Non-human animals comprising a genetically modified endogenous igha locus and methods of making the same

Abstract

The present disclosure provides a non-human animal that displays clinical signs of a disease associated with IgA deposition (e.g., IgA nephropathy) and comprises in its genome a genetically modified endogenous Igha locus comprising a heterologous gene encoding a human IgA (e.g., IgAl) heavy chain constant region or fragment thereof. The present disclosure also provides a method of making the non-human animal or a use of the non-human animal.

Description

TECHNICAL FIELD

[0001] The present invention relates generally to transgenic non-human animals that can be used to study diseases associated with IgA deposition. More specifically, the present invention relates to transgenic non-human animals comprising a genetically modified endogenous IgA locus, as well as methods of making the transgenic non-human animals or uses of the transgenic non-human animals. BACKGROUND

[0002] IgA is the main pathogenic antibody isotype in IgA nephropathy (IgAN) and comes in three forms, monotypic IgA, secretory IgA, and IgA-containing complexes. Serum IgA concentrations are elevated in IgAN subjects compared to healthy volunteers (Wang M, Lv J, Zhang X, et al. Kidney Int Rep 2020; 5: 165-172). IgAN is characterized by the presence of IgA dominant or codominant immune deposits within the glomerulus and elevated galactose-deficient IgA1 (or “Gd-IgA1”). A ‘multiple hit’ hypothesis has been proposed to explain the pathogenesis of IgAN, with galactose-deficient IgA1 (Gd-IgA1) defined as the first hit. Gd-IgA1 is thought to form nephritogenic high-molecular weight immune complexes to induce kidney damage (Novak J, Barratt J, Julian BA, et al. Semin Nephrol 2018; 38: 461-476). Only great apes and humans have the O-glycosylated IgA1 subclass. Therefore, humanized IgA1 animals can be helpful to study the pathogenesis of IgAN. A fully human IgA1 knock-in (alKI) mouse model was generated and backcrossed with human CD89 Tg mice to develop a model of IgAN-like disease (Duchez S, Amin R, Cogne N, et al. Proc Natl Acad Sci U S A 2010; 107: 3064-3069; Berthelot L, Papista C, Maciel TT, et al. The Journal of experimental medicine 2012; 209: 793-806). However, due to limitations in gene editing technology, human IgA1 replaced the mouse IgM heavy chain in this alKI mouse (Wehbi B, Oblet C, Boyer F, et al. J Am Soc Nephrol 2019; 30: 1238-1249). Human IgA1 and mouse IgA coexist in alKI mice, but IgM is absent, which can interfere with the study of the pathogenesis of IgAN. Some classic models of IgAN usually show limited renal histological changes with or without genetic manipulation.

[0003] Therefore, there is a need for new animal models of IgAN to understand the mechanisms behind this disease and to develop therapeutic approaches. SUMMARY

[0004] In one aspect, the present disclosure provides a method of making a genetically modified non-human animal. Specifically, the method comprises: (i) modifying the genome of a non-human animal to incorporate a nucleotide comprising a heterologous gene encoding a human IgAl heavy chain constant region or fragment thereof in its endogenous Igha locus; and (ii) treating the non-human animal produced in step (i) with a formulation comprising an immunostimulant.

[0005] In some embodiments, step (i) comprises introducing a nucleotide comprising the heterologous gene into a pluripotent cell of the non-human animal, thereby obtaining a non-human animal pluripotent cell comprising the heterologous gene, and using the non-human animal pluripotent cell comprising the heterologous gene to make a non-human animal. In some embodiments, the nucleotide comprising the heterologous gene is introduced into the pluripotent cell of the non-human animal by using a CRISPR / Cas system. In some embodiments, the CRISPR / Cas system comprises a Cas9 protein and a guide RNA targeting a guide RNA target sequence within the endogenous Igha locus. In some embodiments, the pluripotent cell is an embryonic stem (ES) cell. In some embodiments, a segment or all of the endogenous non-human Igha gene at the endogenous non-human Igha locus has been deleted and replaced with the nucleotide comprising the heterologous gene. In some embodiments, the non-human animal does not express an endogenous non-human IgA heavy chain constant region.

[0006] In some embodiments, the nucleotide comprising the heterologous gene is operably linked to an endogenous regulatory element (e.g., a promoter, an enhancer, a silencer, etc.) of the non-human animal.

[0007] In some embodiments, the nucleotide comprising the heterologous gene is a genomic fragment of a human IGHA1 gene. In some embodiments, the heterologous gene comprises both human IGHA1 coding and non-coding sequences. In some embodiments, the non-human animal produces a chimeric IgAl whose heavy chain comprises a non-human animal variable region and a human constant region or fragment thereof. In some embodiments, the human IgAl heavy chain constant region of the non-human animal comprises an O-glycosylated hinge region. In some embodiments, the heterologous gene is a human IGHA1 gene encoding a human IgAl heavy chain constant region or fragment thereof.

[0008] In some embodiments, the formulation comprises an immunostimulant and an antigen. In some embodiments, the formulation consists of an immunostimulant. In some embodiments, the immunostimulant is selected from the group consisting of an aluminum adjuvant, an emulsion adjuvant, a biological adjuvant, and a saponin.

[0009] In some embodiments, the aluminum adjuvant is selected from the group consisting of aluminum hydroxide (Al(OH)3), aluminum phosphate, aluminum hydroxyphosphate, and amorphous aluminum hydroxyphosphate sulfate (AAHS). In some embodiments, the aluminum adjuvant is aluminum hydroxide.

[0010] In some embodiments, the emulsion adjuvant is an oil-in-water emulsion adjuvant or a water-in-oil emulsion adjuvant. In some embodiments, the water-in-oil emulsion adjuvant is selected from the group consisting of complete Freund’s adjuvant (CFA), incomplete Freund’s adjuvant, MF59, and AS03.

[0011] In some embodiments, the biological adjuvant is selected from the group consisting of a lipopolysaccharide or derivative thereof, a toll-like receptor (TLR) agonist (e.g., a TLR4 ligand, a TLR7 ligand, a TLR8 ligand, a TLR9 ligand), an immunostimulatory oligonucleotide (e.g., a DNA or dsRNA comprising CpG), a cytokine (e.g., IL-12), an interferon, an endotoxin, and a lipid (e.g., lipid A or an analog thereof). In some embodiments, the biological adjuvant is a lipopolysaccharide or derivative thereof.

[0012] In some embodiments, the antigen comprises a rhamnose source. In some embodiments, the rhamnose source is a Lactobacillus casei cell wall extract (LCWE).

[0013] In some embodiments, in step (ii), the non-human animal produced in step (i) is treated with a formulation comprising LCWE emulsified with CFA.

[0014] In some embodiments, in step (ii), the formulation is injected intraperitoneally into the non-human animal. In some embodiments, the formulation is injected intraperitoneally into the non-human animal at a continuous low dose. In some embodiments, the formulation is injected intraperitoneally into the non-human animal at a dose of less than 1 pg LCWE and / or less than 5 pg CFA per gram of the non-human animal produced in step (i).

[0015] In some embodiments, the formulation is administered to the non-human animal for at least one month. In some embodiments, the formulation is administered to the non-human animal for at least three months. In some embodiments, the formulation is administered to the non-human animal produced in step (i) at an interval of three times per week for the first two weeks and once per week for the next two weeks for a period of one month. In some embodiments, the formulation is administered to the non-human animal produced in step (i) at an interval of once every two weeks for a period of three months.

[0016] In some embodiments, the non-human animal is a rodent, a mammal, or a non-human primate. In some embodiments, the rodent is a mouse or a rat.

[0017] In another aspect, the present disclosure provides a non-human animal produced by the methods described herein. In some embodiments, the non-human animals provided herein display increased levels of galactose-inadequate IgA proteins. In some embodiments, the non-human animals display increased levels of galactose-inadequate IgA proteins in serum or intestinal mucus.

[0018] In some embodiments, a segment or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha locus has been deleted and replaced with a nucleotide comprising the heterologous gene. In some embodiments, the non-human animal does not express an endogenous non-human IgA heavy chain constant region.

[0019] In some embodiments, the nucleotide comprising the heterologous gene is operably linked to an endogenous regulatory element (e.g., a promoter, an enhancer, a silencer, etc.) of the non-human animal. In some embodiments, the nucleotide comprising the heterologous gene is a genomic fragment of a human IGHA1 gene. In some embodiments, the heterologous gene comprises both human IGHA1 coding sequences and non-coding sequences. In some embodiments, the non-human animal produces a chimeric IgAl whose heavy chain comprises a non-human animal variable region and a human constant region or fragment thereof. In some embodiments, the human IgA heavy chain constant region of the non-human animal comprises an O-glycosylated hinge region. In some embodiments, the heterologous gene is a human IGHA1 gene encoding a human IgAl heavy chain constant region or fragment thereof.

[0020] In some embodiments, the non-human animal is a rodent, a mammal, or a non-human primate. In some embodiments, the rodent is a mouse or a rat.

[0021] In another aspect, the present disclosure provides the use of a non-human animal described herein as an animal model for a disease associated with IgA deposition.

[0022] In some embodiments, the diseases associated with IgA deposition are selected from the group consisting of: IgA nephropathy, herpetic dermatitis, and Henlein-Schönlein purpura. (Also known as IgA vasculitis), Kawasaki disease, Henoch-Schönlein purpura nephritis, IgA vasculitis-related kidney injury, IgA rheumatoid factor-positive rheumatoid arthritis, IgA-mediated anti-GBM disease, or IgA-mediated ANCA-associated vasculitis. In some embodiments, the disease associated with IgA deposition is IgA nephropathy, IgA vasculitis, or Kawasaki disease.

[0023] In another aspect, this disclosure provides a method for evaluating the efficacy of a candidate drug for treating or preventing diseases associated with IgA deposition, the method comprising: providing a non-human animal as described herein; administering the candidate drug to the non-human animal; and evaluating whether the candidate drug inhibits one or more symptoms of the disease compared to a non-human animal control that has not been administered the candidate drug.

[0024] In another aspect, this disclosure provides a method for identifying candidate drugs for treating or preventing diseases associated with IgA deposition, the method comprising: providing a non-human animal as described herein; administering the candidate drug to the non-human animal; and assessing whether the candidate drug inhibits one or more symptoms of the disease compared to a non-human animal control that has not been administered the candidate drug. Attached Figure Description

[0025] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure. This disclosure can be better understood by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0026] Figure 1 shows IGHA1 + / + Mouse development. (A) Illustration of the genomic loci of human IGHA1, mouse Igha, and humanized IGHA1, not to scale. The mouse Igha gene (including the entire coding region) was deleted and replaced by the human IGHA1 gene. (B) Diluted WT, IGHA1 + / - and IGH1 + / + Mouse IgA and human IgA were detected in mouse and human serum by immunoblotting. (CD)WT, IGH1 + / - and IGH1 + / + Immunohistochemical staining of mouse IgA and human IgA1 in Peyer patches and small intestine of mice. (E)BCR seq shows immunoglobulin isotypes in IgA1. + / + It is expressed in the terminal ileum tissue with PP in WT mice. (F)IGHA1+ / + Frequency of J gene usage of hlgAl and mouse IgA in mice. (G) CDR3 sequence length distribution of hlgAl or IgA in three IGHAl + / + and WT mice was uniform. mlgA, mouse IgA; hlgAl, human IgAl; Homo, human.

[0027] Figure 2 Pathogen exposure was shown to promote production of hlgAl in multiple organs. (A) Detection of human IgAl on 1 : 1000 diluted GF, SPF, CV housed IGHAl + / + mouse sera and 1 : 5000 diluted human sera. (B) Serum hlgAl and Gd-IgAl levels of the above three groups at 4 months of age. (C) mRNA expression of IGHAl in SP, ILN, PP, MLN of GF, SPF and CV housed IGHAl + / + mice as determined by RT-qPCR. Relative gene expression was normalized by GAPDH. (D-E) Typical confocal images and mean fluorescence intensity (AU) of hlgAl + cells in SI lamina propria. Scale bar = 50um.

[0028] Figure 3 It was shown that LCWE antigen induced production of hlgAl in IGHAl + / + mice. (A) LCWE plus CFA or PBS was administered intraperitoneally to 2 month old IGHAl + / + mice and observation was extended to 8 months of age. (B) IGHAl + / + mice produced LCWE specific hlgAl and Gd-IgAl. (C-E) IGHAl + / + mice in order. (F) Serum hlgAl was purified and either coomassie protein stained or immunoblotted with anti-hlgAl antibody. (G-H) Serum hlgAl, Gd-IgAl and hlgAl containing complex levels of LCWE and PBS induced IGHAl + / + mice. The average number of GalNAc per HR was equal, while the Gal / GalNAc ratio decreased with increasing antigenic stimulation.

[0029] Figure 4 It was shown that TD and TI pathways were responsible for enhanced mucosa derived Gd-IgAl. (A) IGHAl + / +hlgAl and Gd-IgAl concentrations were higher in the small intestinal mucus of mice. (B) Immunofluorescence staining of hlgAl and Gd-IgAl showed enhanced LCWE-induced IGHAl + / + Gd-IgAl in the lamina propria of the small intestine of mice + Plasma cells. Scale bar = 50 um. (C-F) mRNA expression of BAFF, APRIL, TGFβ and iNOS was increased by LCWE stimulation. Relative gene expression was normalized by GAPDH.

[0030] Figure 5 Gd-IgAl was shown to trigger severe complement activation and pathological lesions in the kidney. (A) Immunostaining of CD31, human IgAl and C3, and Gd-IgAl showed that LCWE-induced IGHAl + / + In mice, human IgAl was deposited with C3 in the kidney mesangial region. Scale bar = 50 um. (B-C) Semi-quantitative grading of hlgAl and C3 deposition in the glomerular mesangial region. (D) Representative micrographs of PAS and Masson staining showed that LCWE-induced IGHAl + / + Mesangial expansion, hypercellularity and tubulointerstitial fibrosis in the kidney of mice. Scale bar = 50 um. Electron micrographs of glomeruli in each group showed that LCWE-induced IGHAl + / + Electron-dense deposits in the mesangial region in mice. Scale bar = 1 um.

[0031] Figure 6 Human IGHAl gene was shown to be expressed in IGHAl + / + In mice to replace mouse IgA. (A) Detection of WT, IGHAl + / - and IGHAl + / + In mice. (B) Mouse IgA and human IgAl genes were measured by RT-qPCR in WT, IGHAl + / - and IGHAl + / + In mice. (C) WT, IGHAl + / - and IGHAl + / + In mice. (D-E) Serum mouse IgA and human IgAl levels were measured by ELISA in WT, IGHAl + / - and IGHAl + / +mRNA expression in the spleen and small intestine of mice. Relative gene expression was normalized relative to GAPDH expression. (FI) Markers derived from WT and IGH were obtained using APC / Cy7-conjugated B220, FITC-conjugated anti-mouse IgA, and AF647-conjugated anti-human IgA1. + / - and IGH1 + / + Cells from the spleen, small intestine, bone marrow, and lymph nodes of mice show IgA. + B220 + B cell counting frequency.

[0032] Figure 7 IGHA1 was demonstrated + / + Mice can produce a variety of immunoglobulins, just like WT mice. (A)IGHA1 + / + The frequency of the V gene usage for hIgA1 in mice and IgA in WT mice. (B) Venn diagram showing IGH1 + / + Cluster analysis of IGH in WT mice. (C)IGHA1 + / + Cluster distribution of the top 100 IGH clusters in WT mice. Percentages represent the proportion of the top 100 IGH clusters in all detected IGH.

[0033] Figure 8 IGHA1 kittens were shown at 4 months old and were raised with GF, SPF, and CV. + / + Mouse serum contains hIgA1 complex ( Figure 8 A), IgM and IgG( Figure 8 The level of B).

[0034] Figure 9 Demonstrates the reduction of IGH1 through LCWE stimulation + / + Mouse weight ( Figure 9 A), and sequentially LCWE and PBS-induced IGH1 + / + Serum IgM and IgG levels in mice Figure 9 B).

[0035] Figure 10 This paper presents a representative example of extractive ion chromatography for the analysis of O-glycopeptides in the hIgA1 hinge region.

[0036] Figure 11 H&E staining of the cardiac vessels and thoracic and abdominal aorta of mice induced by PBS and LCWE was shown. Figure 11 A), and assessed urinary albumin / creatinine ratio, serum creatinine, and serum urea nitrogen in PBS- and LCWE-induced mice. Figure 11 B).

[0037] Figure 12Serum immunoglobulin levels in 5-month old mice injected i.p. with CFA are shown.

[0038] Figure 13 Immunofluorescence of kidney cryosections from 7.5-month old mice injected i.p. with CFA is shown.

[0039] Figure 14 PAS histological staining results of kidneys from 4-month and 5-month old mice injected i.p. with CFA are shown.

[0040] Figure 15 PAS histological staining results of kidneys from 6-month and 7.5-month old mice injected i.p. with CFA are shown.

[0041] Figure 16 Comparison of SCr and ACR levels in 5-month and 7.5-month old mice injected i.p. with CFA is shown.

[0042] Figure 17 Serum immunoglobulin levels in 4-month and 7-month old mice treated with different routes of administration are shown.

[0043] Figure 18 Immunofluorescence of kidney cryosections from 7-month old mice treated with different routes of administration is shown.

[0044] Figure 19 PAS histological staining results of kidneys from 7-month old mice treated with different routes of administration are shown.

[0045] Figure 20 Immunohistochemical staining of mouse IgA and human IgAl in spleen from WT, IGHA1 + / - (i.e., KIWT) and IGHA1 + / + (i.e., KIKI) mice is shown.

[0046] Figure 21 A-D show semi-quantitative scoring of immunofluorescence images and PAS staining micrographs by a specialized pathologist. (A) Human IgAl intensity; (B) Mouse IgA intensity; (C) C3 intensity; (D) Mesangial cell hyperplasia score.

[0047] Figure 22A -B shows serum human IgA levels for each group treated with PBS, saponin, Al(OH)3, LPS, IFA, CFA and LCWE-CFA, respectively.

[0048] Figure 23A-B shows serum human IgA-mouse IgG complex levels for each group treated with PBS, saponin, Al(OH)3, LPS, IFA, CFA, and LCWE-CFA, respectively.

[0049] Figure 24A -B shows urine albumin-creatinine ratio (ACR) for each group treated with PBS, saponin, Al(OH)3, LPS, IFA, CFA, and LCWE-CFA, respectively.

[0050] Figure 25 Deposition of human IgA (hlgA) and C3 in each group treated with PBS, saponin, Al(OH)3, LPS, IFA, CFA, and LCWE-CFA, respectively, and PAS staining, as measured by immunofluorescence and PAS histological staining of the kidneys, are shown.

[0051] Figure 26A -C shows semi-quantitative scoring of immunofluorescence images and PAS staining micrographs by a professional pathologist. (A) Mouse IgM intensity; (B) human IgA intensity; (C) C3 deposition intensity. DETAILED DESCRIPTION

[0052] Particular features (including method steps) of the present application are referred to in the summary of the application above and in the detailed description below and in the claims and in the drawings. It should be understood that the disclosure of the application in this specification includes all possible combinations of particular features recited in the specification. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment or a particular claim of the application, that feature can also be used, mutatis mutandis, in combination with other particular aspects and embodiments of the application and / or in the context of other particular aspects and embodiments of the application and can be used generally in the application.

[0053] Where reference is made in this text to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (unless the context excludes such a possibility) and the method can include one or more other steps before, between, or after the defined steps (unless the context excludes such a possibility).

[0054] All publications, patents, and patent applications cited in this specification are incorporated by reference herein as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference and incorporated herein by reference in their entirety for the disclosure and description they provide, in conjunction with the disclosure provided in the specification itself. Any citation of any publication is for the disclosure and description provided therewith at the time of application and should not be taken as an admission that the prior publication was prior art to the disclosure of this application. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.

[0055] In general, the terms used herein are to be understood in the manner consistent with their meaning in the art. Specific definitions for certain terms are provided herein and below; the meaning of these and other terms will be apparent to one of skill in the art in light of the context in which such terms are used. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0056] I. Definitions

[0057] It should be understood that, as used in this document and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a pluripotent cell” is a reference to one or more pluripotent cells and includes equivalents thereof known to those skilled in the art, and the like.

[0058] As used herein, the term “comprising” and its grammatical equivalents are used herein to mean that other components, ingredients, steps, or the like can optionally be present in addition to those specifically recited. Thus, for example, a method that “comprises” (or, that “comprises the step of”) steps A, B, and C can consist of (i.e., contain only) steps A, B, and C, or can additionally contain one or more other steps.

[0059] The term “or” as used in a claim is used to mean “and / or” unless explicitly indicated to refer to alternatives selected from the group or alternatives which are mutually exclusive of each other. As used herein, “another” can mean at least a second or more.

[0060] As used herein, the term "locus" refers to the specific location of a gene, DNA sequence, polypeptide-encoding sequence, or position on a chromosome of a genome of an organism. A known locus can include known genetic information, such as one or more polymorphic marker sites. For example, an "Igha locus" can refer to the specific location of the Igha gene, Igha DNA sequence, immunoglobulin heavy chain constant region alpha-encoding sequence, or Igha position on a chromosome of a genome of an organism that has been identified as the location at which such sequences reside. An "Igha locus" can include regulatory elements of the Igha gene, including, for example, enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), or combinations thereof.

[0061] As used herein, the term "gene" refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes coding sequences (i.e., sequences that encode a particular product). In some embodiments, a gene can also include non-coding sequences. In some particular embodiments, a gene can include both coding (e.g., exonic) sequences and non-coding (e.g., intronic) sequences. In some embodiments, a gene can include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intronic sequences, e.g., that can control or influence one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.). For the sake of clarity, it should be noted that the term "gene" as used in the present application generally refers to a portion of a nucleic acid that encodes a polypeptide; the term can optionally encompass regulatory sequences, as will be clear to one of ordinary skill in the art in context. This definition is not intended to exclude application of the term "gene" to non-protein-encoding expression units, but rather to clarify that, in most instances, the term as used in the present document refers to a nucleic acid that encodes a polypeptide.

[0062] As used herein, a "coding sequence" or sequence that "encodes" a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo, e.g., when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence are generally determined by the start of the translation initiation codon and the end of the translation termination codon. A coding sequence can include, but is not limited to, cDNA from a virus, a prokaryotic or eukaryotic mRNA, a genomic DNA sequence from a virus or prokaryote, and even synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0063] The term "operably linked" refers to an arrangement of components wherein so described are configured to perform their usual function. In the case of a promoter, a promoter operably linked to a coding sequence will direct expression of the coding sequence. The promoter or other control element need not be contiguous with the coding sequence, just so long as it functions to direct its expression. For example, intervening untranslated but still transcribed sequences can exist between the promoter sequence and the coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0064] As used herein, the term "IgA" refers to one of the antibody (immunoglobulin) isotypes, which is also known as immunoglobulin alpha. As used herein, IgA is also referred to as "IgA antibody" or "IgA type antibody". In serum, IgA is mainly present in the form of monomeric IgA (serotype IgA), and IgAl is the major component. When secreted into the mucosa, IgA is present in the form of polymeric IgA (secretory IgA or SIgA), which is a dimer or higher polymer.

[0065] As used herein, the term "Igha gene" (also known as immunoglobulin heavy chain constant region alpha) encodes the constant region of IgA heavy chain. Specifically, the term "Ighal gene" or "IGHA1 gene" is also known as immunoglobulin heavy chain constant region alpha 1, which encodes the constant region of IgAl heavy chain.

[0066] The human IGHAl gene is located on human chromosome 14, and an exemplary genomic sequence can be found under NCBI Gene ID No. 3493 (assembly GRCh38.p14; location NC_000014.9 (c105708664-105707168), and its sequence is set forth in SEQ ID NO: 19). Another exemplary genomic sequence of the human IGHAl gene is set forth in SEQ ID NO: 33 (IGHAl-202 (ENST00000641837.1 from Ensembl)). In some embodiments, the wild-type human IgAl heavy chain constant region encoded by the human IGHAl gene has been assigned UniProtKB / Swiss-Prot: P01876.2 (the sequence of which is set forth in SEQ ID NO: 20). In some embodiments, the wild-type human IgAl heavy chain constant region encoded by the human IGHAl gene has been assigned UniProtKB: A0A286YEY1 (the sequence of which is set forth in SEQ ID NO: 34). At least five isoforms of the human IgAl heavy chain constant region are known in the art, and have been assigned GenBank: AAF03879.1 (the sequence of which is set forth in SEQ ID NO: 21), GenBank: AAT74070.1 (the sequence of which is set forth in SEQ ID NO: 22), GenBank: AAX09631.2 (the sequence of which is set forth in SEQ ID NO: 23), GenBank: AAX09630.2 (the sequence of which is set forth in SEQ ID NO: 24), and GenBank: ABI26625.1 (the sequence of which is set forth in SEQ ID NO: 25), respectively.

[0067] The mouse Igha gene is located on mouse chromosome 12 and an exemplary genomic sequence can be found under NCBI Gene ID No. 238447 (NC_000078.7 reference GRCm39 C57BL / 6J (c113223856-113219824), and its sequence is shown in SEQ ID NO: 26). The wild-type mouse IgA heavy chain constant region encoded by the mouse Igha gene has been assigned UniProtKB / Swiss-Prot: P01878.1 (the sequence of which is shown in SEQ ID NO: 27). At least five isotypes of the mouse IgA heavy chain constant region are known in the art and have been assigned GenBank: AAL15539.1 (the sequence of which is shown in SEQ ID NO: 28), GenBank: AAL15541.1 (the sequence of which is shown in SEQ ID NO: 29), GenBank: AAL15540.1 (the sequence of which is shown in SEQ ID NO: 30), GenBank: AAL15543.1 (the sequence of which is shown in SEQ ID NO: 31), and GenBank: AAL15542.1 (the sequence of which is shown in SEQ ID NO: 32), respectively.

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[0069]

[0070]

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[0073]

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[0075]

[0076] As used herein, the term “endogenous locus” or “endogenous gene” refers to a genetic locus found in a parent or reference organism prior to the introduction of the alterations, disruptions, deletions, insertions, modifications, substitutions, or replacements described herein. In some embodiments, an endogenous locus includes all or a portion of a sequence found in nature. In some embodiments, an endogenous locus is a wild-type locus. In some embodiments, a reference organism is a wild-type organism. In some embodiments, a reference organism is an engineered organism. In some embodiments, a reference organism is a laboratory-bred organism (whether wild-type or engineered). For example, an “endogenous Igha locus” of a non-human animal (e.g., a mouse or rat) refers to a native Igha locus naturally occurring in the non-human animal (e.g., a mouse or rat).

[0077] The terms “wild-type” or “WT” include an entity having a structure and / or activity found in a normal (as opposed to mutated, diseased, altered, etc.) state or environment. Wild-type genes and polypeptides often exist in a variety of different forms (e.g., alleles).

[0078] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and refer to polymeric forms of nucleotides of any length (deoxyribonucleotides (DNA) or ribonucleotides (RNA) or their analogs) in either single- or double-stranded form. Polynucleotides can have any three-dimensional structure, and can perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, short or long single- stranded RNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule can be linear or circular. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or a deoxyinosine residue (see, Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0079] The terms "protein," "polypeptide," and "peptide" are used interchangeably and refer to polymers of amino acids. The proteins, polypeptides, or peptides described herein can contain naturally occurring amino acids, or can contain non-naturally occurring amino acids, or analogs or mimetics of amino acids. The proteins, polypeptides, or peptides described herein can be obtained by any method known in the art, such as, but not limited to, by natural isolation, recombinant expression, chemical synthesis, and the like.

[0080] As used herein, the term "amino acid" refers to an organic compound containing an amino (-NH2) and carboxyl (-COOH) functional group, and a side chain specific to each amino acid. Amino acid names are also represented in the disclosure as standard one-letter or three-letter codes, summarized below.

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[0082]

[0083] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain sequences from any organism, including immunoglobulin heavy chain constant region sequences. Unless otherwise specified, a heavy chain variable region includes three heavy chain complementarity determining regions (CDRs) and four FR regions. Fragments of a heavy chain include CDRs and FRs, and combinations thereof. A typical heavy chain has, following the variable region (from N- to C-terminus), a CHI domain, a hinge, a CH2 domain, a CH3 domain, and a CH4 domain (in the case of IgM or IgE). Functional fragments of a heavy chain include fragments that are capable of specifically recognizing an epitope (e.g., recognizing an epitope with a KD in the micromolar, nanomolar, or picomolar range), capable of being expressed and secreted from a cell, and including at least one CDR.

[0084] The term "heterologous" refers to an agent or entity from a different source. For example, when used in reference to a polypeptide, gene, or gene product present in a particular cell or organism, the term clarifies that the relevant polypeptide or fragment thereof, gene or fragment thereof, or gene product or fragment thereof: 1) is artificially engineered; 2) is introduced into the cell or organism (or a precursor thereof) by artifice (e.g., by genetic engineering); and / or 3) is not naturally produced by or present in the relevant cell or organism (e.g., the relevant cell type or organism type). As used herein, the term "heterologous" also includes a polypeptide or fragment thereof, gene or fragment thereof, or gene product or fragment thereof that is normally present in a particular native cell or organism, but has been modified, e.g., by mutation or substitution, under the control of a non-native, and, in some embodiments, non-endogenous, regulatory element (e.g., a promoter).

[0085] The term "fragment," when referring to a protein, means a protein that is shorter or has fewer amino acids than a full-length protein. The term "fragment," when referring to a nucleic acid, means a nucleic acid that is shorter or has fewer nucleotides than a full-length nucleic acid. When referring to a fragment of a protein, the fragment can be, for example, an N-terminal fragment (i.e., removal of a portion of the C-terminus of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminus of the protein), or an internal fragment (i.e., removal of a portion of each of the N-terminus and the C-terminus of the protein). When referring to a fragment of a nucleic acid, the fragment can be, for example, a 5' fragment (i.e., removal of a portion of the 3' end of the nucleic acid), a 3' fragment (i.e., removal of a portion of the 5' end of the nucleic acid), or an internal fragment (i.e., removal of a portion of each of the 5' end and the 3' end of the nucleic acid).

[0086] In the context of inserting a nucleic acid sequence into a cell, the term "introducing" refers to "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid sequence into a cell where the nucleic acid sequence can be present on a transitory basis or can be incorporated into the genome of the cell (e.g., chromosomal, plasmid, plastid, or mitochondrial DNA), transformed as an autonomous replicon. The nucleic acid sequence can be introduced into the cell using any method known in the art. A variety of techniques for transforming animal cells can be employed, including, for example: microinjection, retroviral-mediated gene transfer, electroporation, transfection, and the like (see, e.g., Keown et al., Methods in Enzymology 1990, 185:527-537).

[0087] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or light chain derived from one species and the remainder of the heavy chain and / or light chain derived from a different species. In illustrative examples, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0088] The term "non-human animal" refers to any organism that is not a human. For example, a non-human animal can be a domesticated animal, such as a cow, pig, sheep, goat, poultry, or horse; or a rodent, such as a rat or mouse; or a primate, such as an ape, monkey, chimpanzee, gorilla, orangutan, baboon; or a domesticated animal, such as a dog or cat.

[0089] The terms "5' regulatory region" and "3' regulatory region" as used herein include regulatory elements found in the 5' upstream region and 3' downstream region of a gene. The term "regulatory element" includes both 5' transcriptional regulatory sequences, including promoter, enhancer and suppressor elements, and 3' transcriptional regulatory sequences, including transcriptional termination sequences. The term "regulatory element" also includes regulatory sequences in the 5' untranslated region (5' UTR) and 3' UTR that can affect the efficiency of transcription and stability of the transcript, as well as the initiation of translation.

[0090] As used herein, a “CRISPR-Cas guide RNA” or “guide RNA,” “gRNA,” or “sgRNA” refers to an RNA that directs sequence-specific binding of a CRISPR complex to a target sequence. Typically, a guide RNA comprises (i) a guide sequence having sufficient complementarity to a target polynucleotide sequence to hybridize with the target sequence and (ii) a trans-activating cr (tracr) mate sequence. A guide RNA can further comprise a tracr RNA fused at the 3’ end, resulting in a single chimeric guide RNA. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment can be determined by using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, Burrows-Wheeler Transform based algorithms (such as Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, 11, 10, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence can be assessed by any suitable assay. For example, components of a CRISPR system sufficient to form a CRISPR complex, including a guide sequence to be tested, can be provided to a host cell having a corresponding target sequence, such as by transfecting with vectors encoding the components of the CRISPR sequence, and then assessing preferential cleavage within the target sequence.Similarly, cleavage of a target polynucleotide sequence can be assessed in vitro by providing the target sequence, components of the CRISPR complex including the guide sequence to be tested, and a control guide sequence that is different from the test guide sequence, and comparing the rate of binding or cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.

[0091] As used herein, the term "embryonic stem (ES) cell" refers to a pluripotent cell isolated from the inner cell mass of a developing blastocyst, or progeny of such cells. The "ES cell" can be derived from any organism. ES cells can be derived from mammals, including mice, rats, rabbits, guinea pigs, goats, pigs, cows, monkeys, and humans. In particular non-limiting examples, the cells are human or non-human (e.g., murine) cells. Without being bound by theory, ES cells can give rise to the various cells (skeletal, muscle, brain cells, etc.) that are present in vivo, provided they are exposed to conditions that favor development of these cell types. Methods for producing murine ES cells can be found in U.S. Patent 5,670,372, which is incorporated herein by reference. Methods for producing human ES cells can be found in U.S. Patent No. 6,090,622, WO 00 / 70021, and WO 00 / 27995, which are incorporated herein by reference.

[0092] II. Methods of Making Genetically Modified Non-Human Animals

[0093] In one aspect, the present disclosure provides a method of making a non-human animal comprising in its genome a genetically modified endogenous Igha locus comprising a heterologous gene encoding a human IgA (e.g., IgAl) heavy chain constant region or fragment thereof. Specifically, the present disclosure provides a method of making a genetically modified non-human animal, the method comprising: (i) modifying the genome of a non-human animal to incorporate into its endogenous Igha locus a nucleotide comprising a heterologous gene encoding a human IgA heavy chain constant region or fragment thereof; and (ii) treating the non-human animal produced in step (i) with a formulation comprising an immunostimulatory agent.

[0094] Step (i)

[0095] In some embodiments, step (i) of the methods provided herein comprises introducing a nucleotide comprising a heterologous gene encoding a human IgA heavy chain constant region or fragment thereof into a pluripotent cell of a non-human animal, thereby obtaining a non-human animal pluripotent cell comprising the heterologous gene, and using the non-human animal pluripotent cell comprising the heterologous gene to make a non-human animal.

[0096] Nucleotides comprising a heterologous gene encoding a human IgA heavy chain constant region or fragment thereof can be prepared by using methods well known in the art. For example, nucleotide molecules can be prepared as part of a larger plasmid. Such preparation allows for the cloning and isolation of the correct construct in a highly efficient manner known in the art. Various methods employed in the preparation of plasmids and transformation of host organisms are known in the art (see, e.g., Molecular Cloning A Laboratory Manual, 2nded., Sambrook et al., eds., Cold Spring Harbor Laboratory Press, 1989). Additionally, yeast artificial chromosomes (YACs) can be used to isolate, clone, and transfer entire loci of the desired heterologous gene encoding a human IgA heavy chain constant region or fragment thereof. Alternatively, bacterial artificial chromosome (BAC) libraries (see, e.g., Genome BAC Library from Invitrogen, Carlsbad Calif.) can provide nucleic acid sequences of the desired heterologous gene encoding a human IgA heavy chain constant region or fragment thereof as well as regulatory sequences.

[0097] The prepared transgenic construct can be introduced into a pluripotent cell (e.g., an ES cell) using any method known in the art. Various techniques for transforming mammalian cells can be used in the present application, including, for example: microinjection, retrovirus-mediated gene transfer, electroporation, transfection, and the like (see, e.g., Gordon, Inti. Rev Cytol., 115: 171 (1989); Keown et al., Methods Enzymol., 185: 527-537 (1990); Mansour et al., Nature, 336: 348-352 (1988)).

[0098] Methods for modifying the genome of a non-human animal (e.g., a pig, a cow, a rodent, a chicken, etc.) include, for example, using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or Cas proteins (i.e., CRISPR / Cas system) to modify the genome to include a heterologous gene encoding a human IgA heavy chain constant region or fragment thereof as described herein. Guidance for methods for modifying the germline genome of a non-human animal can be found, for example, in U.S. Patent Publication Nos. US20150376628A1, US20160145646A1, and US20160177339A1, all of which are incorporated by reference herein.

[0099] In some embodiments, the nucleotides comprising the heterologous gene are introduced into the pluripotent cell of the non-human animal by using a CRISPR / Cas system. In some embodiments, the CRISPR / Cas system is a CRISPR / Cas9 system. In some embodiments, the CRISPR / Cas9 system comprises a Cas9 protein and a guide RNA targeting a guide RNA target sequence within the endogenous Igha locus. In some embodiments, the CRISPR / Cas9 system used in the present application comprises a Cas9 protein and one, two or more, three or more, or four or more guide RNAs targeting a guide RNA target sequence within the endogenous Igha locus. For example, the CRISPR / Cas9 system used in the present application comprises a Cas9 protein and a guide RNA comprising any one, two, three, or four of the sequences as set forth in SEQ ID NOs: 9-12. In some embodiments, the CRISPR / Cas system is a CRISPR / Cas13 system. In some embodiments, the CRISPR / Cas13 system comprises a Cas13 protein and a guide RNA targeting a guide RNA target sequence within the endogenous Igha locus. In some embodiments, the CRISPR / Cas13 system used in the present application comprises a Cas13 protein and one, two or more, three or more, or four or more guide RNAs targeting a guide RNA target sequence within the endogenous Igha locus.

[0100] The pluripotent cells described herein can include undifferentiated cells having the ability to develop into more than one differentiated cell type. Such pluripotent cells can be, for example, embryonic stem (ES) cells or ES-like cells, such as induced pluripotent stem (iPS) cells. In some embodiments, the pluripotent cell is an embryonic stem (ES) cell. ES cells include embryonic-derived pluripotent cells that are capable of contributing to any tissue of a developing embryo when introduced into an embryo. ES cells can be derived from the inner cell mass of a blastocyst and are capable of differentiating into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm). An ES cell having a desired nucleotide incorporated in the endogenous Igha locus can be selected. In some embodiments, one or more copies of the nucleotide can be incorporated at one or more specific sites; and in some embodiments, one copy of the nucleotide can be incorporated at one specific site.

[0101] ES cells are typically obtained from pre-implantation embryos cultured in vitro (see, e.g., Evans, M.J. et al., Nature 292: 154-156 (1981); Bradley, M.O. et al., Nature 309: 255-258 (1984); Gossleer et al., Proc. Natl. Acad. Sci. USA 83: 9065-9069 (1986); Robertson et al., Nature 322: 445-448 (1986)). ES cells are cultured and prepared for introduction of the transgene construct using methods well known in the art (see, e.g., Teratocarcinomas and Embryonic Stem cells: a Practical Approach, Robertson, ed., IRL Press (1987); Bradley et al., Current Topics in Devel. Biol. 20: 357-371 (1986); Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1986); Thomas et al., Cell 51: 503 (1987); Koller et al., Proc. Natl. Acad. Sci. USA 88: 10730 (1991); Donn et al., Transgenic Res. 1: 101 (1992); Veis et al., Cell 75: 229 (1993)). ES cells into which the desired heterologous gene encoding a human IgA heavy chain constant region or fragment thereof has been inserted are derived from embryos or blastocysts of the same species as the developing embryo or blastocyst into which they are to be introduced. ES cells are typically selected for their ability to integrate into the inner cell mass and contribute to the germ line of an individual when introduced into the embryo of a host animal at the blastocyst stage of development. Thus, any ES cell line having this ability is suitable for use in the practice of the present application.

[0102] The selected ES cells are then injected into an embryo of the animal (suitable for blastocyst or other stage of development for purposes of generating a live animal) to generate a chimera (see, e.g., Teratocarcinomas and Embryonic Stem Cells: Methods and Applications, EJ. Robertson, ed., Oxford IRL Press, pp 113-152 (1987)). Alternatively, the selected cells can be aggregated with dissociated embryonic cells to form an aggregated chimera. The chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and allowed to come to term. The chimeric offspring contains the desired human gene in its germ cells, which can be used to breed animals in which all cells of the animal contain the desired human gene. ES cells having the desired nucleotides incorporated into the endogenous Igha locus are then used as donor ES cells. In some embodiments, an embryo comprising the donor ES cells is implanted into a pseudopregnant female to generate a F0 non-human animal.

[0103] In other embodiments, a non-human animal comprising a genetically modified endogenous Igha locus comprising a heterologous gene in its genome can be prepared without the use of ES cells. For example, the genome of a non-ES cell (e.g., a fibroblast or an induced pluripotent cell) can be modified based on conventional transformation methods (e.g., electroporation), and the modified genome of such non-ES cell can be transferred to a suitable recipient cell, such as an oocyte, by using nuclear transfer techniques. The modified cell (e.g., the modified oocyte) is then incubated under suitable conditions to form an embryo. See, e.g., Han et al., Methods Enzymol. 476:171-184 (2010) and Zhou et al., Science 302:1179 (2003).

[0104] In some embodiments, the non-human animal is a rodent, a mammal, or a non-human primate. In some embodiments, the non-human animal is a male animal. In some embodiments, the non-human animal is a female animal. In some embodiments, the non-human animal is a mouse or a rat.

[0105] In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a mouse of the C57BL strain (e.g., a C57BL strain selected from the group consisting of C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / 01a). In other embodiments, the rodent is a mouse of the 129 strain (e.g., a 129 strain selected from the group consisting of 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129 / SvJae, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2) (see, e.g., Festing et al. (1999), Mammalian Genome 10:836; Auerbach et al. (2000), Biotechniques 29(5): 1024-1028, 1030, 1032). In some embodiments, the rodent is a mouse that is a mix of the aforementioned 129 strains and the aforementioned C57BL / 6 strains. In certain embodiments, the mouse is a mix of the aforementioned 129 strains (i.e., hybrid), or a mix of the aforementioned C57BL strains, or a mix of C57BL strains and 129 strains. In certain embodiments, the mouse is a mix of C57BL / 6 strains and 129 strains. In particular embodiments, the mouse is a VGF1 strain, also known as F1H4, which is a hybrid of C57BL / 6 and 129. In other embodiments, the mouse is a BALB strain, e.g., a BALB / c strain. In some embodiments, the mouse is a BALB strain and a mix of another of the aforementioned strains.

[0106] In some embodiments, the rodent is a rat. In certain embodiments, the rat is selected from the group consisting of Wistar rat, LEA strain, Sprague Dawley strain, Fischer strain, F344, F6, and Dark Agouti. In other embodiments, the rat is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0107] In some embodiments, a segment or all of the endogenous non-human animal IgHa gene at the endogenous non-human animal IgHa locus has been deleted. In some embodiments, a segment or all of the endogenous non-human animal IgHa gene at the endogenous non-human animal IgHa locus has been deleted and replaced with nucleotides comprising a heterologous gene encoding a human IgA heavy chain constant region or fragment thereof. In some embodiments, the non-human animals described herein do not express an endogenous non-human IgA heavy chain constant region.

[0108] Preferably, replacement of the endogenous non-human animal IgHa gene with a corresponding human IGHAl gene can be achieved by homologous recombination (see, e.g., Allen et al., Eur J Neurosci 17, 1881-1895 (2003); Makita et al., Am J Physiol Renal Physiol 294, F542-F553 (2008)). Replacement-type targeting constructs are typically used for homologous gene replacement. To make a replacement-type targeting construct, the nucleic acid sequence of the desired human IGHAl gene is flanked between two homologous recombination sequences derived from the non-human animal genomic sequence. Double crossover between the homologous recombination sequences of the targeting construct and the non-human animal genomic sequence results in targeted integration of the nucleic acid sequence of the desired human IGHAl gene into the locus of the corresponding non-human animal IgHa gene in an animal cell. Typically, the homologous recombination sequences of the targeting construct include sequences that flank a segment of the endogenous non-human animal IgHa gene, such that homologous recombination results in concomitant deletion of the segment of the endogenous non-human animal IgHa gene and homologous integration of the segment of the desired human IGHAl gene (see Roebroek et al., Trangenic Mouse Methods and Protocols, Hofker and van Deursen, eds., Methods in Mol Biol, 693:257-275 (2011)). The entire or a segment of the endogenous non-human animal IgHa gene can be replaced by the desired human IGHAl gene by a single targeting event or by multiple targeting events that sequentially replace individual exons. One or more selectable markers (e.g., positive or negative selectable marker genes) can be used in the targeting construct. It is typically preferred that the selectable marker be located in an intronic region of the desired human IGHAl gene.

[0109] In some embodiments, replacing the endogenous non-human animal Igha gene with a corresponding human IGHAl gene can be achieved using a site-specific recombination system (e.g., a Cre / LoxP system or a Flp / FRT system; see Roebroek et al., Methods in Transgenic Mice, Hofker and van Deursen, eds., Methods in Molecular Biology, 693:257-275 (2011)). As a starting point for this strategy, two recombinase recognition sequences (e.g., LoxP, FRT, or attB / attP sequences) are introduced into an appropriate non-human animal cell (e.g., an ES cell) by a first homologous recombination event, such that the two introduced recombinase recognition sequences flank the endogenous non-human animal Igha gene. In the first homologous recombination event, a selectable marker gene (e.g., HygTK) is typically introduced into the non-human animal cell, allowing for positive selection (e.g., by hygromycin B) and negative selection (e.g., by ganciclovir). A transgenic construct is prepared that carries the desired human IGHAl gene corresponding to the endogenous non-human animal Igha gene, such that the desired human IGHAl gene is flanked by the two recombinase recognition sites. The transgenic construct is subsequently introduced into the targeted non-human animal cell resulting from the first homologous recombination event. A source of recombinase enzyme is provided to trigger site-specific recombination, which results in the exchange of the endogenous animal Igha gene with the desired human IGHAl gene. The recombinase recognition sequences can remain present in the locus after the exchange, and are preferably not present in the protein-coding region.

[0110] In some embodiments, the heterologous gene encoding a human IgAl heavy chain constant region or fragment thereof is a genomic fragment of a human IGHAl gene. In some embodiments, the heterologous gene comprises only the human IGHAl coding sequence. In some embodiments, the heterologous gene comprises both the human IGHAl coding sequence and non-coding sequences. In some embodiments, the heterologous gene comprises the entire human IGHAl coding sequence from ATG to STOP, with 5' and 3' untranslated regions and intervening introns in between. In some embodiments, the human IgAl heavy chain constant region of the non-human animal comprises an O-glycosylated hinge region. In some embodiments, the heterologous gene is a human IGHAl gene encoding a human IgAl heavy chain constant region or fragment thereof.

[0111] A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or 5' regulatory region containing a promoter is said to be operably linked to a coding sequence if it influences the transcription of the coding sequence.

[0112] In some embodiments, a heterologous gene encoding a human IgA heavy chain constant region or fragment thereof is inserted into the same genetic locus as the corresponding endogenous non-human animal Igha gene, such that expression of the heterologous gene is controlled by the transcriptional regulatory elements of the corresponding endogenous non-human animal Igha gene. The heterologous gene can be comprised in nucleotides. In some embodiments, the nucleotides are operably linked to an endogenous regulatory element (e.g., a promoter, enhancer, silencer, etc.) of the non-human animal. Examples of such transcriptional regulatory elements include, but are not limited to, promoters, enhancers, silencers, and initiation signals. In some embodiments, a humanized transgenic non-human animal containing a human IGHA1 gene can be made such that the protein coding sequence of the human IGHA1 gene is inserted into the locus of the endogenous non-human animal Igha gene. The insertion can be performed by recombination such that the protein coding sequence of the introduced human IGHA1 gene replaces the corresponding protein coding sequence of the non-human Igha gene. This allows expression of the introduced human IGHA1 gene to be controlled by the transcriptional regulatory elements of the endogenous non-human animal Igha gene.

[0113] In some embodiments, the nucleotides can additionally comprise a 3' regulatory region that is operably linked to the heterologous gene encoding a human IgAl heavy chain constant region or fragment thereof.

[0114] In some embodiments, the 3' regulatory region comprises a 3' UTR. In some embodiments, the 3' regulatory region comprises a 3' UTR of the heterologous gene encoding a human IgAl heavy chain constant region or fragment thereof.

[0115] In some embodiments, the 3' regulatory region comprises a sequence upstream of the 3' UTR of the heterologous gene. In some embodiments, the 3' regulatory region comprises a nucleotide sequence immediately upstream of the 3' UTR of the heterologous gene and is at least 50 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, at least 1000 bp, or longer in length (e.g., up to 2500-4000 bp).

[0116] In some embodiments, the 3' regulatory region comprises a sequence downstream of the 3' UTR of the heterologous gene. In some embodiments, the 3' regulatory region comprises a nucleotide sequence immediately downstream of the 3' UTR of the heterologous gene and is at least 50 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, at least 1000 bp, or longer in length (e.g., up to 2500-4000 bp).

[0117] In some embodiments, the nucleotide can additionally comprise a 5' regulatory region operably linked to the heterologous gene encoding the human IgAl heavy chain constant region or fragment thereof.

[0118] In some embodiments, the 5' regulatory region comprises a 5' UTR. In some embodiments, the 5' regulatory region comprises a 5' UTR of the heterologous gene encoding the human IgAl heavy chain constant region or fragment thereof.

[0119] In some embodiments, the 5' regulatory region comprises a sequence upstream of the 5' UTR of the heterologous gene. In some embodiments, the 5' regulatory region comprises a nucleotide sequence immediately upstream of the 5' UTR of the heterologous gene and is at least 50 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, at least 1000 bp, or longer in length (e.g., up to 2500-4000 bp).

[0120] In some embodiments, the 5' regulatory region comprises a sequence downstream of the 5' UTR of the heterologous gene. In some embodiments, the 5' regulatory region comprises a nucleotide sequence immediately downstream of the 5' UTR of the heterologous gene and is at least 50 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, at least 1000 bp, or longer in length (e.g., up to 2500-4000 bp).

[0121] In some embodiments, the modification to the non-human animal genome or the incorporation of the nucleotide into the non-human animal causes a structural change to the endogenous Igha locus. In some embodiments, a segment or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha locus is silenced. In some embodiments, a segment or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha locus has been deleted and replaced with a nucleotide comprising a heterologous gene.

[0122] A gene is "silenced" when expression of a normal protein product from the gene is inhibited. A gene is typically silenced by disrupting the genomic sequence of the gene. A gene is disrupted when a DNA segment is positioned and recombines with the endogenous homologous sequence. Disruption can include insertion, missense, frameshift, deletion, or substitution, or replacement, or any combination thereof, of DNA sequence. Insertion includes insertion of an entire gene when the disruption can alter the normal gene product by partially or completely inhibiting production of the normal gene product. In preferred embodiments, the disruption is a null disruption such that the gene is not significantly expressed.

[0123] The term "replace" or "replacement" as used herein means that in a transgenic non-human animal into which a desired human gene is introduced, the endogenous non-human animal gene corresponding to the introduced human gene is silenced such that the introduced human gene functionally replaces the corresponding animal gene. For example, in a humanized transgenic mouse into which a human Ighal gene is introduced, the endogenous murine Igha gene is further silenced such that the introduced human IgA1 protein functionally replaces the murine IgA1 protein.

[0124] In some embodiments, the modification to the non-human animal genome results in a functional change to the endogenous Igha locus. In some embodiments, the non-human animal does not express an endogenous non-human IgA heavy chain constant region. In some embodiments, the non-human animal produces a chimeric IgA1 whose heavy chain comprises a non-human animal variable region and a human constant region or fragment thereof.

[0125] Step (ii)

[0126] In step (ii) of the methods provided herein for making a genetically modified non-human animal, the non-human animal produced in step (i) above is treated with a formulation comprising an immunostimulatory agent.

[0127] An "immunostimulatory agent" is any substance that stimulates the immune system by inducing activation of or increasing the activity of any component of the immune system, particularly immune effector cells. An immunostimulatory agent can be proinflammatory (e.g., when treating an infection or cancer) or anti-inflammatory (e.g., when treating an autoimmune disease).

[0128] In some embodiments, the immunostimulatory agent comprises an adjuvant-type immunostimulatory agent, such as an APC Toll-like receptor agonist or a costimulatory / cell adhesion membrane protein. Examples of Toll-like receptor agonists include costimulatory / adhesion proteins, such as CD80, CD86, and ICAM-1. In some embodiments, the immunostimulatory agent used in the present application is selected from the group consisting of an aluminum adjuvant, an emulsion adjuvant, a biological adjuvant, and a saponin.

[0129] In some embodiments, the aluminum adjuvant is selected from the group consisting of aluminum hydroxide (Al(OH)3), aluminum phosphate, aluminum hydroxyphosphate, and amorphous aluminum hydroxyphosphate sulfate (AAHS). In some embodiments, the aluminum adjuvant is aluminum hydroxide.

[0130] In some embodiments, the emulsion adjuvant is an oil-in-water emulsion adjuvant or a water-in-oil emulsion adjuvant. In some embodiments, the emulsion adjuvant is selected from the group consisting of Freund’s complete adjuvant (CFA), Freund’s incomplete adjuvant, MF59, and AS03. In some embodiments, the immunostimulant used in the present application is CFA. Various commercial CFAs are available to the public, for example, products purchased from Sigma-Aldrich, Merck, etc. In some embodiments, the CFA used in the present application is purchased from SIGMA (catalog # F5881). In some embodiments, the CFA used in the present application contains Mycobacterium tuberculosis, paraffin oil, and mannide monooleate. In some embodiments, the CFA used in the present application contains 0.5-5 mg (e.g., 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, or 5 mg) of heat-killed and dried Mycobacterium tuberculosis, 0.1-1 mL (e.g., 0.5 mL, 0.85 mL, 1 mL) of paraffin oil, and 0.05-1 mL (e.g., 0.1 mL, 0.15 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1 mL) of mannide monooleate per mL of CFA. In some embodiments, the CFA used in the present application contains 1 mg of heat-killed and dried Mycobacterium tuberculosis (H37Ra, ATCC 25177), 0.85 mL of paraffin oil, and 0.15 mL of mannide monooleate per mL of CFA.

[0131] In some embodiments, the biological adjuvant is selected from the group consisting of a lipopolysaccharide or a derivative thereof, a toll-like receptor (TLR) agonist (e.g., a TLR4 ligand, a TLR7 ligand, a TLR8 ligand, a TLR9 ligand), an immunostimulatory oligonucleotide (e.g., a DNA or dsRNA comprising CpG), a cytokine (e.g., IL-2, IL-7, IL-12, IL-15, and IL-23) or a variant thereof, an interferon (e.g., IFN-a or IFN-g), a colony-stimulating factor (e.g., M-CSF and GM-CSF), a tumor necrosis factor, an endotoxin, and a lipid (e.g., lipid A or an analog thereof). In some embodiments, the biological adjuvant is a lipopolysaccharide or a derivative thereof.

[0132] In some embodiments, the formulation used to treat the non-human animal produced in step (i) comprises an immunostimulant and an antigen.

[0133] The term "antigen" relates to an agent comprising an epitope that can generate an immune response. In particular, the term "antigen" includes proteins and peptides. In some embodiments, the antigen is a disease-related antigen, such as a viral antigen or a bacterial antigen, and the epitope is derived from such an antigen.

[0134] The term "viral antigen" refers to any viral component having antigenic properties, i.e. capable of eliciting an immune response in an individual. The viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0135] The term "bacterial antigen" refers to any bacterial component having antigenic properties, i.e. capable of eliciting an immune response in an individual. The bacterial antigen can be derived from the cell wall or cytoplasmic membrane of the bacterium. In some embodiments, the antigen used in the present application is derived from a cell wall extract of the bacterium. In some embodiments, the antigen comprises a rhamnose source. In some embodiments, the rhamnose source is a Lactobacillus casei cell wall extract (LCWE).

[0136] In some embodiments, the non-human animal produced in step (i) is treated with a formulation comprising LCWE emulsified with CFA. Detailed treatment with LCWE can be performed using routine means in the art (see Duong T T, Silverman E D, Bissessar M V, et al. International immunology. 2003(1): 15; Noval Rivas et al. Immunity. 2019(3): 51; Suganuma E, Sato S, Honda S, et al. Experimental Animals. 2020, 69(2)).

[0137] In some embodiments, the formulation consists of an immunostimulant. For example, the non-human animal produced in step (i) is treated with CFA alone. The inventors of the present disclosure unexpectedly found that treating the non-human animal produced in step (i) comprising a humanized Igha locus with an immunostimulant (e.g., CFA) alone (e.g., without an antigen) is sufficient to produce a genetically modified non-human animal that displays clinical signs of a disease associated with IgA deposition (e.g., IgAN), for example, displays elevated IgAl, elevated galactofaultive IgAl (Gd-IgAl), IgAl / C3 glomerular deposition, mesangial hypercellularity, endocapillary cellularity, segmental sclerosis, and / or crescents, as compared to a wild-type non-human animal.

[0138] The formulation comprising an immunostimulant can be administered to the non-human animal produced in step (i) by any route known in the art, for example, by parenteral routes (including subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection) or non-parenteral routes (including transdermal, oral, intranasal, intraocular, sublingual, rectal, or topical). In some embodiments, in step (ii), the formulation is administered intraperitoneally to the non-human animal produced in step (i).

[0139] Without being bound by any theory, but it is believed that sustained delivery (e.g., sustained intraperitoneal delivery) or slow release of the formulation comprising an immunostimulant to the non-human animal produced in step (i) is advantageous for making a genetically modified non-human animal that displays clinical signs of a disease associated with IgA deposition (e.g., IgAN).

[0140] As used herein, the term “sustained delivery” refers to the continuous delivery of the formulation comprising an immunostimulant in vivo over a period of time, preferably at least a few days, a week or several weeks, a month or several months, after administration.

[0141] In some embodiments, the formulation is intraperitoneally injected into the non-human animal produced in step (i) in a continuous dose. In some embodiments, the formulation is intraperitoneally injected into the non-human animal produced in step (i) in a continuous low dose. In some embodiments, the formulation comprises LCWE, and the formulation is intraperitoneally injected into the non-human animal produced in step (i) in a dose of less than 1 pg (e.g., less than 0.1 pg, less than 0.2 pg, less than 0.3 pg, less than 0.4 pg, less than 0.5 pg, less than 0.6 pg, less than 0.7 pg, less than 0.8 pg, less than 0.9 pg) LCWE per gram of the non-human animal. In some embodiments, the formulation comprises or consists of CFA, and the formulation is intraperitoneally injected into the non-human animal produced in step (i) in a dose of less than 5 pL (e.g., less than 4 pL, less than 3 pL, less than 2 pL, less than 1.9 pL, less than 1.8 pL, less than 1.7 pL, less than 1.6 pL, less than 1.5 pL, less than 1.4 pL, less than 1.3 pL, less than 1.2 pL, less than 1.1 pL, less than 1 pL, less than 0.9 pL, less than 0.8 pL, less than 0.7 pL, less than 0.6 pL, less than 0.5 pL, less than 0.4 pL, less than 0.3 pL, less than 0.2 pL, less than 0.1 pL) CFA per gram of the non-human animal. In some embodiments, the formulation comprises LCWE emulsified with CFA, and the formulation is intraperitoneally injected into the non-human animal produced in step (i) in a dose of less than 1 pg (e.g., less than 0.1 pg, less than 0.2 pg, less than 0.3 pg, less than 0.4 pg, less than 0.5 pg, less than 0.6 pg, less than 0.7 pg, less than 0.8 pg, less than 0.9 pg) LCWE and less than 5 pL (e.g., less than 4 pL, less than 3 pL, less than 2 pL, less than 1.9 pL, less than 1.8 pL, less than 1.7 pL, less than 1.6 pL, less than 1.5 pL, less than 1.4 pL, less than 1.3 pL, less than 1.2 pL, less than 1.1 pL, less than 1 pL, less than 0.9 pL, less than 0.8 pL, less than 0.7 pL, less than 0.6 pL, less than 0.5 pL, less than 0.4 pL, less than 0.3 pL, less than 0.2 pL, less than 0.1 pL) CFA per gram of the non-human animal.

[0142] In some embodiments, the formulation is administered to the non-human animal produced in step (i) for at least one month. In some embodiments, the formulation is administered to the non-human animal produced in step (i) for at least two months. In some embodiments, the formulation is administered to the non-human animal produced in step (i) for at least three months. In some embodiments, the formulation is administered to the non-human animal produced in step (i) for at least four months.

[0143] In some embodiments, the formulation is administered to the non-human animal produced in step (i) at an interval of three times per week for the first two weeks and once per week for the next two weeks (i.e., a total of eight times over a one-month period). In some embodiments, the formulation is administered to the non-human animal produced in step (i) at an interval of once every two weeks (i.e., a total of six times over a three-month period). In some embodiments, the formulation is administered to the non-human animal produced in step (i) starting at 8 weeks of age and ending at 5 months of age, 6 months of age, 6.5 months of age, 7 months of age, 7.5 months of age, or 8 months of age.

[0144] In some embodiments, the non-human animal described above is a rodent, a mammal, or a non-human primate. In some embodiments, the rodent is a mouse or a rat.

[0145] III. Genetically modified non-human animals

[0146] In another aspect, the present disclosure provides a non-human animal produced by the methods described herein. In another aspect, the present disclosure also provides a non-human cell or non-human genome produced by the methods described herein.

[0147] In some embodiments, the non-human animals, cells, or genomes provided herein comprise in their genome a genetically modified endogenous Igha locus comprising a heterologous gene encoding a human IgA heavy chain constant region or fragment thereof, wherein the non-human animal displays clinical signs of a disease associated with IgA deposition (e.g., IgA nephropathy). In some embodiments, the non-human animals, cells, or genomes provided herein comprise in their genome a genetically modified endogenous Igha locus comprising a heterologous gene encoding a human IgA heavy chain constant region or fragment thereof, wherein the non-human animal displays clinical signs of IgA nephropathy.

[0148] In some embodiments, the non-human animals provided herein display histological lesions of IgA nephropathy. In some embodiments, the non-human animals provided herein display one or more histological lesions selected from the group consisting of: mesangial hypercellularity, endocapillary hypercellularity, segmental sclerosis, interstitial inflammation / tubular atrophy, and crescents.

[0149] In some embodiments, the non-human animals provided herein display increased levels of galactose-deficient IgA proteins. In some embodiments, the non-human animals provided herein display increased levels of galactose-deficient IgA proteins in serum or intestinal mucus. In some embodiments, an “increased level” of galactose-deficient IgA proteins refers to an increase in the level of galactose-deficient IgA proteins compared to a reference level of galactose-deficient IgA proteins obtained from a wild-type counterpart non-human animal. The level of galactose-deficient IgA proteins can be determined by methods well known in the art, for example, Irabu H, et al., J Immunol Res. 2020 May 21;2020:4284379; Suzuki H., Clin Exp Nephrol. 2019 Jan;23(l):26-31; and Neufeld M, et al., J Am Acad Dermatol. 2019 Mar 19. pii: S0190-9622(19)30443-8, all of which are incorporated by reference herein in their entireties.

[0150] In some embodiments, the non-human animals described herein display at least a 10-fold increase (e.g., at least an 11-fold, at least a 12-fold, at least a 13-fold, at least a 14-fold, at least a 15-fold, at least a 16-fold, at least a 17-fold, at least a 18-fold, at least a 19-fold, at least a 20-fold, at least a 25-fold, or at least a 30-fold increase) in galactose-deficient IgA proteins. In some embodiments, the non-human animals described herein display a 10- to 20-fold increase in galactose-deficient IgA proteins with different kidney pathology phenotypes, suggesting that changes in glycosylation are closely related to the function of human IgA, but not to the function of non-human (e.g., mouse) IgA under the same stimuli.

[0151] As described above, the methods of making a genetically-modified non-human animal provided herein can result in an alteration of the structure and / or function of an endogenous Igha locus in the non-human animal. In some embodiments, a segment or all of an endogenous non-human animal Igha gene at an endogenous non-human animal Igha locus has been deleted and replaced with a nucleotide comprising a heterologous gene. In some embodiments, the nucleotide comprising a heterologous gene is operably linked to an endogenous regulatory element (e.g., a promoter, enhancer, silencer, etc.) of the non-human animal. In some embodiments, the nucleotide comprising a heterologous gene is a genomic fragment of a human IGHA1 gene. In some embodiments, the heterologous gene comprises both a human IGHA1 coding sequence and non-coding sequence. In some embodiments, the heterologous gene is a human IGHA1 gene that encodes a human IgA1 heavy chain constant region or fragment thereof. In some embodiments, the human IgA heavy chain constant region of a non-human animal provided herein comprises an O-glycosylated hinge region. In some embodiments, the non-human animal does not express an endogenous non-human IgA heavy chain constant region. In some embodiments, a non-human animal provided herein produces a chimeric IgA1 whose heavy chain comprises a non-human animal variable region and a human constant region or fragment thereof.

[0152] In some embodiments, the non-human animal described above is a rodent, a mammal, or a non-human primate. In some embodiments, the rodent is a mouse or a rat.

[0153] IV. Uses of Genetically-Modified Non-Human Animals

[0154] A non-human animal provided herein, e.g., a non-human animal comprising a genetically-modified endogenous Igha locus in the genome, the endogenous Igha locus comprising a heterologous gene that encodes a human IgA heavy chain constant region or fragment thereof, can be used as an animal model for a disease associated with IgA deposition, wherein the non-human animal displays clinical signs of the disease associated with IgA deposition (e.g., IgA nephropathy).

[0155] In another aspect, the disclosure further provides a method for evaluating the efficacy of a candidate drug for treating or preventing a disease associated with IgA deposition, the method comprising: providing a non-human animal described herein; administering the candidate drug to the non-human animal; and evaluating whether the candidate drug inhibits one or more symptoms of the disease as compared to a control non-human animal that was not administered the candidate drug. If the candidate drug inhibits one or more symptoms of the disease as compared to the control non-human animal that was not administered the candidate drug, the candidate drug will be considered effective for treating or preventing a disease associated with IgA deposition.

[0156] In additional aspects, the present disclosure provides a method for identifying a candidate drug for treating or preventing a disease associated with IgA deposition, the method comprising: providing a non-human animal described herein; administering the candidate drug to the non-human animal; and assessing whether the candidate drug inhibits one or more symptoms of the disease as compared to a control non-human animal that was not administered the candidate drug. If the candidate drug inhibits one or more symptoms of a disease associated with IgA deposition (e.g., IgA nephropathy), the candidate drug will be considered useful for treating or preventing a disease associated with IgA deposition.

[0157] In some embodiments, candidate drugs that can be evaluated or identified using the non-human animals provided herein include candidate inhibitors of IgA (e.g., IgAl), such as, but not limited to, small molecule inhibitors, nucleic acid-based inhibitors (e.g., siRNAs, ribozymes, antisense constructs, etc.), antigen binding proteins (e.g., antibodies or antigen binding fragments thereof), blocking peptides / peptide inhibitors, IgA proteases or functional variants thereof, fusion proteins comprising IgA proteases or functional variants thereof.

[0158] In some embodiments, the disease associated with IgA deposition is selected from the group consisting of IgA nephropathy, dermatitis herpetiformis, Henoch-Schonlein purpura (also known as IgA vasculitis), Kawasaki disease, purpura nephritis, IgA vasculitis renal injury, IgA rheumatoid factor positive rheumatoid arthritis, IgA-mediated anti-GBM disease, or IgA-mediated ANCA-associated vasculitis. In some embodiments, the disease associated with IgA deposition is IgA nephropathy, IgA vasculitis, or Kawasaki disease. In some embodiments, the disease associated with IgA deposition is IgA nephropathy.

[0159] Examples

[0160] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and evaluate the compositions and / or methods claimed herein and are intended to be purely exemplary and are not intended to be limiting to the disclosure.

[0161] Materials and methods

[0162] Animals and housing conditions

[0163] All animal experiments were approved by the Laboratory Animal Care and Use Committee of Peking University First Hospital (number 201994 and number 2021114). Mice were bred and raised under barrier conditions for validation of IGHA1 + / - and IGHA1+ / + Human IgAl expression in mice. To assess the impact of different pathogen exposure on IGHA1 + / + mice, three breeding groups were established. First, IGHA1 + / + mice were germ-free (GF) purified and their offspring were raised under germ-free isolation conditions. Faecal samples were regularly checked for their GF status using microscopic examination and culture techniques. Second and third, IGHA1 + / + mice were raised under specific pathogen-free (SPF) conditions, similar to conventional (CV) bred animals. At 4 weeks of age, IGHA1 + / + mice were either raised under barrier conditions or transferred to non-barrier conditions. At 16 weeks of age, mice were euthanized and their kidneys were collected for histological examination after embedding in optimal cutting temperature compound or fixation in 10% formalin. In addition, IGHA1 TM mice raised under barrier conditions were injected intraperitoneally with 2 pg / g CFA or PBS emulsified 0.5 pg / g LCWE, three times a week for the first 2 weeks and once a week for the following 2 weeks. The observation period was extended until mice reached 8 months of age.

[0164] Urine, terminal serum, spleen (SP), small intestine (SI), inguinal lymph nodes (ILN), mesenteric lymph nodes (MLN), Peyer’s patch lymph nodes (PP), femur and small intestine mucus diluted in 3 mL PBS were collected for further analysis.

[0165] Polymerase chain reaction (PCR) and real-time quantitative (RT-qPCR)

[0166] IGHA1 gene was identified from mouse tail or ear clip DNA by Mouse Direct PCR Kit (B40015, Bimake). RNA was extracted from mouse tissues based on TRIzol (15596026, Invitrogen) or RNA simple total RNA extraction kit (DP419, TIANGEN), followed by GoScript TM Reverse Transcription (A2801, Promega) to cDNA. cDNA and primers were combined with PowerUp TM SYBR TM Green Master Mix (A25742, Invitrogen) for RT-qPCR analysis on a LightCycler® 480 Instrument (Roche). GAPDH was used as an internal reference gene. Primers are listed in Table 1 below.

[0167] Table 1. RT-qPCR primers

[0168] Table 1. RT-qPCR primers

[0169] Serum enzyme-linked immunosorbent assay (ELISA) and Western blot

[0170] Serum mouse IgA, IgG, IgM, human IgA1, human IgA1-mouse IgG complex, and polymer IgA (pIgA) complex levels binding to CD89 were determined by ELISA. Plates were coated overnight with goat F(ab')2 anti-mouse Ig (1012-01, SouthernBiotech), F(ab')2 fragment of goat anti-human IgA (109-006-011, Jackson Immuno), or recombinant human CD89 protein (10414-H08H, Sino Biological). After blocking for 2 hours at room temperature (RT) with 1% bovine serum albumin (BSA) in phosphate-buffered saline with 0.1% Tween (PBST), diluted sera and standards were added to the plates for a 1-hour RT incubation. Mouse IgA, IgG, and IgM were then detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgA, IgG, and IgM antibodies (1040-05, 1030-05, and 1020-05, SouthernBiotech), HRP-conjugated mouse anti-human IgA1 (9130-05, SouthernBiotech), HRP-conjugated goat anti-human IgA alpha chain (ab98558, Abeam), or HRP-conjugated goat anti-mouse IgG (1030-05, SouthernBiotech), respectively. And to detect LCWE-specific hIgA1 and Gd-IgA1, plates were coated with 2.5 pg / mL LCWE, and HRP-conjugated mouse anti-human IgA1 (9130-05, SouthernBiotech), rat monoclonal anti-human Gd-IgA1 antibody (10777, Immuno-Biological Laboratories) plus HRP-conjugated polyclonal rabbit anti-rat IgG (RS030226, Immunoway Biotechnology Company) were used for detection.

[0171] All ELISA tests described above were detected by a tetramethylbenzidine liquid substrate system. The optical density was measured at 450 nm with a wavelength correction at 570 nm. Serum galactose-deficient IgA1 (Gd-IgA1) levels were measured using a Gd-IgA1 assay kit (27600, Immuno-Biological Laboratories) according to the manufacturer's protocol.

[0172] Serum (0.02 μΐ / sample) was loaded with non-reducing loading buffer onto gradient SDS-PAGE (8012011, BioSci, Shenzhen, Guangdong, China) and run at 150 V for 2 hours; subsequently, they were transferred onto 0.45 μιη PVDF membranes. For N-acetylgalactosamine residues (GalNAc) detection, 2 μg of FPLC purified hlgA was desialidated by neuraminidase (GK80040, Agilent) as a sample. After blocking with 5% skim milk, the membranes were incubated with HRP-conjugated goat anti-mouse IgA (1040-05, SouthernBiotech) or HRP-conjugated goat anti-human IgA a chain (ab98558, Abcam) for IgA detection at room temperature for 1 hour. The membranes were detected by enhanced chemiluminescence.

[0173] Urine analysis

[0174] Urine albumin was determined by ELISA. Plates were coated with goat anti-mouse albumin (A90-134A, Bethyl Laboratories) overnight. After blocking with 1% BSA in PBST for 2 hours at RT, diluted urine and standards were added to the plates for 1 hour RT incubation. Then, horseradish peroxidase (HRP)-conjugated anti-mouse albumin (A90-134P, Bethyl Laboratories) was used as detection antibody. Optical density was measured as described above. Urinary creatinine levels were measured using a creatinine assay kit (DICT-500; BioAssay Systems, Hayward, CA, USA). Urinary albumin / creatinine ratio (ACR, mg / g) was used to define kidney function.

[0175] Flow cytometry assays

[0176] SP, PP, MLN and ILN were ground and flushed with PBS containing 2% fetal bovine serum (FBS) to single cells from bone marrow (BM). Then, the suspension was filtered through a 100 pm cell strainer. Red blood cells of spleen, bone marrow and whole blood were lysed by RBC lysis buffer (420301, Biolegend) and then the cells were washed in PBS containing 2% FBS. Small intestine was opened longitudinally, cut into small pieces and washed in RPMI at 200 rpm for 30 minutes. Subsequently, the washed tissue was digested in 20 ml of RPMI containing collagenase IV (V900893, Sigma), dispase II (04942078001M, Roche) and deoxyribonuclease I (SLBF7798V, Sigma) at 37°C for 35 minutes. Then, the digested tissue was filtered through a 100 pm cell strainer and washed with PBS containing 2% FBS at least 2 times. Lymphocytes of the digested intestinal tissue were isolated by gradient centrifugation with Histopaque-1083 (10831, Sigma).

[0177] The single cell suspension was blocked with TruStain FcX (101320, Biolegend) and stained with APC / Cy7 conjugated B220 (RA3-6B2, Biolegend), FITC conjugated anti-mouse IgA (1040-30, SouthernBiotech) and AF647 coupled anti-human IgA1 (9130-31, SouthernBiotech). After washing three times, the cells were resuspended in PBS containing 2% FBS for data collection by flow cytometer (Verse, BD). In the B220 + In the cell gate, the percentage of mouse IgA or human IgA1 expression was analyzed.

[0178] LCWE preparation

[0179] Cell wall extracts of L. casei (ATCC 11578) were obtained as previously described (Wan F, Wang H, Wang M, et al. J Pathol 2022;257(3):262-273). Briefly, bacteria were harvested during the exponential growth phase while growing in MRS broth (BD). Bacteria were incubated with 4% sodium dodecyl sulfate (SDS) overnight. Cell wall debris was washed at least 8 times to remove residual SDS. The treated cell wall debris was sonicated for 2 hours by cooling in dry ice / ethanol bath. After sonication, cell wall debris was centrifuged at 38000 rpm for 1 hour at 4°C and the supernatant was harvested. Total rhamnose content of cell wall was determined by colorimetric phenol-sulfuric extraction technique as previously described (Lehman TJ, Walker SM, Mahnovski V, et al. Arthritis Rheum 1985;28:652-659). LCWE used in the IgA nephropathy deposition mouse model was emulsified with CFA as previously disclosed (Wan F, Wang H, Wang M, et al. J Pathol 2022;257:262-273).

[0180] Tissue fixation, staining and histopathology

[0181] Fresh kidney tissues were embedded in the compound of optimal cutting temperature and sectioned at 2 pm. The frozen sections were stained with the following antibodies overnight: Dylight 488-conjugated anti-human IgA (ab98553, Abcam), AF488-conjugated anti-mouse IgA (1040-30, SouthernBiotech), rat anti-mouse CD31 (553370, BD Pharmingen) plus AlexaFluor 555-conjugated anti-rat IgG (ab150166, Abcam), and rabbit anti-C3 / C3b antibody (ab200999, Abcam) plus cy3-conjugated anti-rabbit IgG (A0516, Beyotime Biotechnology). After sealing with DAPI Fluoromount-G (0100-20, SouthernBiotech), the sections were imaged with a confocal microscope (LSM780, Zeiss).

[0182] Kidneys, small intestines and spleens were collected, fixed in 10% formalin solution and embedded in paraffin blocks. All sections for histopathology and immunohistochemistry were 2.5 pm thick. Kidneys were stained with hematoxylin and eosin or periodic acid-Schiff or Masson according to standard protocols. After antigen retrieval, intestines and spleens with Peyer's patches lymph nodes were stained with HRP-conjugated anti-human IgA or HRP-conjugated anti-mouse IgA. After standard procedure in DAB chromogenic system, sections were counterstained with hematoxylin and mounted with balsam. For small intestine and kidney Gd-IgA1 staining, deparaffinated paraffin sections were antigen retrieved with 0.05% bacterial protease subtilisin A (P5380, Sigma-Aldrich) for 30 min at room temperature. Non-specific binding was blocked with 3% bovine serum albumin in phosphate-buffered saline blocking solution. Sections were incubated with rat monoclonal anti-human Gd-IgA1 antibody (10777, Immunobio) for 1 h at 37°C, then with Alexa Fluor 555-conjugated goat anti-rat IgG (ab150166, Abcam) for 30 min at 37°C. They were also stained with Dylight 488-conjugated anti-human IgA (ab98553, Abeam) and DAPI Fluoromount-G (0100-20, SouthernBiotech) and then imaged with a confocal microscope (LSM 780, Zeiss).

[0183] Immunofluorescence semi-quantification of the kidneys was assessed by fluorescence intensity in the mesangial area. Mean fluorescence intensity of the small intestine was assessed by the ratio of positive integrated density to villi area.

[0184] Electron microscopy

[0185] Kidney tissues of treated mice were cut into small pieces (about 1 mm 3 ), fixed in phosphate buffer containing 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide and embedded in Epon 812 resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a transmission electron microscope (JEM-1230; JEOL, Tokyo, Japan).

[0186] B cell receptor repertoire sequencing (BCR seq)

[0187] Approximately 1 cm terminal ileum tissue with PP was washed in PBS three times and then preserved in RNA (AM7020, Invitrogen). RNA extraction, library preparation, and data analysis for high-throughput sequencing were performed by Seqhealth Technology Co., Ltd. (Wuhan, China). Total RNA was extracted from samples using TRIzol (15596018, Invitrogen) and DNA digestion by deoxyribonuclease I (EN0521, Thermo Scientific). RNA quality was determined by checking A260 / A280 with a spectrophotometer (ND-ONEC-W, Thermo Scientific). RNA integrity was confirmed by 1.5% agarose gel electrophoresis. Finally, the qualified RNA was quantified with Qubit® 3 Fluorometer (Q33216, Invitrogen) using Qubit® RNA HS Assay Kit (Q32855, Invitrogen). TM One C RNA quality was determined by checking A260 / A280 with a spectrophotometer (ND-ONEC-W, Thermo Scientific). RNA integrity was confirmed by 1.5% agarose gel electrophoresis. Finally, the qualified RNA was quantified with Qubit® 3 Fluorometer (Q33216, Invitrogen) using Qubit® RNA HS Assay Kit (Q32855, Invitrogen). TM 3 Fluorometer (Q33216, Invitrogen) using Qubit® RNA HS Assay Kit (Q32855, Invitrogen). TM RNA quality was determined by checking A260 / A280 with a spectrophotometer (ND-ONEC-W, Thermo Scientific). RNA integrity was confirmed by 1.5% agarose gel electrophoresis. Finally, the qualified RNA was quantified with Qubit® 3 Fluorometer (Q33216, Invitrogen) using Qubit® RNA HS Assay Kit (Q32855, Invitrogen).

[0188] According to the manufacturer's instructions, using KC-Digital TM Stranded BCR-seq Library Prep Kit (DT0815-02, Seqhealth Technology Co., Ltd.) was used for BCR sequencing library preparation with approximately 2 pg of total RNA from each sample. By using 8 random base unique molecular identifiers (UMIs) to label pre-amplified cDNA molecules, the kit eliminated the bias of duplication in PCR and sequencing steps. The library products corresponding to 250-500 bp were enriched, quantified, and finally sequenced on NovaSeq® 6000 (Illumina) with 2 x 150 paired-end reads. Sequencing. Raw sequencing data were first filtered by SOAPnuke (version 1.6.0), and then the clean reads were clustered first according to UMI sequences, in which reads with the same UMI sequence were grouped into the same cluster. The deduplicated consensus sequences were used for BCR-seq analysis. They were mapped to the international ImMunoGeneTics (IMGT) database using MiXCR software (version 3.0.3) to obtain V, D, and J segment rearrangements and CDR3 sequences. P < 0.05 or fold change > 2 were used to screen for significantly differentially expressed genes. The diversity of BCR is determined by the diversity of the complementarity determining region (CDR). When composed of the same V-D-J gene, the CDR3 sequence with the largest functional epitope change of the antibody is called the same clone, and then Shannon's entropy and Simpson's index are introduced to verify the diversity of BCR by the number of non-repeated clones.

[0189] Shannon's entropy:

[0190] Simpson's index:

[0191] Quantitative analysis of O-glycopeptides in IgAl hinge region (HR) using LC-MS analysis

[0192] End serum was diluted to 1 ml with phosphate buffered saline (PBS) solution for hIgA1 purification. In the FPLC system, IgA1 fraction was purified using HiTrap NHS activated HP affinity chromatography column (17-0716-01, Cytiva) coupled with F(ab')2 fragment of goat anti-human IgA antibody (109-006-011, Jackson Immuno). The column was washed with PBS (pH = 7.3), and the fraction was eluted with glycine (pH = 2.7). The eluted liquid was immediately neutralized with tris buffer (pH = 9.0), then pooled and concentrated by ultrafiltration with 30 kDa regenerated cellulose membrane (UFC8030, Ultra-4, Miliipore).

[0193] Purified serum hIgA1 was loaded with non-reducing loading buffer to gradient SDS-PAGE (8012011, BioSci, Shenzhen, Guangdong, China) and electrophoresed at 150 V for 1.5-2 hours. Then the gel was stained by Coomassie protein staining (ab119211, Abcam) and the gel containing monomeric and dimeric hIgA1 was cut for O-glycosylation analysis.

[0194] Strips were cut into smaller pieces (1 mm x 1 mm) and washed with 50 mM ammonium bicarbonate at 37 °C. Gel pieces were shrunk in 100% acetonitrile (ACN) three times and rehydrated in small volumes (10 μΐ) of 50 mM AmBic with 10 mM DTT, 50 mM AmBic with 55 mM iodoacetamide, or 50 mM AmBic, respectively. The small volume solvents were then digested with trypsin at 37 °C for 18 hours, followed by PNGase F treatment at 37 °C for 4 hours. Glycosylated peptides were further enriched by a commercial HILIC column, dried and reconstituted in 10 μΐ, of 0.1% formic acid prior to analysis.

[0195] Peptides were first digested with Thermo Scientific EASY-nLC TM 1200 nano LC system (Thermo Fisher, San Jose) using a trap-elute mode, followed by an ejection into a Thermo Scientific Orbitrap Eclipse mass spectrometer (Thermo Fisher, San Jose) provided with a FAIMS Pro (Thermo Fisher, San Jose). Solvent A was water with 0.1% formic acid, while solvent B was 80% ACN with 0.1% formic acid. After a 3 minute load at a flow rate of 10 microliters / minute in a trap column (Thermo Scientific Acclaim PepMap 100 C18, 75 μιη*2 cm, 3 μιη, ), all peptides were further eluted in an analytical column (Thermo Scientific Acclaim PepMap RSLC, 75 μιη*25 cm, 1.9 μιη, ) at a flow rate of 250 nanoliters / minute using a gradient of 8% to 30% solvent B for 127 minutes and 30% to 90% solvent B for 16 minutes. This was followed by a 14 minute wash.

[0196] Full scan mass spectra were recorded in positive ion mode with a resolution of 60000 (at 200 m / z) over a scan range of 350 to 2000 m / z. The AGC target and maximum injection time were set to 4e5 and 50 milliseconds. dd-MS2 scans used a maximum 1 second setting with 30 second dynamic exclusion. Data were collected using FAMIS Pro at -45 V and -65 V. EThcD spectra were obtained using calibrated charge dependent ETD parameters and 27% NCE HCD supplemental activation with a resolution set to 30000 (at 200 m / z), AGC target set to 4e5, and maximum injection time set to 400 milliseconds.

[0197] Using ByonicTM (Version 3.8.13) and Byologic R Software (Version 3.8-11-x64; Protein Metrics Inc.) was used to extract glycopeptide fragment data from raw files with mass tolerances of ±10 and ±20 ppm for precursor and fragment ions, respectively. Protein Metrics 7.8-Linked Glycan Library was designated as O-glycan modification to analyze intact O-glycopeptides, which contain the HR peptide HYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR and O-glycan. A false discovery rate (FDR) of 1% was applied. The total area under the curve (AUC) in the extracted ion chromatogram (XIC) of the O-glycopeptides of interest was recorded. The content of each glycopeptide was described by the ratio of AUC to total glycopeptide content. The average HR O-glycan composition was then calculated for each sample.

[0198] Statistical analysis

[0199] Continuous normal distribution variables are expressed as mean ± standard deviation (SD). Non-normal distribution variables are represented by median and interquartile spacing (IQR). Differences between two or more groups of normally distributed data were compared using T-test or analysis of variance (ANOVA) and least significant difference (LSD) post-hoc multiple comparisons, supplemented by linear trend test.

[0200] IBM SPSS Statistics 26 software (SPSS Company, Chicago, Illinois, USA) and GraphPad Prism 9 software (GraphPad Software, San Diego, California) were used for all statistical methods and figures. P < 0.05 was considered statistically significant in a two-sided test.

[0201] Example 1. IGHAl + / + Generation of mice

[0202] In this study, the inventors constructed human immunoglobulin heavy chain constant region alpha 1 knock-in (IGHA1 + / + ) mice and investigated the effects of different levels of pathogen exposure on hIgA1 expression and glycosylation. IGH A1 + / +Mice were generated by GemPharmatech Co. Ltd Company (Nanjing, China) and the targeting strategy is shown in Figure 1A Briefly, IGHAl + / + Mice were generated using the CRISPR / Cas9 system (Cong L, Ran FA, Cox D, et al. Science (New York, NY) 2013; 339(6121): 819-823) using Cas9 mRNA, sgRNA (the sequences of sgRNAs are shown in Table 2 below) and donors (which were co-injected into C57BL / 6J heterozygotes using a microinjection method). In other words, a human IGHAl genomic fragment (the nucleic acid sequence of which is shown in SEQ ID NO: 33) replaced the mouse Igha locus. The amino acid sequence of the human IgAl heavy chain constant region expressed by the humanized mice is shown in SEQ ID NO: 34. Subsequently, the embryos were implanted into pseudopregnant females to generate C57BL / 6J F0 mice. The mice were screened by PCR analysis using specific primers (the primer sequences are shown in Table 3 below). The assay also determined the IGHAl genotype and confirmed the transmission of the humanized IGHAl allele through the germline. The F0 mice were then backcrossed onto C57BL / 6J mice for two generations to obtain homozygosity for replacement of the endogenous mouse Igha gene with the human IGHAl gene.

[0203] Table 2. gRNA sequences

[0204]

[0205]

[0206] Table 3. Genotype primers

[0207]

[0208] PCR analysis of tail DNA from representative WT, IGHAl + / - and IGHAl + / + mice confirmed successful replacement of the mouse Igha gene with the human IGHAl gene Figure 6 A). Expression of hIgAl and mouse IgA mRNA in white blood cells from each group indicates that human IgAl can be correctly translated in IGHAl + / - and IGHAl + / + mice Figure 6 B). Circulating levels of human IgAl protein in IGHAl + / + mice are comparable to levels of mouse IgA protein in WT controls Figure 6C). As expected, in IGHA1 + / + Mouse IgA was not detected in mice. Western blot analysis of serum samples from each group confirmed that chimeric IgA1 was expressed as a complete immunoglobulin (IgA1). Figure 1B ).

[0209] Most IgA is secreted by IgA plasma cells in mucosa-associated lymphoid tissue. To assess the normal expression of the human IgHA1 gene in immune and mucosal organs, WT, IgHA1 + / - and IGH1 + / + The levels of mouse IgA and human IgA1 in the spleen were examined in mice. Figure 6 D) and small intestine ( Figure 6 mRNA expression in E). Related to IGH1 + / + The mouse blood cells did not contain mouse IgA, consistent with IGH1. + / - and IGH1 + / + Normal production of chimeric human IgA1 was observed in mice. Flow cytometry analysis of multiple organs (represented as B220+ cells) showed that the human IgA1 gene plays a role in IgA1 production. + / - and IGH1 + / + It is expressed in the spleen, small intestine, bone marrow, and lymph nodes of mice, but not in WT mice. Figure 6 FI). These results indicate that IGHA1 + / - and IGH1 + / + The mouse IgA expression system was matched with that of WT mice.

[0210] Immunohistochemical analysis of tissues containing mouse IgA and human IgA revealed that, compared with WT mice, IGHA1... + / - and IGH1 + / + The localization patterns of antibody-secreting cells in mouse lymphatic and mucosal tissues are similar. Plasma cells are located around rare Peyer's cluster lymph nodes. Figure 1C ) and the intestinal lamina propria and in the intestinal recesses ( Figure 1D Overall, the human IGHA1 gene is fully expressed, thus replacing IGHA1. + / + Mouse IgA in mice.

[0211] To verify IGHA1 + / +The inventors sequenced the B cell receptor (BCR) repertoire in terminal ileum tissue from the Peyer’s patches of mice for their ability to produce a diverse repertoire of immunoglobulins through variable, diversity, and joining gene (V-D-J) rearrangement, somatic hypermutation (SHM), and class switch recombination (CSR). Using specific primers to amplify immunoglobulin subtypes (hlgAl, IgA, IgG, IgM, IgD, IgE), the inventors found that IGHAl + / + Mice exhibited a similar immunoglobulin sub-subtype composition to WT mice Figure 1E ) IGHAl + / + The predominant subtype in the terminal ileum tissue of mice and WT mice was hlgAl (>90%) and IgA, respectively. The inventors defined identical V-D-J genes as clonotypes and assessed BCR diversity using Shannon entropy and Simpson’s index, which measure the number of non-redundant clonotypes. Higher values of Shannon entropy and Simpson’s index indicate greater diversity of BCRs in a sample (Feutrill, A. and M. Roughan, Entropy (Basel), 2021. 23(8)). The diversity distribution in hlgAl + / + Mice was similar to mouse IgA in WT mice, but with a slight increase in the absolute number of hlgAl clonotypes. The frequency of V and J gene usage in hlgAl was also similar to the frequency in mouse IgA Figure 1F , Figure 7 A). Furthermore, complementarity determining region 3 (CDR3) sequences, which exhibit the greatest functional epitope diversity and range typically from 8 to 20 amino acids, showed a uniform normal distribution in hlgAl and IgA Figure 1G ) IGHAl + / + BCR clusters were increased in mice, but there was no significant bias in clustering analysis of immunoglobulin heavy chain (IGH) as revealed by a Venn diagram that uncovered 3055 common IGH sequences Figure 7 B). The distribution of the top 100 IGH clusters was uniform and there was no significant difference between the two strains Figure 7 C). Overall, IGHAl + / + Mice successfully performed class switch recombination and affinity maturation to synthesize a variety of immunoglobulin types similar to WT mice to cope with the complexity of mucosal immunity.

[0212] Example 2. LCWE-induced IGHA1 + / + Generation and characterization of mice

[0213] Pathogen exposure promotes hlgAl production in multiple organs

[0214] IgA is the most abundant antibody isotype produced by humans, found primarily in mucosal regions, but also in the blood ( M. and The Role of IgA in the Pathogenesis of IgA Nephropathy. Int J Mol Sci, 2019. 20(24)). In humans, IgA can exist in three main forms: monomer (mIgA), polymer (mainly dimer; pIgA, dIgA), and IgA-containing complex. All GF, SPF, and CV-fed IGHAl + / + Mice synthesized all three forms of hIgAl Figure 2 A-B, Figure 8 A). And as the level of pathogen exposure increased, IGHAl + / + Both mIgA (approximately 160 kD) and pIgA, especially dIgA (approximately 340 kD), in mouse serum increased. The feeding conditions also changed the serum Gd-IgAl levels Figure 2 B). IGHAl + / + The elevated Gd-IgAl production in mice suggests that infection has an impact on Gd-IgAl export.

[0215] To determine the source of the elevated serum hIgAl, the inventors measured the mRNA expression level of IGHAl in lymphocytes from SP, PP, MLN, and ILN Figure 2 C). RT-qPCR results showed that IGHAl mRNA expression in both systemic and mucosal sites of hIgAl production increased in the order of GF, SPF, and CV-fed mice. Interestingly, the inventors found that in systemic sites such as SP and ILN, SPF-fed mice had comparable IGHAl expression to GF, while the expression level was comparable to CV in mucosal sites such as PP and MLN. Perhaps because in SPF conditions, IGHAl + / + Mice established commensal bacteria in mucosal sites, but not enough pathogen stimulation for large amounts of systemic hIgAl production.

[0216] In humans and mice, nearly 80% of the total plasma cells in the body are located in the intestinal mucosa, where they secrete dimeric IgA (Tezuka, H. and T. Ohteki, Regulation of IgA Production by Intestinal Dendritic Cells and Related Cells. Frontiers in Immunology, 2019.10: p.1891). hIgA1 is rarely detected in the SI lamina propria of GF mice. + Cells. The inventors recognized that when IGHA1 was colonized with symbiotic bacteria... + / + In mice, SI hIgA + The mean fluorescence intensity (MFI) of plasma cells increased significantly. Figure 2 These results indicate that elevated serum hIgA1 originates from multiple immune tissues, with both systemic and mucosal sites involved in this process.

[0217] LCWE antigen induces an increase in serum Gd-IgAl and hlgAl -containing complex levels in IGHAl+ / +mice

[0218] The LCWE used in the IgA kidney deposition mouse model was emulsified with Freund's complete adjuvant (CFA) as previously disclosed (Wan F, Wang H, Wang M et al., Journal of Pathology 2022; 257:262-273). The LCWE was administered to 2-month-old IgA1 mice raised under barrier conditions. + / + Mice were intraperitoneally injected with LCWE (emulsified with CFA) or PBS eight times and observed until they reached 8 months of age. Figure 3 A, Figure 9A). Gd-IgA1 has been identified as an initial trigger in the pathogenesis of IgA nephropathy (Wyatt, R.J. and B.A. Julian, IgA nephropathy. N Engl J Med, 2013. 368(25): p. 2402-14; Lai, K.N., et al., IgA nephropathy. Nat Rev Dis Primers, 2016. 2: p. 16001; Novak, J., et al., Aberrant Glycosylation of the IgA1 Molecule in IgA Nephropathy. Semin Nephrol, 2018. 38(5): p. 461-476), and it is well known that circulating immune complexes containing IgA are pathogenic, which is the most common form of kidney deposition (Zhang, X., et al., Poly-IgA Complexes and Disease Severity in IgA Nephropathy. Clin J Am Soc Nephrol, 2021. 16(11): p. 1652-1664; He, J.W., et al., Perspectives on how mucosal immune responses, infections and gut microbiome shape IgA nephropathy and future therapies. Theranostics, 2020. 10(25): p. 11462-11478). Upon LCWE antigen stimulation, IGHAl + / + Mouse export of LCWE-specific hlgAl and Gd-IgAl Figure 3 B). Serum Gd-IgAl absolute values and relative proportion to hlgAl were significantly increased in LCWE-induced IGHAl + / + (Also referred to as “IGHA + / + -LCWE” in this application) mice Figure 3 C-D). And hlgAl -mouse IgG complexes and CD89-bound plgA complexes developed to even higher levels upon stimulation even at 8 months of age Figure 3 E). Serum IgM increased with age and IgG peaked immediately upon stimulationFigure 9 B).

[0219] Purification of PBS and LCWE-induced IGHAl + / + Serum hIgAl from mice, stained with anti-hIgAl antibodies and verified hIgAl proteins in the gel Figure 3 F) The hIgAl gel band was then excised and sent for LC-MS analysis of hIgAl HR O-glycans Figure 10 ) LCWE-induced IGHAl + / + The Gal / GalNAc ratio in IgAl HR from mice was decreased while the number of GalNAc residues remained unchanged Figure 3 G-H).

[0220] In summary, both Gd-IgAl and hIgAl immune complexes were continuously elevated under strong mucosal stimulation, which mimicked the first to third hits of the ‘multiple hit’ hypothesis.

[0221] TD and TI pathways are responsible for the enhanced mucosa-derived Gd-IgAl

[0222] The terminal ileum is where the PP are concentrated, serving as the main site for antigen sampling and immune induction in gut-associated lymphoid tissue (GALT)50. LCWE-induced IGHAl + / + hIgAl and Gd-IgAl concentrations were higher in the small intestinal mucus from mice Figure 4 A). To understand the process of Gd-IgAl + enhancement in the SI lamina propria Figure 4 B), mRNA levels were analyzed in the terminal ileum. Initial B cells that undergo class switch recombination to produce IgAl in the gut are regulated by T cell-dependent (TD) and T cell-independent (TI) immune responses (Tezuka, H., and T. Ohteki, Regulation of IgA production by intestinal dendritic cells and related cells. Front Immunol, 2019. 10: p. 1891). TGFp is influenced by both TD and TI pathways, and the inventors observed a thousand-fold increase in transcription in the LCWE-induced group compared to the other three groups Figure 4E). And through the TI pathway, IgAl class switching occurs directly in response to BAFF and APRIL secreted by DCs activated by PP and Toll-like receptor ligands in the lamina propria (Barratt, J., et al., Why Target the Gut to Treat IgA Nephropathy? Kidney Int Rep, 2020. 5(10): p. 1620-1624; Coppo, R., The Gut-Renal Connection in IgA Nephropathy. Semin Nephrol, 2018. 38(5): p. 504-512). BAFF and APRIL mRNA levels increase in terminal ileal tissue under LCWE stimulation Figure 4 C-D). Transcription of inducible nitric oxide (NO) synthase (iNOS) is also elevated Figure 4 F), which is expressed in DCs and macrophages and mediates massive NO production, inducing TGFp receptors on B cells in the TD pathway and BAFF and APRIL expression on DCs in the TI pathway (Tezuka, H. and T. Ohteki, Regulation of IgA production by intestinal dendritic cells and related cells. Front Immunol, 2019. 10: p. 1891). That is, inflammatory cytokine transcription levels are elevated in both TD and TI pathways, leading to pathogen-stimulated IGHAl + / + Mice produce more Gd-IgAl secreting plasma cells in the gut.

[0223] Gd-IgAl triggers severe complement activation and pathological lesions in the kidney

[0224] In line with the gradient increase in serum hIgAl and hIgAl complex-containing levels, the inventors observed that hIgAl, especially Gd-IgAl, in LCWE-induced IGHAl + / + Deposits in the mesangial region of the glomerulus of mice showing continuous and stable human IgAl (positive rate: 100%) mesangial deposits with C3 co-deposits (positive rate: 80%) until 8 months old Figure 5 A). Compared to the PBS group, LCWE-induced IGHAl + / + Mice inhibited the increase in semi-quantitative scores of C3 and hIgAl Figure 5 B-C). Histology showed a significant increase in mesangial expansion, hypercellularity, and tubulointerstitial fibrosis. Electron microscopy showed LCWE-induced IGHAl + / + Typical mesangial electron-dense deposits in mice Figure 5D). Unlike the Kawasaki disease mouse model (Noval Rivas, M., et al., Intestinal Permeability and IgA Provoke Immune Vasculitis Linked to Cardiovascular Inflammation. Immunity, 2019. 51(3): p. 508-521. e6), LCWE-induced IGHA1 + / + Mice did not have cardiac vascular or aortic inflammation Figure 11 A). Compared to the early reported LCWE-induced wild-type mice (C3 positivity rate: 30%) (Wan, F., et al., Sustained release of Lactobacillus casei cell wall extract can induce a continuous and stable IgA deposition model. Journal of Pathology, 2022. 257(3): p. 262-273), LCWE-induced IGHA1 + / + Mice had more severe complement C3 activation, mesangial expansion, and endocapillary hypercellularity (see Table 4 below). But there were no significant differences in clinical markers such as serum creatinine, urea nitrogen, or urine albumin-creatinine ratio by stimulation Figure 11 B).

[0225] Table 4. Pathological features of PBS-induced IGHA1 + / + Mice, LCWE-induced WT (C57BL / 6), and IGHA1 + / + Mice, LCWE-induced WT (C57BL / 6), and IGHA1

[0226] M1 E1 S1 T1 C1 IGHAl -PBS (n = 12) 9(75％) 5(41.7％) 0 0 1(8.3％) WT-LCWE (n = 8) 5(62.5％) 2(25％) 0 0 0 IGHAl -LCWE (n = 12) 11(91.7％) 12(100％) 0 0 3(25％)

[0227] Discussion

[0228] Insights from IgAN models can provide valuable information on various aspects of IgAN pathogenesis and contribute to the development of IgAN-specific drugs (Suzuki, H. and Y. Suzuki, Murine Models of Human IgA Nephropathy. Kidney Res. Clin. Pract., 2018. 38(5): p. 513-520). Mucosal polymeric IgA and Gd-IgAl are important in the pathogenesis of IgAN (Schena, F.P. and S.N. Cox, Biomarkers and Precision Medicine in IgA Nephropathy. Kidney Res. Clin. Pract., 2018. 38(5): p. 521-530). The novel IgAN mouse model described herein can further deepen our understanding of the disease. The inventors successfully constructed IGHAl + / + mouse model. Under pathogen exposure and enhanced antigen stimulation, mucosal hypersecretion and HR hypogalactosylated hlgAl showed significant kidney deposition, with stronger effects on activating complement and initiating inflammatory damage.

[0229] IGHAl + / + The mouse model successfully expressed chimeric hlgAl with human IgAl heavy chain constant region and mouse variable region but without mouse IgA. During the immune response, B lymphocyte progenitor cells rearrange V(D)J gene segments to generate the antigen-binding region of BCR (Cooper, M.D. and M.N. Alder, The evolution of adaptive immune systems. Cell, 2006. 124(4): p. 815-22), SHM and CSR to generate high-affinity antibodies with biological effector functions (Tezuka, H. and T. Ohteki, Regulation of IgA production by intestinal dendritic cells and related cells. Front. Immunol., 2019. 10: p. 1891). BCR seq enables IGHAl + / +Mouse and WT mice have identical immunoglobulin gene structure. Compared to aKI mice that lack mouse IgM and have mouse IgA (Duchez, S., et al., Premature replacement of mu with alpha immunoglobulin chains impairs lymphopoiesis and mucosal homing but promotes plasma cell maturation. Proc Natl Acad Sci U S A, 2010. 107(7): p. 3064-9), IGHAl + / + Mice can better defend against pathogens and mimic human mucosal immunity. In humans, 75-90% of serum IgA is monomeric, 10-15% is in polymer form, and 1% is bound in circulating immune complexes (Braun, A. and M. Thurnheer, The role of IgA in mucosal immunity. Front Immunol, 2019. 10: p. 1791). M. and IgA in the pathogenesis of IgA nephropathy. Int J Mol Sci, 2019. 20(24)). The third hit of the IgAN ‘multiple hit’ hypothesis is the formation of Gd-IgAl -containing immune complexes, which can form as self-aggregates, in complex with anti-Gd-IgAl antibodies (IgG or IgA) or soluble CD89 (Selvaskandan, H., J. Barratt, and C.K. Cheung, Immunological drivers of IgA nephropathy: Exploring the mucosa-kidney link. Int J Immunogenet, 2022. 49(1): p. 8-21). The levels of poly-IgA immune complexes are significantly higher in IgAN patients compared to healthy controls (Zhang, X., et al., Poly-IgA Complexes and Disease Severity in IgA Nephropathy. Am J Kidney Dis, 2021. 78(11): p. 1652-1664). Mice do not express CD89, but the inventors can use recombinant CD89 affinity probes as an ex vivo tool to measure IGHAl + / +Poly-hIgAl levels in mouse sera. With stronger pathogen exposure, not only hIgAl-IgG complexes, but also poly-hIgAl complexes binding to CD89, levels significantly increased. Moreover, serum Gd-IgAl levels are reported to be elevated in up to 90% of IgAN patients from different cohorts worldwide (Zhang, H., and J. Barratt, Is IgA nephropathy the same disease in different parts of the world? Semin Immunopathol, 2021. 43(5): p. 707-715). The present inventors verified that IGHAl + / + The hIgAl hinge region of mice is O-glycosylated. And upon LCWE stimulation, HR qualitatively showed a reduction in Gal modification. Moreover, serum Gd-IgAl levels were quantitatively elevated five-fold compared to controls. The present inventors report for the first time an elevated Gd-IgAl mouse model in the world.

[0230] Chimeric IgAl in IGHAl + / +Functions and roles in mice are related to symbiotic flora and inflammatory responses (Monteiro, R.C., Role of IgA and IgA fc receptors in inflammation. J Clin Immunol, 2010. 30(1): p. 1-9; Wilmore, J.R., et al., Commensal Microbes Induce Serum IgA Responses that Protect against Polymicrobial Sepsis. Cell Host Microbe, 2018. 23(3): p. 302-311. e3). Emerging evidence confirms a key role of mucosal immunity in the pathogenesis of IgAN (Gesualdo, L., V. Di Leo, and R. Coppo, The mucosal immune system and IgA nephropathy. Semin Immunopathol, 2021. 43(5): p. 657-668).Current experimental results in IgAN-like mouse models support the view that mucosal polymers of IgA by mucosal exposure to food and microbial antigens can induce IgAN-like injury in glomeruli (McCarthy, D. D., et al., Mice overexpressing BAFF develop a commensal flora-dependent, IgA-associated nephropathy. J Clin Invest, 2011. 121(10): p. 3991-4002; Kano, T., et al., Nasal-associated lymphoid tissue is the major induction site for nephritogenic IgA in murine IgA nephropathy. Kidney Int, 2021. 100(2): p. 364-376; Monteiro, R.C. and Y. Suzuki, Are there animal models of IgA nephropathy? Semin Immunopathol, 2021. 43(5): p. 639-648). In secondary lymphoid organs including lymph nodes, SPs, PP, and other MALT (such as GALT, T, and B cells) are present and interact with entrapped antigens (Megha, K.B. and P.V. Mohanan, Role of immunoglobulin and antibodies in disease management. Int J Biol Macromol, 2021. 169: p. 28-38). IGHAl + / + Human IgAl expression in mice was enhanced in these immunological induction sites. However, hIgA + and Gd-IgAl + Plasma cells were abundantly present in the SI lamina propria of IGHAl + / + Mice, which can reflect germinal center (GC) independent or shorter GC involvement in LCWE-induced IgA +B cell production (Seifert, M. and R. Küppers, Human memory B cells. Leukemia, 2016. 30(12): p. 2283-2292). The mRNA levels in the terminal ileum explained that not only the TD but also the TI pathway affected the synthesis of mucosal hlgAl. Thus, LCWE-induced IGHAl + / + Mice are very suitable for studying the mucosa- kidney axis of IgA nephropathy.

[0231] LCWE-induced mice exhibited pathological features of IgAl and C3 deposits. By 8 months of age, our model presented histological lesions of IgAN-like mesangial hypercellularity, intracapillary hypercellularity, segmental sclerosis, interstitial inflammation, tubular atrophy, and a few mice showed crescent formation. Compared to the early reported LCWE-induced wild-type mice (Wan, F., et al., Sustained release of Lactobacillus casei cell wall extract can induce a continuous and stable IgA deposition model. Journal of Pathology, 2022. 257(3): p. 262-273), LCWE-induced IGHAl + / + Mice have a stronger ability to activate pathogenic Gd-IgAl complement, so they have more severe complement C3 activation, mesangial expansion, and intracapillary hypercellularity compared to WT-LCWE mice or PBS-LCWE mice.

[0232] In summary, the inventors established the first mouse model with elevated serum Gd-IgAl in the world. In the future, the animal model will not only be used to explore the mechanisms of IgA nephropathy affecting genetic and environmental factors, but also will help to explore new therapeutic strategies or promote the development of new drugs. Our group has already used LCWE-induced IGHAl + / + Mice explored the in vivo effect of chimeric fusion of IgA protease with Fc (Xie, X., et al., Chimeric Fusion between Clostridium Ramosum IgA Protease and IgG Fc Provides Long-Lasting Clearance of IgA Deposits in Mouse Models of IgA Nephropathy. Journal of the American Society of Nephrology, 2022. 33(5): p. 918-935). Therefore, this model has great prospects in advancing IgAN drug development and promoting clinical translation.

[0233] Example 3. Immunostimulation with different immunostimulants

[0234] At baseline, 2-month-old IGHAl + / + Mice were injected intraperitoneally with Freund's complete adjuvant (CFA, Cat# F5881) alone, once every two weeks for a total of 6 injections, at a dose of 2 μg / g mouse weight per injection. Samples were taken from mice at 5 months of age to 7.5 months of age.

[0235] The results show that the levels of human IgAl, mouse IgG, mouse IgM, and human IgAl-mouse IgG complex in the serum of 5-month-old mice were significantly increased Figure 12 ) compared to the control mice.

[0236] The positive rates of human IgA, C3, IgM, and IgG measured by immunofluorescence in kidney frozen sections of 7.5-month-old IGHAl + / + Mice (mice #6-14, a total of 9 mice) were 100%, 88.9%, 100%, and 33.3% respectively Figure 13 ).

[0237] In the PAS pathological staining assay of the kidney, one 4-month-old mouse (mouse #1) was found to have increased matrix in the mesangial region of the glomerulus; 5-month-old mice (mice #2-3) were found to have increased matrix and cells, significantly increased endothelial cells, and segmental glomerulosclerosis Figure 14 ) in the mesangial region of the glomerulus. In addition, 6-month-old mice (mice #4-5) were found to have mild proliferation in the mesangial region of the glomerulus, and the lesions were regressed compared to the 5-month-old mice. For 7.5-month-old mice (mice #6-14), the lesions entered the chronic stage, and proliferative lesions were found in the mesangial region of the glomerulus in all 9 mice. For example, mouse #6 was found to have increased matrix and cells in the mesangial region of the glomerulus and segmental glomerulosclerosis Figure 15 ).

[0238] The levels of serum creatinine (SCR) and urine albumin-creatinine ratio (ACR) were measured, and no statistically significant difference was found between 5-month-old mice (6 needles) and 7.5-month-old mice. However, the ACR value of the mouse with a relatively severe kidney pathological phenotype (e.g., mouse #6) was significantly higher than that of the other test mice and the control mice Figure 16 ).

[0239] In summary, stimulation with CFA alone also caused elevated serum IgA and complex levels in IGHAl humanized mice, and IgA deposition rates reached 100% in the mesangial region of glomeruli in 7.5-month-old mice. The pathological changes in the kidney could be divided into an acute phase and a chronic phase. During the administration period (5-month-old), the matrix and cells increased in the mesangial region of glomeruli, and the endothelial cells increased significantly; after the administration was stopped (6-month-old), the lesions regressed. The pathological changes in the kidney subsequently entered the chronic phase, and the matrix and cells increased in the mesangial region of glomeruli in 7.5-month-old mice, and glomerular segmental sclerosis was found. The urine albumin / creatinine ratio (ACR) of mice with relatively severe disease increased significantly.

[0240] Other types of immune stimulants were also tested in the examples, such as saponin, aluminum hydroxide (Al(OH)3), lipopolysaccharide (LPS), Freund’s Incomplete Adjuvant (IFA), CFA, and LCWE emulsified with CFA (LCWE-CFA). All of these immune stimulants are commercially available, such as saponin (Sigma, Catalog # SAE0073), aluminum hydroxide (Thermo, Catalog # 77161), lipopolysaccharide (Sigma, Catalog # L2630), Freund’s Incomplete Adjuvant (Sigma, Catalog # F5506), and CFA (Sigma, Catalog # F5881). The administration schedule is described in Table 5 below. For each group, at baseline, 2-month-old IGHAl humanized mice were injected intraperitoneally with the corresponding test sample, and the mice were housed under barrier conditions. + / + Mice were injected intraperitoneally three times per week for the first 2 weeks, and then once per week for the following 2 weeks. The observation period was extended until the mice reached 8 months of age. + / + Mice were injected intraperitoneally three times per week for the first 2 weeks, and then once per week for the following 2 weeks. The observation period was extended until the mice reached 8 months of age.

[0241] Table 5. Administration schedule

[0242]

[0243]

[0244] Serum human IgA levels in each group before and after stimulation are shown in Figure 22A and Figure 22B As shown in Figure 22A and Figure 22B In the LCWE-CFA group, serum human IgA levels increased significantly and persistently, and also remained at a relatively high level. In the Al(OH)3, CFA, and IFA groups, serum human IgA levels also increased significantly, but decreased at a later stage.

[0245] Serum human IgA-mouse IgG complex levels in each group before and after stimulation are shown in Figure 23A andFigure 23B As shown. Figure 23A and Figure 23B As shown, the serum human IgA-mouse IgG complex levels were significantly increased in each of the Al(OH)3, IFA, CFA, and LCWE-CFA groups, with the LCWE-CFA group reaching the highest level.

[0246] Before and after stimulation, the urinary albumin-creatinine ratio (ACR) for each group was as follows: Figure 24A and Figure 24B As shown. Figure 24A and Figure 24B As shown, there was no significant difference in urinary ACR among the groups.

[0247] The deposition of human IgA (hIgA) and C3 in each group was measured by immunofluorescence and PAS pathological staining of the kidneys, as well as PAS staining as shown in the figure. Figure 25 As shown. Figure 25 As shown, the positivity rate and intensity of hIgA in the LCWE-CFA group were higher than all other groups, with the CFA group ranking second. Similarly, the positivity rate and intensity of C3 deposition in the Al(OH)3 group were higher than all other groups, with the LCWE-CFA group ranking second. Furthermore, increased matrix, widening of the glomerular mesangial region, and a significant increase in endothelial cells were observed in the Al(OH)3, CFA, and LCWE-CFA groups, and crescent formation was observed in some mice.

[0248] The semi-quantitative scores of immunofluorescence images and photomicrographs of mice in each group are also shown in Table 6 and Figure 26 below. As shown in Figure 26, there was no significant difference in the intensity of IgM in mice among each group ( Figure 26A The LCWE-CFA group had higher levels of human IgA (hIgA) than any other group, and the CFA group ranked second. Figure 26B The intensity of C3 deposition in the Al(OH)3 group was higher than that in all other groups, and the LCWE-CFA group ranked second. Figure 26C ).

[0249] Table 6. Pathological staining scores of kidneys in 8-month-old mice (based on IgAN MEST-C scores)

[0250]

[0251]

[0252] Example 4. Immunostimulation with different administration routes (intraperitoneal vs intranasal)

[0253] At baseline, towards 2-month-old IGHA1 + / +Mice were administered PBS or CFA-emulsified LCWE (inLCWE or ipLCWE group) intranasally or intraperitoneally over a one-month period, three times a week for the first two weeks and once a week for the following two weeks. Samples were obtained from 7-month-old mice.

[0254] like Figure 17 As shown, the results indicated that, compared to the PBS group, the serum levels of human IgA1 and mouse IgG in 4-month-old mice in the ipLCWE group were significantly increased; for the inLCWE group, the level of human IgA1 tended to increase, but there was no significant difference compared to the PBS group. The serum levels of human IgA1, mouse IgG, mouse IgM, and human IgA1-mouse IgG complexes in 7-month-old mice in the ipLCWE group were significantly increased. For example, the level of human IgA1 in the ipLCWE group was twice that in the PBS group. However, there were no significant differences in these biomarkers between the inLCWE and PBS groups. Figure 17 ).

[0255] For example, in frozen sections of the kidneys of 7-month-old mice, the positivity rate of human IgA1 in the inLCWE group was 75% by immunofluorescence assay, which was lower than the 100% positivity rate in the ipLCWE group. The positivity rates of C3 in the inLCWE group and the ipLCWE group were 12.5% ​​and 40%, respectively. Figure 18 ).

[0256] In PAS pathological staining of the kidneys of 7-month-old mice, no obvious pathological changes were found in the inLCWE group, while an increase in matrix and cells in the glomerular mesangial region was found in the ipLCWE group. Figure 19 ).

[0257] In summary, comparing the inLCWE and ipLCWE groups, the results showed that at the end of the intranasal administration period (4 months old) in the inLCWE group, serum IgA levels increased, and the IgA deposition rate in the glomerular mesangial region of 7-month-old mice was 75%, lower than the 100% deposition rate in the ipLCWE group. The positivity rates and intensities of IgA and C3 in the inLCWE group were also lower than those in the ipLCWE group. Furthermore, no pathological changes in the kidneys were observed in the inLCWE group.

[0258] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principles and spirit of the invention and its modifications, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing genetically modified rodents, the method comprising: (i) Modifying the genome of rodents to enhance their endogenous... Igha The locus incorporates a nucleotide containing a heterologous gene encoding a human IgA heavy chain constant region containing an O-glycosylated hinge region; and (ii) Using a mixture containing Freund's complete adjuvant (CFA) and Lactobacillus casei ( Lactobacillus casei The rodents produced in step (i) of the formulation treatment of cell wall extract (LCWE); in, The rodent endogenous [product / product] has been deleted. Igha All endogenous rodents at the gene locus Igha The gene is replaced with the nucleotide containing the heterologous gene; the rodent does not express the endogenous non-human IgA heavy chain constant region and shows increased levels of galactose-deficient IgA protein.

2. The method of claim 1, wherein step (i) comprises introducing a nucleotide containing the heterologous gene into the rodent pluripotent cell to obtain a rodent pluripotent cell containing the heterologous gene, and preparing rodents using the rodent pluripotent cell containing the heterologous gene.

3. The method of claim 2, wherein the nucleotide containing the heterologous gene is introduced into the pluripotent cell of the rodent using a CRISPR / Cas system.

4. The method of claim 3, wherein the CRISPR / Cas system comprises a Cas9 protein and a guide RNA, the guide RNA targeting the endogenous Igha Guide RNA target sequence within the locus.

5. The method of claim 2, wherein the pluripotent cell is an embryonic stem (ES) cell.

6. The method of claim 1, wherein the nucleotide of the heterologous gene is operatively linked to an endogenous regulatory element of the rodent.

7. The method according to claim 6, wherein the endogenous regulatory element is a promoter, enhancer, or silencer.

8. The method of claim 1, wherein the nucleotide comprising the heterologous gene is human. IGH1 A segment of the genome of a gene.

9. The method of claim 1, wherein the heterologous gene comprises human... IGH1 Both encoded and non-coded sequences.

10. The method of claim 1, wherein the rodent produces chimeric IgA1, the heavy chain of the chimeric IgA1 comprising a rodent variable region and a human constant region.

11. The method of claim 1, wherein the heterologous gene is a human gene encoding the human IgA1 heavy chain constant region. IGH1 Gene.

12. The method of claim 1, wherein in step (ii), the rodent produced in step (i) is treated with a formulation comprising LCWE emulsified with CFA.

13. The method of claim 1, wherein in step (ii), the preparation is injected intraperitoneally into the rodent.

14. The method of claim 1, wherein the formulation is injected intraperitoneally into the rodent at consecutive low doses.

15. The method of claim 1, wherein the formulation is injected intraperitoneally into the rodent at a dose of less than 1 μg LCWE and / or less than 5 μL CFA per gram of the rodent.

16. The method of claim 1, wherein the formulation is applied to the rodent for at least three months.

17. The method of claim 1, wherein the formulation is applied to the rodents produced in step (i) at an interval of three times a week for the first two weeks and once a week for the next two weeks for a period of one month; or the formulation is applied to the rodents produced in step (i) at an interval of once every two weeks for a period of three months.

18. The method of claim 1, wherein the rodent exhibits clinical signs of IgA nephropathy.

19. The method of claim 1, wherein the rodent exhibits increased levels of galactose-deficient IgA protein in serum or intestinal mucus.

20. The method according to any one of the preceding claims, wherein the rodent is a mouse or a rat.

21. The uses of rodents as animal models of IgA nephropathy, among which, Endogenous in the genome of the rodent Igha All endogenous rodents at the gene locus Igha The gene was deleted and replaced with nucleotides containing a heterologous gene encoding the human IgA heavy chain constant region containing an O-glycosylated hinge region; it was replaced with nucleotides containing Freund's complete adjuvant (CFA) and Lactobacillus casei (…). Lactobacillus casei The rodents were treated with a formulation of cell wall extract (LCWE); The rodents did not express the endogenous non-human IgA heavy chain constant region and exhibited clinical signs of IgA nephropathy, showing increased levels of galactose-deficient IgA protein.

22. The use according to claim 21, wherein the rodent exhibits increased levels of galactose-deficient IgA protein in serum or intestinal mucus.

23. Use of rodents prepared according to any one of claims 1 to 20 in evaluating the efficacy of candidate drugs for the treatment or prevention of IgA nephropathy, said evaluation comprising: Provided a rodent prepared by the method according to any one of claims 1 to 20, said rodent exhibiting clinical signs of IgA nephropathy; The candidate drug is administered to the rodents; and the rodent control is evaluated to determine whether the candidate drug inhibits one or more symptoms of IgA nephropathy compared to a rodent control that has not been administered the candidate drug.

24. Use of rodents prepared according to any one of claims 1 to 20 in the identification of candidate drugs for the treatment or prevention of IgA nephropathy, the identification comprising: Provided a rodent prepared by the method according to any one of claims 1 to 20, said rodent exhibiting clinical signs of IgA nephropathy; The candidate drug is administered to the rodents; and the rodent control is evaluated to determine whether the candidate drug inhibits one or more symptoms of IgA nephropathy compared to a rodent control that has not been administered the candidate drug.

25. The use according to claim 23 or 24, wherein the rodent exhibits increased levels of galactose-deficient IgA protein in serum or intestinal mucus.

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