11, wherein the sequence encoding the corresponding region of human GCGR is immediately after endogenous 5’-UTR.
The animal of any one of claims 10-12, wherein the sequence encoding a region of endogenous GCGR comprises the full-length coding sequence of endogenous GCGR (e.g., a nucleic acid sequence encoding amino acids 1-485 of SEQ ID NO: 1) .
The animal of any one of claims 10-12, wherein the sequence encoding a region of endogenous GCGR comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14, or a part thereof, of the endogenous GCGR gene.
The animal of any one of claims 10-14, wherein the replaced sequence starts with the start codon and ends with the stop codon of the endogenous mouse GCGR gene.
The animal of any one of claims 10-15, wherein the animal is heterozygous with respect to the replacement at the endogenous GCGR gene locus.
The animal of any one of claims 10-15, wherein the animal is homozygous with respect to the replacement at the endogenous GCGR gene locus.
A method for making a genetically-modified, non-human animal, comprising:

replacing in at least one cell of the animal, at an endogenous GCGR gene locus, a sequence encoding a region of endogenous GCGR with a sequence encoding a corresponding region of human GCGR.
The method of claim 18, wherein the sequence encoding the corresponding region of human GCGR comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14, or a part thereof, of a human GCGR gene.
The method of claim 18 or 19, wherein the sequence encoding the corresponding region of human GCGR comprises a portion of exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and a portion of exon 14, of a human GCGR gene.
The method of any one of claims 18-20, wherein the sequence encoding the corresponding region of human GCGR encodes amino acids 1-477 of SEQ ID NO: 2.
The method of any one of claims 18-21, wherein the sequence encoding a region of endogenous GCGR comprises exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, and/or exon 14, or a part thereof, of the endogenous GCGR gene.
The method of any one of claims 18-22, wherein the animal is a mouse, and the sequence encoding a region of endogenous GCGR starts within exon 2 and ends within exon 14 of the endogenous mouse GCGR gene.
A method of making a genetically-modified animal cell that expresses a human or chimeric GCGR, the method comprising:

replacing at an endogenous GCGR gene locus, a nucleotide sequence encoding a region of endogenous GCGR with a nucleotide sequence encoding a corresponding region of human GCGR, thereby generating a genetically-modified animal cell that includes a nucleotide sequence that encodes the human or chimeric GCGR, wherein the animal cell expresses the human or chimeric GCGR.
The method of claim 24, wherein the animal is a mouse.
The method of claim 24 or 25, wherein the nucleotide sequence encoding the human or chimeric GCGR is operably linked to an endogenous GCGR regulatory region, e.g., promoter.
The animal of any one of claims 1-17, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.
The animal of claim 27, wherein the additional human or chimeric protein is programmed cell death protein 1 (PD-1) , Toll-like receptor (TLR) , CD40, tumor necrosis factor receptor superfamily member 9 (4-1BB) , glucagon-like peptide-1 receptor (GLP 1R) , programmed cell death ligand 1 (PD-L1) , IL4, IL6, B7 Homolog 3 (B7-H3) , T-Cell immunoreceptor with Ig and ITIM Domains (TIGIT) , or CD28.
The method of any one of claims 18-26, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein.
The method of claim 29, wherein the additional human or chimeric protein is PD-1, TLR, CD40, 4-1BB, GLP1R, PD-L1, IL4, IL6, B7-H3, TIGIT, or CD28.
A method of determining effectiveness of a GCGR modulator for treating a metabolic disorder (e.g., diabetes) , comprising:

a) administering the GCGR modulator to an animal of any one of claims 1-17;

b) optionally, administering glucose to the animal; and

c) determining blood glucose level of the animal.
A method of determining effectiveness of a GCGR modulator for reducing blood glucose level, comprising:

a) administering the GCGR modulator to an animal of any one of claims 1-17;

b) optionally, administering glucose to the animal; and

c) determining blood glucose level of the animal.
The method of claim 31 or 32, further comprising: comparing the blood glucose level of the animal with blood glucose level of a reference animal, wherein the reference animal is not administered with the GCGR modulator.
A method of determining effectiveness of a GCGR modulator for increasing glucagon (e.g., free glucagon) level, increasing GLP-1, and/or decreasing insulin level, comprising:

a) administering the GCGR modulator to an animal of any one of claims 1-17; and

b) determining glucagon (e.g., free glucagon) level, GLP-1 level, and/or insulin level in the serum of the animal.
The method of claim 34, further comprising: comparing the glucagon (e.g., free glucagon) level and/or insulin level in the serum of the animal with glucagon (e.g., free glucagon) level and/or insulin level in the serum of a reference animal, wherein the reference animal is not administered with the GCGR modulator.
The method of any one of claims 31-35, wherein the GCGR modulator is an anti-GCGR antibody or antigen-binding fragment thereof.
The method of claim 36, wherein the anti-GCGR antibody or antigen-binding fragment thereof is an anti-human GCGR antibody.
The method of any one of claims 31-35, wherein the GCGR modulator is a drug (e.g., a small-molecule drug) targeting GCGR.
The method of any one of claim 31-38, wherein the animal is induced to make a diet-induced obesity (DIO) model.
A protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following:

(a) an amino acid sequence set forth in SEQ ID NO: 1 or 2;

(b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 1 or 2;

(c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1 or 2;

(d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 1 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

(e) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 1 or 2.
A nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following:

(a) a sequence that encodes the protein of claim 40;

(b) SEQ ID NO: 3, 4, 5, 6, 7, 8, 32, or 33;

(c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 32, or 33;

(d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 32, or 33; and

(e) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 32, or 33.