Non-human animals containing genetically modified endogenous IgA locus and methods for producing them

Abstract

This disclosure provides a non-human animal that exhibits clinical signs of IgA deposition-related disease (e.g., IgA nephropathy) and whose genome contains a genetically modified endogenous Igha locus that includes a heterologous gene encoding the human IgA (e.g., IgA1) heavy chain constant region or a fragment thereof. This disclosure also provides a method for producing or using a non-human animal.

Description

[Technical Field]

[0001] This invention relates to transgenic non-human animals that are generally useful for research on IgA deposition-related diseases. More specifically, this invention relates to transgenic non-human animals containing a genetically modified endogenous IgA locus, and to methods for producing the same or using the same. [Background technology]

[0002] IgA is the major pathogenic antibody isotype in IgA nephropathy (IgAN) and exists in three forms: monoIgA, secretory IgA, and IgA-containing complexes. Compared to healthy volunteers, serum IgA concentrations were elevated in individuals with IgAN (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 co-dominant immunodeposits in the glomeruli and elevated levels of galactose-deficient IgA1 (also known as Gd-IgA1). To elucidate the pathogenesis of IgAN, the "multi-hit" hypothesis has been proposed, with galactose-deficient IgA1 (Gd-IgA1) defined as the initial hit. Gd-IgA1 is thought to induce renal damage by forming a macromolecular immune complex that causes nephropathy (Novak J, Barratt J, Julian BA, et al., Semin Nephrol 2018; 38:461-476). Only primates and humans possess the O-glycosylated IgA1 subclass. Therefore, humanized IgA1 animals may be useful in elucidating the pathogenesis of IgAN. To develop an IgAN-like disease model, a fully human IgA1 knock-in (α1KI) mouse model was created and backcrossed with human CD89 transgenic mice (Duchez S, Amin R, Cogne N, et al., Proc Natl Acad Sci USA 2010; 107:3064-3069; Berthelot L, Papista C, Maciel TT, et al., The Journal of experimental medicine 2012; 209:793-806). However, due to the limited gene editing technology available, human IgA1 was used to substituted the mouse IgM heavy chain in these α1KI mice (Wehbi B, Oblet C, Boyer F, et al., J Am Soc Nephrol 2019; 30:1238-1249). α1KI mice possessed both human IgA1 and mouse IgA, but lacked IgM, which may hinder research into the pathogenesis of IgAN.Several classical IgAN models typically show limited histological changes in the kidney, whether or not they are subjected to genetic manipulation.

[0003] Therefore, to understand the mechanisms leading to this disease and develop therapeutic approaches, novel IgAN animal models are needed. SUMMARY OF THE INVENTION

[0004] In one aspect, the present disclosure provides a method for generating a genetically modified non-human animal. Specifically, the method includes: (i) modifying the genome of a non-human animal to incorporate a nucleotide containing a heterologous gene encoding a human IgA heavy chain constant region or a fragment thereof into the endogenous Igha locus of the non-human animal; and (ii) treating the non-human animal generated in step (i) with a formulation containing an immune stimulant.

[0005] In some embodiments, step (i) includes introducing a nucleotide containing a heterologous gene into pluripotent cells of a non-human animal to obtain non-human animal pluripotent cells containing the heterologous gene, and generating a non-human animal using the non-human animal pluripotent cells containing the heterologous gene. In some embodiments, the nucleotide containing the heterologous gene is introduced into pluripotent cells of a non-human animal by a CRISPR / Cas system. In some embodiments, the CRISPR / Cas system includes a Cas9 protein and a guide RNA targeting a guide RNA target sequence within the endogenous Igha locus. In some embodiments, the pluripotent cells are embryonic stem (ES) cells. In some embodiments, a part or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha locus is deleted and replaced with a nucleotide containing a 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 containing the heterologous gene is operably linked to an endogenous regulatory element (e.g., a promoter, enhancer, silencer, etc.) of the non-human animal.

[0007] In some embodiments, the nucleotides containing the heterologous gene are genomic fragments of the human IGHA1 gene. In some embodiments, the heterologous gene contains both the human IGHA1 coding sequence and the non-coding sequence. In some embodiments, the non-human animal produces chimeric IgA1 in which the heavy chain contains a non-human variable region and a human constant region or fragment thereof. In some embodiments, the human IgA1 heavy chain constant region of the non-human animal contains an O-glycosylated hinge region. In some embodiments, the heterologous gene is a human IGHA1 gene encoding the human IgA1 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 aluminum adjuvants, emulsion adjuvants, biological adjuvants, and saponins.

[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 lipopolysaccharides or derivatives thereof, Toll-like receptor (TLR) agonists (e.g., TLR4 ligand, TLR7 ligand, TLR8 ligand, and TLR9 ligand), immunostimulatory oligonucleotides (e.g., DNA or dsRNA containing CpG), cytokines (e.g., IL-12), interferons, endotoxins, and lipids (e.g., lipid A or its analogues). In some embodiments, the biological adjuvant is a lipopolysaccharide or a derivative thereof.

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

[0013] In some embodiments, in step (ii), the non-human animals prepared in step (i) are treated with a formulation containing LCWE emulsified with CFA.

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

[0015] In some embodiments, the formulation is administered to non-human animals for at least one month. In some embodiments, the formulation is administered to non-human animals for at least three months. In some embodiments, the formulation is administered to the non-human animals prepared in step (i) three times a week for the first two weeks, and then once a week for the following two weeks, for one month. In some embodiments, the formulation is administered to the non-human animals prepared in step (i) once every two weeks for three months.

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

[0017] In another embodiment, the present disclosure provides non-human animals prepared by the methods described herein. In some embodiments, elevated levels of galactose-deficient IgA protein have been observed in the non-human animals provided herein. In some embodiments, elevated levels of galactose-deficient IgA protein have been observed in serum or intestinal mucus of the non-human animals.

[0018] In some embodiments, part or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha locus is deleted and replaced with nucleotides containing heterologous genes. In some embodiments, the non-human animal does not express the endogenous non-human IgA heavy chain constant region.

[0019] In some embodiments, the nucleotides containing the heterologous gene are operably ligated to endogenous regulatory elements of a non-human animal (e.g., promoters, enhancers, silencers, etc.). In some embodiments, the nucleotides containing the heterologous gene are genomic fragments of the human IGHA1 gene. In some embodiments, the heterologous gene contains both the human IGHA1 coding sequence and the non-coding sequence. In some embodiments, the non-human animal produces chimeric IgA1 in which the heavy chain contains a non-human 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 contains an O-glycosylated hinge region. In some embodiments, the heterologous gene is a human IGHA1 gene encoding the human IgA1 heavy chain constant region or fragment thereof.

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

[0021] In a further embodiment, the present disclosure provides the use of non-human animals described herein as animal models for IgA deposition-related diseases.

[0022] In some embodiments, IgA deposition-related diseases are selected from the group consisting of IgA nephropathy, herpetiform dermatitis, Henoch-Schönlein purpura (also known as IgA vasculitis), Kawasaki disease, Henoch-Schönlein purpura nephritis, renal impairment due to IgA vasculitis, IgA rheumatoid factor-positive rheumatoid arthritis, IgA-mediated anti-GBM disease, or IgA-mediated ANCA-associated vasculitis. In some embodiments, the IgA deposition-related disease is IgA nephropathy, IgA vasculitis, or Kawasaki disease.

[0023] In yet another aspect, the Disclosure provides a method for evaluating the efficacy of a candidate drug for treating or preventing an IgA deposition-related disease, comprising: preparing 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 control non-human animal that has not been administered the candidate drug.

[0024] In yet another aspect, the Disclosure provides a method for identifying candidate drugs for treating or preventing IgA deposition-related diseases, comprising: preparing a non-human animal as described herein; administering a candidate drug to the non-human animal; and evaluating whether the candidate drug inhibits one or more symptoms of the disease compared to a control non-human animal that has not been administered the candidate drug.

[0025] The drawings below 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 drawings in conjunction with the detailed description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0026] [Figure 1A]Figure 1 shows the development of IGHA1+ / + mice. (A) This figure shows the genomic loci of human IGHA1, mouse Igha, and humanized IGHA1 (not to scale). The mouse Igha gene (including the entire coding region) is deleted and replaced with the human IGHA1 gene. (B) Mouse IgA and human IgA were examined by immunoblotting in diluted WT, IGHA1+ / -, and IGHA1+ / + mice, as well as human serum. (C-D) Immunohistochemical staining of mouse IgA and human IgA1 in Peyer's patches and the small intestine of WT, IGHA1+ / -, and IGHA1+ / + mice. (E) BCR seq shows the immunoglobulin subtype expressed in terminal ileal tissue with Peyer's patches in IGHA1+ / + and WT mice. (F) This shows the frequency of J gene use of hIgA1 in IGHA1+ / + mice and the frequency of J gene use of mouse IgA in WT mice. (G) The CDR3 sequence length distribution of hIgA1 or IgA was uniform across three IGHA1+ / + mice and three WT mice, respectively. mIgA: mouse IgA; hIgA1: human IgA1; Homo: human. [Figure 1B] the above [Figure 1C] the above [Figure 1D] the above [Figure 1E] the above [Figure 1F] the above [Figure 1G] the above [Figure 2]Figure 2 shows that pathogen exposure promotes hIgA1 production in multiple organs. (A) Human IgA1 was detected by Western blotting using serum (1:1000 dilution) from IGHA1+ / + mice housed in GF, SPF, and CV, and human serum (1:5000 dilution). (B) Serum hIgA1 and Gd-IgA1 levels of 4-month-old mice from the three groups above are shown. (C) mRNA expression of IGHA1 in SP, ILN, PP, and MLN of IGHA1+ / + mice housed in GF, SPF, and CV was measured by RT-qPCR. Relative gene expression was normalized with GAPDH. (D-E) Typical confocal images and mean fluorescence intensity (AU) of hIgA1+ cells in the SI mucosal lamina propria are shown. Scale = 50 μm. [Figure 3] Figure 3 shows elevated levels of serum Gd-IgA1 and hIgA1-containing complexes in LCWE antigen-induced IGHA1+ / + mice. (A) 2-month-old IGHA1+ / + mice were intraperitoneally injected with CFA or PBS in addition to LCWE, and the observation period was extended to 8 months. (B) IGHA1+ / + mice produced LCWE-specific hIgA1 and Gd-IgA1. (C-E) Show the levels of serum hIgA1-containing complexes, Gd-IgA1-containing complexes, and hIgA1-containing complexes in LCWE and PBS-induced IGHA1+ / + mice, respectively. (F) Serum hIgA was purified, stained with Coomassie protein, or immunoimaged with anti-hIgA1 antibody. (G-H) The mean number of GalNAcs per HR was similar in LCWE and PBS-induced IGHA1+ / + mice, but the Gal / GalNAc ratio decreased under strong antigen stimulation. [Figure 4]Figure 4 shows that the TD and TI pathways play a role in enhancing mucosal-derived Gd-IgA1. (A) The concentrations of hIgA1 and Gd-IgA1 in the small intestinal mucus of LCWE-induced IGHA1+ / + mice were higher. (B) Immunofluorescence staining of hIgA1 and Gd-IgA1 enhanced Gd-IgA1+ plasma cells in the lamina propria of the small intestinal mucosa of LCWE-induced IGHA1+ / + mice. Scale = 50 μm. (C~F) mRNA expression of BAFF, APRIL, TGFβ, and iNOS was increased by LCWE stimulation. Relative gene expression was normalized by GAPDH. [Figure 5] Figure 5 shows that Gd-IgA1 caused severe complement activation and pathological damage in the kidney. (A) Immunostaining of CD31, human IgA1, and C3, as well as Gd-IgA1, shows that human IgA1, along with C3, was deposited in the renal mesangial region in LCWE-induced IGHA1+ / + mice. Scale = 50 μm. (B-C) Semi-quantitative grades of hIgA1 and C3 deposited in the glomerular mesangial region are shown. (D) Representative micrographs of PAS staining and Masson staining show mesangial dilation, cell hyperplasia, and tubulointerstitial fibrosis in LCWE-induced IGHA1+ / + mice. Scale = 50 μm. Electron micrographs of glomeruli from each group show electron-dense deposits in the mesangial region of LCWE-induced IGHA1+ / + mice. Scale = 1 μm. [Figure 6]Figure 6 shows that in IGHA1+ / + mice, the human IGHA1 gene was fully expressed in place of mouse IgA. (A) Detection of the IGHA1 gene in WT, IGHA1+ / -, and IGHA1+ / + mice by PCR analysis using specific primers covering the 5' or 3' arm of the knocked-in IGHA1 gene and the mouse Igha gene. (B) Expression of mouse IgA and human IgA1 genes in WT, IGHA1+ / -, and IGHA1+ / + mice by RT-qPCR. (C) Serum levels of mouse IgA and human IgA1 in WT, IGHA1+ / -, and IGHA1+ / + mice. (D-E) mRNA expression of mouse IgA and human IgA in the spleen and small intestine of WT, IGHA1+ / -, and IGHA1+ / + mice was measured by RT-qPCR. Relative gene expression was normalized to GAPDH expression. Cells from the spleen, small intestine, bone marrow, and lymph nodes of (F~I)WT, IGHA1+ / -, and IGHA1+ / + mice were labeled with APC / Cy7-labeled B220, FITC-labeled anti-mouse IgA, and AF647-labeled anti-human IgA1, and the frequency of IgA+B220+ B cells was shown. [Figure 7] Figure 7 shows that IGHA1+ / + mice were able to produce a variety of immunoglobulins, similar to WT mice. (A) Shows the frequency of use of the V gene for hIgA1 in IGHA1+ / + mice and IgA in WT mice. (B) The Venn diagram shows the cluster analysis of IGH in IGHA1+ / + mice and WT mice. (C) Shows the cluster distribution map of the first 100 IGH clusters in IGHA1+ / + mice and WT mice. The percentage represents the proportion of the top 100 IGH clusters out of all detected IGHs. [Figure 8] Figure 8 shows the serum hIgA1-containing complex (Figure 8A), IgM, and IgG (Figure 8B) levels of 4-month-old IGHA1+ / + mice raised on GF, SPF, and CV. [Figure 9] Figure 9 shows the reduced body weight of IGHA1+ / + mice stimulated by LCWE (Figure 9A), and the serum IgM and IgG levels of IGHA1+ / + mice induced by LCWE and PBS (Figure 9B). [Figure 10]Figure 10 shows an example of a typical extracted ion chromatogram from hIgA1 hinge region O-glycopeptide analysis. [Figure 11] Figure 11 shows H&E staining of the cardiovascular and thoracoabdominal aorta of PBS- and LCWE-induced mice (Figure 11A), and urinary albumin / creatinine ratio, serum creatinine, and serum urea nitrogen (Figure 11B) were evaluated in PBS- and LCWE-induced mice. [Figure 12] Figure 12 shows the levels of immunoglobulin in the serum of 5-month-old mice that received intraperitoneal injection of CFA. [Figure 13] Figure 13 shows immunofluorescence of frozen kidney sections from 7.5-month-old mice injected intraperitoneally with CFA. [Figure 14] Figure 14 shows the results of PAS pathological staining of the kidneys of 4-month-old and 5-month-old mice that were injected intraperitoneally with CFA. [Figure 15] Figure 15 shows the results of PAS pathological staining of the kidneys of 6-month-old and 7.5-month-old mice that received intraperitoneal injection of CFA. [Figure 16] Figure 16 shows a comparison of SCr and ACR levels in 5-month-old mice and 7.5-month-old mice that received intraperitoneal injection of CFA. [Figure 17] Figure 17 shows the levels of immunoglobulin in the serum of 4-month-old and 7-month-old mice treated with different routes of administration. [Figure 18] Figure 18 shows immunofluorescence of frozen kidney sections from 7-month-old mice treated with different routes of administration. [Figure 19] Figure 19 shows the results of PAS pathological staining of kidneys from 7-month-old mice treated with different routes of administration. [Figure 20] Figure 20 shows immunohistochemical staining of mouse IgA and human IgA1 in the spleens of WT, IGHA1+ / - (i.e., KIWT), and IGHA1+ / + (i.e., KIKI) mice. [Figure 21]Figures 21A-D show semi-quantitative scores of immunofluorescence images and PAS-stained micrographs performed by specialist pathologists. (A) Human IgA1 intensity; (B) Mouse IgA intensity; (C) C3 intensity; (D) Mesangial hyperplasia score. [Figure 22A] Figures 22A and 22B show the serum human IgA levels of each group treated with PBS, saponin, Al(OH)3, LPS, IFA, CFA, and LCWE-CFA, respectively. [Figure 22B] the above [Figure 23A] Figures 22A and 22B show the levels of the serum human IgA-mouse IgG complex in each group treated with PBS, saponin, Al(OH)3, LPS, IFA, CFA, and LCWE-CFA, respectively. [Figure 23B] the above [Figure 24A] Figures 24A and 24B show the urinary albumin / creatinine ratio (ACR) for each group treated with PBS, saponin, Al(OH)3, LPS, IFA, CFA, and LCWE-CFA, respectively. [Figure 24B] the above [Figure 25] Figure 25 shows the deposition of human IgA (hIgA) and C3 in each group treated with PBS, saponin, Al(OH)3, LPS, IFA, CFA, and LCWE-CFA, respectively, as well as PAS staining. Here, the deposition and PAS staining are measured by immunofluorescence and a PAS pathological staining assay of the kidney. [Figure 26A] Figures 26A-C show semi-quantitative scores of immunofluorescence images and PAS-stained micrographs by a specialist pathologist. (A) Intensity of mouse IgM; (B) Intensity of human IgA; (C) Intensity of C3 deposition. [Figure 26B] the above [Figure 26C] the above [Modes for carrying out the invention]

[0027] [Detailed description of the invention] Certain features of the present invention (including steps of the method) are referenced in the above-mentioned summary of the invention, detailed description of the invention, claims, and accompanying drawings. It should be understood that the disclosure of the present invention herein includes all possible combinations of such specific features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the present invention, or in the context of a particular claim, that feature may be used in general use in the present invention, as far as possible, in combination with and / or in the context of other particular aspects and embodiments of the present invention.

[0028] Where relating herein is a method comprising two or more defined steps, the defined steps may be performed in any order or concurrently (unless the context excludes such possibility), and the method may include one or more other steps performed before any of the defined steps, between two of the defined steps, or after all of the defined steps (unless the context excludes such possibility).

[0029] All publications, patents, and patent applications cited herein are incorporated by reference as if each individual publication, patent, or patent application were specifically and individually incorporated by reference, and are incorporated by reference to disclose and describe methods and / or materials in relation to the cited publications. Any citation of a publication is for its disclosure prior to the filing date, and this disclosure should not be construed as accepting that such publications do not have prior authority based on prior disclosures. Furthermore, the dates of the publications provided may differ from the actual publication dates and may need to be checked independently.

[0030] In general, terms used herein follow their meanings as understood in the art unless otherwise explicitly indicated. Explicit definitions of specific terms are given below. Throughout this specification, the meanings of these and other terms in specific examples will be evident to those skilled in the art from the context. Further definitions of the following and other terms are provided throughout this specification.

[0031] I. Definition When used herein and in the appended claims, the singular forms “a,” “an,” and “the” are understood to encompass multiple referents unless the context explicitly indicates otherwise. For example, a reference to “a pluripotent cell” refers to one or more pluripotent cells, including their equivalents known to those skilled in the art.

[0032] As used herein, the term “comprise” and its grammatical equivalents are used to mean that other components, ingredients, steps, etc., may optionally be present. For example, a method “comprising” steps A, B, and C (i.e., including only steps A, B, and C) may include not only steps A, B, and C but also one or more other steps.

[0033] The use of the term “or” in the claims is intended to mean “and / or” unless it is specifically stated that it means only alternatives or that the alternatives are mutually exclusive; however, this disclosure supports the definition that means only alternatives and “and / or.” As used herein, “another” may mean at least a second one or more.

[0034] As used herein, the term “locus” means a specific location of a gene, DNA sequence, polypeptide-coding sequence, or location on a chromosome in the genome of an organism. Known loci may contain known genetic information, such as one or more polymorphic marker sites. For example, “Igha locus” may mean a specific location of the Igha gene, Igha DNA sequence, immunoglobulin heavy chain constant α-coding sequence, or an Igha location on a chromosome in the genome of an organism in which such sequence is identified. “Igha locus” may include regulatory elements of the Igha gene, such as enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), or combinations thereof.

[0035] As used herein, “gene” means a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes a coding sequence (i.e., a sequence that codes for a particular product). In some embodiments, a gene also includes a non-coding sequence. In some embodiments, a gene includes both coding sequences (e.g., exon sequences) and non-coding sequences (e.g., intron sequences). In some embodiments, a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences that can control or influence one or more aspects of gene expression (e.g., cell type-specific expression, induced expression, etc.). For clarity, as used herein, the term “gene” generally means a portion of a nucleic acid that codes for a polypeptide, and the term may optionally include regulatory sequences, which will be apparent to those skilled in the art from the context. This definition is not intended to exclude the application of the term “gene” to non-protein-coding expression units, but rather to clarify that as used herein, the term often means a nucleic acid that codes for a polypeptide.

[0036] As used herein, a “coding sequence” or a sequence that “codes” a selected polypeptide is a nucleic acid molecule that, for example, is transcribed (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vivo when under the control of an appropriate regulatory sequence (or “regulatory element”). The boundaries of a coding sequence are typically determined by a 5' (amino) terminal start codon and a 3' (carboxy) terminal translation stop codon. A coding sequence may, but is not limited to, viral cDNA, prokaryotic or eukaryotic mRNA, viral genomic DNA sequences, or prokaryotic DNA and synthetic DNA sequences. A transcription termination sequence may be located at the 3' end of the coding sequence.

[0037] The term "operably coupled" refers to the arrangement of elements configured to enable a component described in this way to perform its normal function. In the case of a promoter, a promoter operably coupled to a coding sequence will lead to the expression of the coding sequence. A promoter or other regulatory element does not need to be adjacent to the coding sequence, as long as it functions to lead to the expression of the coding sequence. For example, an untranslated but transcribed intervening sequence can exist between a promoter sequence and a coding sequence, and the promoter sequence can also be considered "operably coupled" to the coding sequence.

[0038] As used herein, the term "IgA" refers to one of the antibody (immunoglobulin) isotypes, also known as immunoglobulin α. As used herein, IgA is also called "IgA antibody" or "IgA-type antibody." In serum, IgA exists mainly as monomeric IgA (serum-type IgA), with IgA1 being the main component. When secreted into mucous membranes, IgA exists as a multimer of dimers or more, called multimeric IgA (secretory IgA or SIgA).

[0039] As used herein, the term "Igha gene" (also known as immunoglobulin heavy chain constant α) encodes the constant region of the IgA heavy chain. In particular, the term "Igha1 gene" or "IGHA1 gene" also means immunoglobulin heavy chain constant α1 and encodes the constant region of the IgA1 heavy chain.

[0040] The human IGHA1 gene is located on human chromosome 14, and an exemplary genome sequence can be identified as NCBI gene ID number 3493 (assembly GRCh38.p14; location NC_000014.9 (c105708664-105707168)), the sequence of which is described in SEQ ID NO: 19. Another exemplary genome sequence of the human IGHA1 gene is described in SEQ ID NO: 33 (IGHA1-202 (ENST00000641837.1 from Ensembl)). In some embodiments, the wild-type human IgA1 heavy chain constant region encoded by the human IGHA1 gene is assigned UniProtKB / Swiss-Prot:P01876.2 (the sequence of which is described in SEQ ID NO: 20). In some embodiments, the wild-type human IgA1 heavy chain constant region encoded by the human IGHA1 gene is assigned UniProtKB:A0A286YEY1 (the sequence of which is described in SEQ ID NO: 34). At least five isoforms of the human IgA1 heavy chain constant region are known in the art and have been assigned the following sequences, respectively: GenBank:AAF03879.1 (sequence described in SEQ ID NO: 21), GenBank:AAT74070.1 (sequence described in SEQ ID NO: 22), GenBank:AAX09631.2 (sequence described in SEQ ID NO: 23), GenBank:AAX09630.2 (sequence described in SEQ ID NO: 24), and GenBank:ABI26625.1 (sequence described in SEQ ID NO: 25).

[0041] The mouse Igha gene is located on mouse chromosome 12, and an exemplary genome sequence can be identified by NCBI gene ID number 238447 (NC_000078.7 Reference GRCm39 C57BL / 6J(c113223856-113219824)), the sequence of which is described in SEQ ID NO: 26. The wild-type mouse IgA heavy chain constant region encoded by the mouse Igha gene is assigned UniProtKB / Swiss-Prot:P01878.1 (the sequence of which is described in SEQ ID NO: 27). At least five isoforms of the mouse IgA heavy chain constant region are known in the art and have been assigned the following sequences: GenBank:AAL15539.1 (sequence described in SEQ ID NO: 28), GenBank:AAL15541.1 (sequence described in SEQ ID NO: 29), GenBank:AAL15540.1 (sequence described in SEQ ID NO: 30), GenBank:AAL15543.1 (sequence described in SEQ ID NO: 31), and GenBank:AAL15542.1 (sequence described in SEQ ID NO: 32). [Table A(1)] [Table A(2)] [Table A(3)] [Table A(4)] [Table A(5)] [Table A(6)] [Table A(7)] [Table A(8)] [Table A(9)] [Table A(10)] [Table A(11)] [Table A(12)]

[0042] As used herein, the terms “endogenous locus” or “endogenous gene” mean a locus that exists in the parent or reference organism prior to the introduction of any modification, disruption, deletion, insertion, alteration, substitution, or replacement described herein. In some embodiments, the endogenous locus includes a sequence found in whole or in part in nature. In some embodiments, the endogenous locus is a wild-type locus. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is a genetically engineered organism. In some embodiments, the reference organism is a laboratory-bred organism (wild-type or genetically engineered). For example, “endogenous Igha locus” in a non-human animal (e.g., mouse or rat) means a naturally occurring Igha locus in a non-human animal (e.g., mouse or rat).

[0043] The term "wild-type" or "WT" includes entities with structures and / or activities found in a normal state or context (as opposed to mutants, diseased individuals, modified individuals, etc.). Wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0044] In this specification, the terms “nucleic acid” and “polynucleotide” are used synonymously and mean polymeric forms of nucleotides of any length in single-stranded or double-stranded form (either deoxyribonucleotide (DNA) or ribonucleotide (RNA), or their analogues). Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Examples of polynucleotides, not limited to these, include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, regulatory regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules may be linear or cyclic. Unless otherwise specified, a particular polynucleotide sequence also implicitly includes its conserved modified variants (e.g., degenerate codon substitutions), alleles, homologous species, SNPs, and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more (or all) selected codons is replaced with a mixed base 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); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0045] The terms "protein," "polypeptide," and "peptide" are used synonymously and refer to polymers of amino acids. The proteins, polypeptides, or peptides described herein may contain naturally occurring amino acids, or amino acids that do not exist naturally, or amino acid analogs or mimetic compounds. The proteins, polypeptides, or peptides described herein can be obtained by any method known in the art, such as natural isolation, recombinant expression, or chemical synthesis, but are not limited to these.

[0046] As used herein, the term "amino acid" means an organic compound containing an amine group (-NH2) and a carboxyl group (-COOH) functional group, and a side chain specific to each amino acid. In this disclosure, the names of amino acids are also represented by the standard one- or three-letter symbols summarized below. [Table B]

[0047] The terms "heavy chain" or "immunoglobulin heavy chain" include immunoglobulin heavy chain sequences containing the constant region sequence, originating from any organism. Unless otherwise specified, the heavy chain variable region includes three heavy chain complementarity-determining regions (CDRs) and four FR regions. Heavy chain fragments include CDRs, FRs, and combinations thereof. A typical heavy chain has, following the variable region (from N-terminus to C-terminus), a CH1 domain, a hinge, a CH2 domain, a CH3 domain, and a CH4 domain (for IgM or IgE). Functional heavy chain fragments include fragments that can specifically recognize epitopes (e.g., recognize epitopes at micromolar, nanomolar, or picomolar KD ranges), can be expressed and secreted from cells, and contain at least one CDR.

[0048] The term “heterogeneous” means preparations or entities originating from different sources. For example, when used in relation to polypeptides, genes, or gene products present in a particular cell or organism, the term makes it clear that the polypeptide or fragment thereof, gene or fragment thereof, or gene product or fragment thereof is 1) artificially manipulated, 2) artificially introduced (e.g., by genetic engineering) into a cell or organism (or its precursor), and / or 3) not produced naturally by or present in the cell or organism (e.g., the cell type or biotype) in question. As used herein, the term “heterogeneous” also includes polypeptides or fragment thereof, genes or fragment thereof, or gene products or fragment thereof that are normally present in a particular natural cell or organism but are modified by mutation or substitution under the control of non-endogenous regulatory elements (e.g., promoters) in some embodiments, for example, that are not naturally related.

[0049] When referring to proteins, the term "fragment" means a protein that is shorter than or has fewer amino acids than a full-length protein. When referring to nucleic acids, the term "fragment" means a nucleic acid that is shorter than or has fewer nucleotides than a full-length nucleic acid. When referring to protein fragments, these fragments may be, for example, N-terminal fragments (i.e., a portion of the C-terminus of a protein removed), C-terminal fragments (i.e., a portion of the N-terminus of a protein removed), or internal fragments (i.e., portions of both the N-terminus and C-terminus of a protein removed). When referring to nucleic acid fragments, these fragments may be, for example, 5' fragments (i.e., a portion of the 3' end of a nucleic acid removed), 3' fragments (i.e., a portion of the 5' end of a nucleic acid removed), or internal fragments (i.e., portions of both the 5' and 3' ends of a nucleic acid removed).

[0050] In the context of inserting nucleic acid sequences into cells, the term “introduce” means “transfection,” “transformation,” or “transduction,” and includes reference to the integration of nucleic acid sequences into cells, where the nucleic acid sequence may be transient within the cell or may be integrated into the cell’s genome (e.g., chromosomes, plasmids, plastids, or mitochondrial DNA) and converted into an autonomous replicon. Nucleic acid sequences can be introduced into cells by any method known in the art. Various techniques can be used to transform animal cells, including, for example, microinjection, retrovirus-mediated gene transfer, electroporation, and transfection (see, e.g., Keown et al., Methods in Enzymology 1990, 185:527-537).

[0051] As used herein, the term “chimera” means an antibody or antigen-binding fragment in which a portion of the heavy chain and / or light chain originates from one species, and the remainder of the heavy chain and / or light chain originates from a different species. In exemplary cases, a chimeric antibody may include a constant region of human origin and a variable region of a non-human animal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0052] The term "non-human animal" refers to any organism other than a human. For example, non-human animals may include livestock such as cattle, pigs, sheep, goats, poultry, or horses; rodents such as rats or mice; primates such as apes, monkeys, chimpanzees, gorillas, orangutans, or baboons; or livestock such as dogs or cats.

[0053] As used herein, the terms “5' regulatory region” and “3' regulatory region” include regulatory elements found in the 5' upstream and 3' downstream regions of a gene. The term “regulatory element” includes transcriptional regulatory sequences that include both 5' transcriptional regulatory sequences such as promoters, enhancers, and suppressor elements, and 3' transcriptional regulatory sequences such as transcription termination sequences. The term “regulatory element” also includes regulatory sequences in the 5' untranslated region (5'UTR) and 3'UTR that may affect transcription efficiency, transcript stability, and translation initiation.

[0054] As used herein, “CRISPR-Cas guide RNA” or “guide RNA,” “gRNA” or “sgRNA” means RNA that guides sequence-specific binding between the CRISPR complex and the target sequence. Typically, a guide RNA comprises (i) a guide sequence that is sufficiently complementary to the target polynucleotide sequence to hybridize with the target sequence, and (ii) a trans-activated cr(tracr) mate sequence. The guide RNA may further contain a tracr RNA fused at the 3' end, resulting in a single chimeric guide RNA. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or higher when optimally aligned using a suitable alignment algorithm. The optimal alignment can be determined using any suitable algorithm for aligning the array, but non-limiting examples include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, and Novoalign (Novocraft). Examples include Technologies, ELAND (Illumina, San Diego, California), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is a nucleotide of approximately 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 in length. In some embodiments, the guide sequence is a nucleotide of approximately 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, 11, 10, or fewer in length. The ability of the guide sequence to guide sequence-specific binding between the CRISPR complex and the target sequence can be evaluated by any suitable assay.For example, sufficient components of the CRISPR system to form a CRISPR complex, including the guide sequence under test, may be provided to host cells having the corresponding target sequence by, for example, transfection with a vector encoding components of the CRISPR sequence, and then evaluating preferential cleavage within the target sequence. Similarly, cleavage of a target polynucleotide sequence may be evaluated in vitro by providing the target sequence, components of the CRISPR complex including the guide sequence under test, and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rates at the target sequence between the reactions of the test guide sequence and the control guide sequence. Other assays are also possible and will be discovered by those skilled in the art.

[0055] As used herein, the term “embryonic stem (ES) cells” means pluripotent cells isolated from the inner cell mass of a developing blastocyst, or their offspring. “ES cells” can originate from any organism. ES cells can originate from mammals, including mice, rats, rabbits, guinea pigs, goats, pigs, cattle, monkeys, and humans. In specific, non-limiting examples, such cells may be human or non-human (e.g., mouse) cells. While not theoretically bound, ES cells can generate various cells present in the body (such as bone cells, muscle cells, and brain cells), provided they are exposed to conditions that promote the development of these cell types. For methods of producing mouse ES cells, see U.S. Patent No. 5,670,372, which is incorporated herein by reference. For methods of producing human ES cells, see U.S. Patent No. 6,090,622, International Publication No. 00 / 70021, and International Publication No. 00 / 27995, which are incorporated herein by reference.

[0056] II. Methods for creating genetically modified non-human animals In one embodiment, the present disclosure provides a method for producing a non-human animal whose genome contains a genetically modified endogenous Igha locus comprising a heterologous gene encoding a human IgA (e.g., IgA1) heavy chain constant region or a fragment thereof. Specifically, the present disclosure provides a method for producing a genetically modified non-human animal, comprising the steps of (i) modifying the genome of the non-human animal to incorporate a nucleotide comprising a heterologous gene encoding a human IgA heavy chain constant region or a fragment thereof into the endogenous Igha locus of the non-human animal, and (ii) treating the non-human animal produced in step (i) with a formulation comprising an immunostimulant.

[0057] Step (i) In some embodiments, step (i) of the method described herein includes introducing a nucleotide containing a heterologous gene encoding a human IgA heavy chain constant region or a fragment thereof into pluripotent cells of a non-human animal to obtain non-human animal pluripotent cells containing the heterologous gene, and producing a non-human animal using the non-human animal pluripotent cells containing the heterologous gene.

[0058] Nucleotides containing heterologous genes encoding the human IgA heavy chain constant region or fragments thereof can be prepared by methods well known in the art. For example, nucleotide molecules can be prepared as part of a larger plasmid. As is well known in the art, such preparation allows for the efficient cloning and isolation of precise constructs. Various methods used for plasmid preparation and transformation of host organisms are well known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd edition, edited by Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Yeast artificial chromosomes (YACs) can also be used to isolate, clone, and transfer entire loci of desired heterologous genes encoding the human IgA heavy chain constant region or fragments thereof. Alternatively, bacterial artificial chromosome (BAC) libraries (see, e.g., the Genome BAC Library of Invitrogen, Inc. (Carlsbad, California)) can provide nucleic acid sequences and regulatory sequences of desired heterologous genes encoding the human IgA heavy chain constant region or fragments thereof.

[0059] The prepared transgene constructs can be introduced into pluripotent cells (e.g., ES cells) using any method known in the art. Various techniques for transforming mammalian cells can be used in the present invention, including, for example, microinjection, retrovirus-mediated gene transfer, electroporation, and transfection (see, for example, Gordon, Intl. Rev Cytol., 115:171 (1989); Keown et al., Methods in Enzymology, 185:527-537 (1990); Mansour et al., Nature, 336:348-352 (1988)).

[0060] Methods for modifying the genomes of non-human animals (e.g., pigs, cattle, rodents, chickens, etc.) include modifying the genome to include heterologous genes encoding the human IgA heavy chain constant region or fragments thereof, as described herein, using, for example, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or Cas proteins (i.e., the CRISPR / Cas system). Guidance for methods for modifying germline genomes of non-human animals can be found, for example, in U.S. Patent Publications 20150376628A1, 20160145646A1, and 20160177339A1, all of which are incorporated herein by reference.

[0061] In some embodiments, nucleotides containing heterologous genes are introduced into pluripotent cells of non-human animals by a CRISPR / Cas system. In some embodiments, the CRISPR / Cas system is the CRISPR / Cas9 system. In some embodiments, the CRISPR / Cas9 system comprises a Cas9 protein and a guide RNA that targets a guide RNA target sequence within an endogenous Igha locus. In some embodiments, the CRISPR / Cas9 system used in this application comprises a Cas9 protein and one, two or more, three or more, or four or more guide RNAs that target a guide RNA target sequence within an endogenous Igha locus. For example, the CRISPR / Cas9 system used in this application comprises a Cas9 protein and a guide RNA containing one, two, three, or four of the sequences described in SEQ ID NOs. 9 to 12. In some embodiments, the CRISPR / Cas system is the CRISPR / Cas13 system. In some embodiments, the CRISPR / Cas13 system comprises a Cas13 protein and a guide RNA that targets a guide RNA target sequence within an endogenous Igha locus. In some embodiments, the CRISPR / Cas13 system used in this application comprises a Cas13 protein and one, two or more, three or more, or four or more guide RNAs that target a guide RNA target sequence within an endogenous Igha locus.

[0062] The pluripotent cells described herein may include undifferentiated cells that have the ability to differentiate into two or more differentiated cell types. Such pluripotent cells may be embryonic stem (ES) cells or ES-like cells, such as induced pluripotent stem (iPS) cells. In some embodiments, the pluripotent cells are embryonic stem (ES) cells. ES cells include embryonic pluripotent cells that, when introduced into an embryo, can contribute to any tissue of the developing embryo. ES cells may originate from the inner cell mass of a blastocyst and can differentiate into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm). ES cells in which a desired nucleotide is incorporated into the endogenous Igha locus are selectable. In some embodiments, one or more nucleotide copies may be incorporated at one or more specific sites. Also, in some embodiments, one nucleic acid copy may be incorporated at one specific site.

[0063] ES cells are typically obtained from preimplantation embryos cultured in vitro (see, for example, Evans, MJ. et al., Nature 292:154-156 (1981); Bradley, MO 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)). To introduce the transgene construct, ES cells are cultured and prepared by methods well known in the art (see, for example, 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); and Veis et al., Cell 75:229 (1993)). ES cells into which a desired heterologous gene encoding the human IgA heavy chain constant region or a fragment thereof is inserted are derived from the same type of embryo or blastocyst as the developing embryo into which the ES cells are introduced. ES cells are typically selected for their ability to integrate into the inner cell mass when introduced into a host animal as an embryo at the blastocyst development stage and contribute to the germline of the individual. For this reason, any ES cell line is suitable for use in carrying out the present invention, as long as it possesses the above ability.

[0064] Next, the selected ES cells are injected into an animal embryo (blastocyst or other developmental stage suitable for producing a viable animal) to create a chimera (see, for example, Bradley, A. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ. Robertson, ed., IRL, Oxford, ppl 13-152 (1987)). Alternatively, the selected cells can be aggregated with dissociated embryonic cells to form aggregated chimeras. The chimeric embryo can then be transplanted into a suitable pseudo-pregnant female rearing animal to produce embryos. Chimeric offspring with the desired human genes in the germ cells can then be used to breed animals in which all cells contain the desired human genes. Next, ES cells with the desired nucleotides incorporated into the endogenous Igha locus are used as donor ES cells. In some embodiments, embryos containing donor ES cells are transplanted into pseudo-pregnant females to produce F0 non-human animals.

[0065] In other embodiments, non-human animals containing genetically modified endogenous Igha loci, including heterologous genes, in their genome can be created without using ES cells. For example, the genome of non-ES cells (e.g., fibroblasts or induced pluripotent cells) can be modified based on conventional transformation methods (e.g., electroporation), and such modified non-ES cell genomes can be transplanted into suitable recipient cells, such as oocytes, by nuclear transfer techniques. The modified cells (e.g., modified oocytes) are then impregnated under appropriate conditions to form an embryo. See, for example, Han et al., Methods in Enzymology 476:171-184 (2010) and Zhou et al., Science 302:1179 (2003).

[0066] In some embodiments, the non-human animal is a rodent, mammal, or 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.

[0067] In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a C57BL mouse selected from the C57BL lineage, such as 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 rodents are 129 lineage mice selected from the group consisting of 129 lineage, e.g., 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129 / SvJae, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2 (see, for example, Festing et al. (1999), Mammalian Genome 10:836; Auerbach et al. (2000), Biotechniques 29(5):1024-1028, 1030, 1032). In some embodiments, the rodents are mice that are a mixture of the 129 strain and the C57BL / 6 strain. In some embodiments, the mice are a mixture of the 129 strain (i.e., hybrid mice), a mixture of the C57BL strain, or a mixture of the C57BL strain and the 129 strain. In some embodiments, the mice are a mixture of the C57BL / 6 strain and the 129 strain. In specific embodiments, the mice are of the VGF1 strain, which is also called F1H4, and is a hybrid of C57BL / 6 and 129. In other embodiments, the mice are of the BALB strain, for example, the BALB / c strain. In some embodiments, the mice are a mixture of the BALB strain and another of the above strains.

[0068] In some embodiments, the rodent is a rat. In one embodiment, the rat is selected from the Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti strains. In other embodiments, the rat is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0069] In some embodiments, part or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha locus is deleted. In some embodiments, part or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha locus is deleted and replaced with nucleotides containing heterologous genes encoding the human IgA heavy chain constant region or a fragment thereof. In some embodiments, the non-human animals described herein do not express the endogenous non-human IgA heavy chain constant region.

[0070] Substitution of an endogenous non-human animal Igha gene with the corresponding human IGHA1 gene is preferably achieved by homologous recombination (see, for example, Allen et al., Eur J Neurosci 17, 1881-1895 (2003); Makita et al., Am J Physiol Renal Physiol 294, F542-F553 (2008)). Homologous gene substitution generally uses a substitutional target construct. To construct a substitutional target construct, two homologous recombination sequences derived from the non-human animal genome sequence are placed on either side of the nucleic acid sequence of the desired human IGHA1 gene. Double crossover between the homologous recombination sequence of the target construct and the non-human animal genome sequence integrates the nucleic acid sequence of the desired human IGHA1 gene as a target into the corresponding non-human animal Igha locus in the animal cell. Typically, the homologous recombination sequence of the target construct includes sequences located on both sides of the endogenous non-human animal Igha gene segment, and therefore homologous recombination results in simultaneous deletion of the endogenous non-human animal Igha gene segment and homologous incorporation of the desired human IGHA1 gene segment (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 partial endogenous non-human animal Igha gene can be replaced with the desired human IGHA1 gene by a single targeting event or by multiple targeting events that sequentially replace individual exons. The targeting construct can use one or more selectable markers (e.g., positive or negative selectable marker genes). Typically, the selectable markers are preferably located in the intron region of the desired human IGHA1 gene.

[0071] In some embodiments, the replacement of an endogenous non-human animal Igha gene with the corresponding human IGHA1 gene can be achieved using site-directed recombination systems (e.g., Cre / LoxP or Flp / FRT systems; generally, see Roebroek et al., Trangenic Mouse Methods and Protocols, edited by Hofker and van Deursen, Methods in Mol Biol, 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 suitable non-human animal cells (e.g., ES cells) by a first homologous recombination event so that these two recombinase recognition sequences are located on either side of the endogenous non-human animal Igha gene. A selectable marker gene (e.g., HygTK) is typically introduced into non-human animal cells during the first homologous recombination event, thereby enabling positive selection (e.g., selection with hygromycin B) and negative selection (e.g., selection with ganciclovir). A transgene construct is prepared containing the desired human IGHA1 gene corresponding to an endogenous non-human animal Igha gene, such that two recombinase recognition sites are located on either side of the desired human IGHA1 gene. This transgene construct is then introduced into target non-human animal cells obtained by the first homologous recombination event. A source of recombinase is provided to induce site-directed recombination, which replaces the endogenous animal Igha gene with the desired human IGHA1 gene. The recombinase recognition sequence may remain at the locus after the exchange, but it is preferable that it is not located in the protein-coding region.

[0072] In some embodiments, the heterogeneous gene encoding the human IgA1 heavy chain constant region or a fragment thereof is a genomic fragment of the human IGHA1 gene. In some embodiments, the heterogeneous gene contains only the human IGHA1 coding sequence. In some embodiments, the heterogeneous gene contains both the human IGHA1 coding sequence and the non-coding sequence. In some embodiments, the heterogeneous gene contains the entire human IGHA1 coding sequence from ATG to STOP, with a 5' untranslated region and a 3' untranslated region, as well as intervening introns. In some embodiments, the human IgA1 heavy chain constant region of a non-human animal includes an O-glycosylated hinge region. In some embodiments, the heterogeneous gene is the human IGHA1 gene encoding the human IgA1 heavy chain constant region or a fragment thereof.

[0073] A nucleic acid sequence is "operably linked" if it has a functional relationship with another nucleic acid sequence. For example, a promoter, or a 5' regulatory region containing a promoter, is considered operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0074] In some embodiments, a heterologous gene encoding a human IgA heavy chain constant region or a fragment thereof is inserted into a locus similar to that of the corresponding endogenous non-human animal Igha gene, such that the expression of the heterologous gene is controlled by a transcriptional regulatory element of the corresponding endogenous non-human animal Igha gene. The heterologous gene may be contained in a nucleotide. In some embodiments, the nucleotide is operably ligated to an endogenous regulatory element of the non-human animal (e.g., a promoter, enhancer, silencer, etc.). 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 constructed such that the protein-coding sequence of this human IGHA1 gene is inserted into the locus of an endogenous non-human animal Igha gene. This 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 makes it possible to control the expression of the introduced human IGHA1 gene using transcriptional regulatory elements of endogenous non-human animal Igha genes.

[0075] In some embodiments, the nucleotide may further include a 3' regulatory region operably ligated to a heterologous gene encoding the human IgA1 heavy chain constant region or a fragment thereof.

[0076] In some embodiments, the 3' regulatory region includes a 3'UTR. In some embodiments, the 3' regulatory region includes a 3'UTR of a heterologous gene encoding the human IgA1 heavy chain constant region or a fragment thereof.

[0077] In some embodiments, the 3' regulatory region includes a sequence located upstream of the 3'UTR of the heterologous gene. In some embodiments, the 3' regulatory region is located immediately upstream of the 3'UTR of the heterologous gene and includes a nucleotide sequence of 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 (e.g., up to 2500-4000 bp).

[0078] In some embodiments, the 3' regulatory region includes a sequence located downstream of the 3'UTR of the heterologous gene. In some embodiments, the 3' regulatory region is located immediately downstream of the 3'UTR of the heterologous gene and includes a nucleotide sequence of 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 (e.g., up to 2500-4000 bp).

[0079] In some embodiments, the nucleotide may further include a 5' regulatory region operably ligated to a heterologous gene encoding the human IgA1 heavy chain constant region or a fragment thereof.

[0080] In some embodiments, the 5' regulatory region includes a 5'UTR. In some embodiments, the 5' regulatory region includes a 5'UTR of a heterologous gene encoding the human IgA1 heavy chain constant region or a fragment thereof.

[0081] In some embodiments, the 5' regulatory region includes a sequence located upstream of the 5'UTR of the heterologous gene. In some embodiments, the 5' regulatory region is located immediately upstream of the 5'UTR of the heterologous gene and includes a nucleotide sequence of 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 (e.g., up to 2500-4000 bp).

[0082] In some embodiments, the 5' regulatory region includes a sequence located downstream of the 5'UTR of the heterologous gene. In some embodiments, the 5' regulatory region is located immediately downstream of the 5'UTR of the heterologous gene and includes a nucleotide sequence of 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 (e.g., up to 2500-4000 bp).

[0083] In some embodiments, modifying the genome of a non-human animal or incorporating nucleotides into a non-human animal alters the structure of the endogenous Igha locus. In some embodiments, some or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha locus is silenced. In some embodiments, some or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha locus is deleted and replaced with nucleotides containing heterologous genes.

[0084] A gene is "silenced" when its ability to express a normal protein product is inhibited. Genes are often silenced by disrupting their genome sequence. A gene is disrupted when a DNA fragment is located and rejoins an endogenous homologous sequence. Disruption may include insertion, missense, frameshift, deletion, substitution, or replacement of a DNA sequence, or any combination thereof. Insertion includes insertion of an entire gene if the disruption can alter the normal gene product by partially or completely inhibiting the production of that normal gene product. In preferred embodiments, disruption is a null disruption such that the gene does not exhibit significant expression.

[0085] As used herein, the terms “substitute” or “replace” mean that in a transgenic non-human animal into which a desired human gene has been introduced, the endogenous non-human animal gene corresponding to the introduced human gene is silenced so that the introduced human gene can functionally replace the corresponding animal gene. For example, in a humanized transgenic mouse into which the human Igha1 gene has been introduced, the endogenous mouse Igha gene is further silenced so that the introduced human IgA1 protein can functionally replace the mouse IgA1 protein.

[0086] In some embodiments, modification of the genome of a non-human animal alters the function of the endogenous Igha locus. In some embodiments, the non-human animal does not express the endogenous non-human IgA heavy chain constant region. In some embodiments, the non-human animal produces chimeric IgA1 in which the heavy chain contains a non-human variable region and a human constant region or a fragment thereof.

[0087] Step (ii) In step (ii) of the method for producing a genetically modified non-human animal provided herein, the non-human animal produced in step (i) is treated with a formulation containing an immunostimulant.

[0088] An "immunostimulant" is a substance that stimulates the immune system by inducing or increasing the activation of one of its components, particularly immune effector cells. Immunostimulants can be pro-inflammatory (e.g., in the treatment of infections or cancer) or anti-inflammatory (e.g., in the treatment of autoimmune diseases).

[0089] In some embodiments, the immunostimulant includes adjuvant-type immunostimulants such as APC Toll-like receptor agonists or costimulatory / cell adhesion membrane proteins. Examples of Toll-like receptor agonists include costimulatory / adhesion proteins such as CD80, CD86, and ICAM-1. In some embodiments, the immunostimulant used in the present invention is selected from the group consisting of aluminum adjuvants, emulsion adjuvants, biological adjuvants, and saponins.

[0090] In some embodiments, the aluminum adjuvant is selected from the group consisting of aluminum hydroxide (Al(OH)3), aluminum phosphate, aluminum hydroxide phosphate, and amorphous aluminum hydroxide phosphate (AAHS). In some embodiments, the aluminum adjuvant is aluminum hydroxide.

[0091] 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 complete Freund's adjuvant (CFA), incomplete Freund's adjuvant, MF59, and AS03. In some embodiments, the immunostimulant used in the present invention is CFA. Various commercially available CFAs are generally available, for example, products purchased from Sigma-Aldrich, Merck, and others. In some embodiments, the CFA used in the present invention is purchased from SIGMA (catalog number: F5881). In some embodiments, the CFA used in the present invention contains Mycobacterium tuberculosis, paraffin oil, and mannidomonoleate. In some embodiments, each 1 mL of CFA used in the present invention contains 0.5 to 5 mg of heat-sterilized and dried Mycobacterium tuberculosis (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), 0.1 to 1 mL of paraffin oil (e.g., 0.5 mL, 0.85 mL, or 1 mL), and 0.05 to 1 mL of mannitol monooleate (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). In some embodiments, each 1 mL of CFA used in the present invention contains 1 mg of heat-sterilized and dried Mycobacterium tuberculosis (H37Ra, ATCC 25177), 0.85 mL of paraffin oil, and 0.15 mL of mannido monooleate.

[0092] In some embodiments, the biological adjuvant is selected from the group consisting of lipopolysaccharides or their derivatives, Toll-like receptor (TLR) agonists (e.g., TLR4 ligand, TLR7 ligand, TLR8 ligand, and TLR9 ligand), immunostimulatory oligonucleotides (e.g., DNA or dsRNA containing CpG), cytokines (e.g., IL-2, IL-7, IL-12, IL-15, and IL-23) or their variants, interferons (e.g., IFN-α or IFN-γ), colony-stimulating factors (e.g., M-CSF and GM-CSF), tumor necrosis factor, endotoxins, and lipids (e.g., lipid A or its analogues). In some embodiments, the biological adjuvant is a lipopolysaccharide or its derivative.

[0093] In some embodiments, the formulation for treating the non-human animal prepared in step (i) comprises an immunostimulant and an antigen.

[0094] The term "antigen" relates to a drug containing an epitope that can elicit an immune response. The term "antigen" includes proteins and peptides in particular. In some embodiments, the antigen is a disease-associated antigen, such as a viral antigen, or a viral antigen, and the epitope is derived from such an antigen.

[0095] The term "viral antigen" refers to any viral component that possesses antigenic properties, i.e., can induce an immune response in an individual. Viral antigens can be viral ribonucleoproteins or envelope proteins.

[0096] The term "bacterial antigen" refers to any bacterial component that possesses antigenic properties, i.e., can induce an immune response in an individual. Bacterial antigens may originate from the cell wall or cytoplasmic membrane of bacteria. In some embodiments, the antigens used in the present invention are derived from bacterial cell wall extracts. In some embodiments, the antigen includes a rhamnose source. In some embodiments, the rhamnose source is Lactobacillus casei cell wall extract (LCWE).

[0097] In some embodiments, the non-human animals prepared in step (i) are treated with a formulation containing LCWE emulsified with CFA. Detailed treatment with LCWE can be carried out using conventional methods in the art (see Duong TT, Silverman ED, Bissessar MV, 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)).

[0098] In some embodiments, the formulation comprises an immunostimulant. For example, the non-human animal prepared in step (i) is treated with CFA alone. The inventors of this disclosure have unexpectedly found that to prepare a genetically modified non-human animal exhibiting clinical signs of IgA deposition-related disease (e.g., IgA nephropathy), such as elevated IgA1, elevated galactose-deficient IgA1 (Gd-IgA1), IgA1 / C3 glomerular deposition, mesangial cell hyperplasia, intracapillary cell proliferation, segmental sclerosis, and / or crescent formation, it is sufficient to treat the non-human animal containing the humanized Igha locus prepared in step (i) with an immunostimulant (e.g., CFA) alone (e.g., without antigen), compared to a wild-type non-human animal.

[0099] The immunostimulant formulation can be administered to the non-human animal prepared in step (i) by any route known in the art, for example, parenteral routes including subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection, or oral routes including transdermal, oral, intranasal, intraocular, sublingual, rectal, or local routes. In some embodiments, in step (ii), the formulation is administered intraperitoneally to the non-human animal prepared in step (i).

[0100] While not bound by any particular theory, sustained delivery (e.g., sustained intraperitoneal delivery) or sustained release of immunostimulant formulations to the non-human animals created in step (i) is considered advantageous for creating genetically modified non-human animals exhibiting clinical signs of IgA deposition-related diseases (e.g., IgAN).

[0101] As used herein, the term “sustained delivery” means that a formulation containing an immunostimulant continues to be delivered into the body over a period of time, preferably for at least several days, a week or several weeks, a month or several months, after administration.

[0102] In some embodiments, the formulation is administered intraperitoneally to the non-human animals prepared in step (i) at a sustained dose. In some embodiments, the formulation is administered intraperitoneally to the non-human animals prepared in step (i) at a sustained low dose. In some embodiments, the formulation contains LCWE and is administered intraperitoneally to the non-human animals prepared in step (i) at a dose of less than 1 μg of LCWE per gram of non-human animal (e.g., less than 0.1 μg, less than 0.2 μg, less than 0.3 μg, less than 0.4 μg, less than 0.5 μg, less than 0.6 μg, less than 0.7 μg, less than 0.8 μg, less than 0.9 μg). In some embodiments, the formulation contains or consists of CFA and is administered by intraperitoneal injection to the non-human animals prepared in step (i) at doses of less than 5 μL of CFA per gram of non-human animals (e.g., less than 4 μL, less than 3 μL, less than 2 μL, less than 1.9 μL, less than 1.8 μL, less than 1.7 μL, less than 1.6 μL, less than 1.5 μL, less than 1.4 μL, less than 1.3 μL, less than 1.2 μL, less than 1.1 μL, less than 1 μL, less than 0.9 μL, less than 0.8 μL, less than 0.7 μL, less than 0.6 μL, less than 0.5 μL, less than 0.4 μL, less than 0.3 μL, less than 0.2 μL, less than 0.1 μL). In some embodiments, the formulation comprises LCWE emulsified with CFA, with less than 1 μg of LCWE per gram of non-human animal (e.g., less than 0.1 μg, less than 0.2 μg, less than 0.3 μg, less than 0.4 μg, less than 0.5 μg, less than 0.6 μg, less than 0.7 μg, less than 0.8 μg, less than 0.9 μg), and CFA The non-human animals prepared in step (i) are administered intraperitoneally in doses of less than 5 μL (for example, less than 4 μL, less than 3 μL, less than 2 μL, less than 1.9 μL, less than 1.8 μL, less than 1.7 μL, less than 1.6 μL, less than 1.5 μL, less than 1.4 μL, less than 1.3 μL, less than 1.2 μL, less than 0.9 μL, less than 0.8 μL, less than 0.7 μL, less than 0.6 μL, less than 0.5 μL, less than 0.4 μL, less than 0.3 μL, less than 0.2 μL, less than 0.1 μL).

[0103] In some embodiments, the formulation is administered to the non-human animals prepared in step (i) for at least one month. In some embodiments, the formulation is administered to the non-human animals prepared in step (i) for at least two months. In some embodiments, the formulation is administered to the non-human animals prepared in step (ii) for at least three months. In some embodiments, the formulation is administered to the non-human animals prepared in step (i) for at least four months.

[0104] In some embodiments, the formulation is administered to the non-human animals prepared in step (i) three times a week for the first two weeks, and then once a week for the following two weeks, for a total of eight doses within one month. In some embodiments, the formulation is administered to the non-human animals prepared in step (i) every two weeks for a total of six doses within three months. In some embodiments, administration of the formulation is started at 8 weeks of age in the non-human animals prepared in step (i) and terminated at 5 months, 6 months, 6.5 months, 7 months, 7.5 months, or 8 months of age.

[0105] In some embodiments, the non-human animal is a rodent, mammal, or non-human primate. In some embodiments, the rodent is a mouse or a rat.

[0106] III. Genetically Modified Non-Human Animals In another aspect, the Disclosure provides non-human animals produced by the methods described herein. In another aspect, the Disclosure also provides non-human cells or non-human genomes produced by the methods described herein.

[0107] In some embodiments, the non-human animals, cells, or genomes provided herein include in their genome a genetically modified endogenous Igha locus containing a heterogene encoding the human IgA heavy chain constant region or a fragment thereof, wherein the non-human animals exhibit clinical signs of IgA deposition-related disease (e.g., IgA nephropathy).

[0108] In some embodiments, the non-human animals provided herein exhibit histological lesions of IgA nephropathy. In some embodiments, the non-human animals provided herein exhibit one or more histological lesions selected from the group consisting of mesangial cell proliferation, intracapillary cell proliferation, segmental sclerosis, interstitial inflammation / tubular atrophy, and crescent formation.

[0109] In some embodiments, elevated levels of galactose-deficient IgA protein have been observed in the non-human animals provided herein. In some embodiments, elevated levels of galactose-deficient IgA protein have been observed in the serum or intestinal mucus of the non-human animals provided herein. In some embodiments, “elevated levels” of galactose-deficient IgA protein mean that the level of galactose-deficient IgA protein is elevated compared to a reference level of galactose-deficient IgA protein obtained from wild-type non-human animals. The level of galactose-deficient IgA protein can be measured by methods well known in the art, such as Irabu H et al., J Immunol Res. 2020 May 21; 2020:4284379; Suzuki H., Clin Exp Nephrol. 2019 Jan; 23(1):26-31 and Neufeld M et al., J Am Acad Dermatol. 2019 May 19. pii:S0190-9622(19)30443-8, all of which are incorporated herein by reference in their entirety.

[0110] In some embodiments, the non-human animals described herein showed at least a 10-fold increase in galactose-deficient IgA protein (e.g., at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, or at least 30-fold). In some embodiments, the 10- to 20-fold increase in galactose-deficient IgA protein with diverse renal pathological phenotypes in the non-human animals described herein suggests that the changes in glycosylation are strongly related to the function of human IgA rather than non-human (e.g., mouse) IgA under similar stimuli.

[0111] As described above, according to the methods for producing genetically modified non-human animals provided herein, the structure and / or function of the endogenous Igha locus in the non-human animal may be altered. In some embodiments, part or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha locus is deleted and replaced with a nucleotide containing a heterologous gene. In some embodiments, the nucleotide containing the heterologous gene is operably ligated to an endogenous regulatory element of the non-human animal (e.g., promoter, enhancer, silencer, etc.). In some embodiments, the nucleotide containing the heterologous gene is a genomic fragment of the human IGHA1 gene. In some embodiments, the heterologous gene contains both the human IGHA1 coding sequence and the non-coding sequence. In some embodiments, the heterologous gene is a human IGHA1 gene encoding the human IgA1 heavy chain constant region or a fragment thereof. In some embodiments, the human IgA heavy chain constant region of the non-human animal provided herein includes an O-glycosylated hinge region. In some embodiments, the non-human animal does not express the endogenous non-human IgA heavy chain constant region. In some embodiments, the non-human animals provided herein produce chimeric IgA1 in which the heavy chain comprises a non-human variable region and a human constant region or a fragment thereof.

[0112] In some embodiments, the non-human animal described above is a rodent, mammal, or non-human primate. In some embodiments, the rodent is a mouse or a rat.

[0113] IV. Use of genetically modified non-human animals Non-human animals provided herein, for example, non-human animals whose genomes contain a genetically modified endogenous Igha locus, including a heterologous gene encoding the human IgA heavy chain constant region or a fragment thereof, exhibit clinical signs of IgA deposition-related diseases (e.g., IgA nephropathy) and can be used as animal models of IgA deposition-related diseases.

[0114] In another aspect, the Disclosure further provides a method for evaluating the efficacy of a candidate drug for treating or preventing an IgA deposition-related disease, comprising: preparing 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 control non-human animal that has not been administered the candidate drug. If one or more symptoms of the disease are inhibited by the candidate drug compared to a control non-human animal that has not been administered the candidate drug, the candidate drug is considered effective for treating or preventing an IgA deposition-related disease.

[0115] In a further embodiment, the Disclosure provides a method for identifying candidate drugs for treating or preventing IgA deposition-related diseases, comprising: preparing 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 control non-human animal that has not been administered the candidate drug. If one or more symptoms of an IgA deposition-related disease (e.g., IgA nephropathy) are inhibited by the candidate drug, the candidate drug is considered useful for treating or preventing an IgA deposition-related disease.

[0116] In some embodiments, candidate drugs that can be evaluated or identified using non-human animals provided herein include, but are not limited to, candidate inhibitors of IgA (e.g., IgA1), such as small molecule inhibitors, nucleic acid-based inhibitors (e.g., siRNA, ribozymes, antisense constructs, etc.), antigen-binding proteins (e.g., antibodies or their antigen-binding fragments), inhibitory peptides / peptide inhibitors, IgA proteases or functional variants thereof, and fusion proteins comprising IgA proteases or functional variants thereof.

[0117] In some embodiments, the IgA deposition-related disease is selected from the group consisting of IgA nephropathy, herpetiform dermatitis, Henoch-Schönlein purpura (also known as IgA vasculitis), Kawasaki disease, Henoch-Schönlein purpura nephritis, renal impairment due to IgA vasculitis, IgA rheumatoid factor-positive rheumatoid arthritis, IgA-mediated anti-GBM disease, or IgA-mediated ANCA-associated vasculitis. In some embodiments, the IgA deposition-related disease is IgA nephropathy, IgA vasculitis, or Kawasaki disease. In some embodiments, the IgA deposition-related disease is IgA nephropathy. [Examples]

[0118] The following examples are provided to those skilled in the art to provide a complete disclosure and description of the methods for preparing and evaluating the compositions and / or methods claimed herein, and are intended to be purely illustrative and not to limit the present disclosure.

[0119] Materials and methods Animals and living conditions All animal experiments were approved by the Laboratory Animal Care and Use Committee of Peking University First Hospital (numbers: 201994 and 2021114). IGHA1 + / - and IGHA1 + / +To verify human IgA1 expression in mice, mice were bred and raised under barrier conditions. Different pathogen exposures were associated with IgA1 expression. + / + To evaluate the effects on mice, three rearing groups were established. First, embryo transfer was performed to introduce IGHA1 + / + We achieved germ-free (GF) status in mice and raised their offspring under sterile, isolated conditions. Fecal samples were regularly examined using microscopy and culture techniques to confirm their GF status. Next, we raised IGHA1 free of specific pathogens (SPF), similar to conventionally raised (CV) animals. + / + Mice were either housed under barrier conditions or moved to non-barrier conditions at 4 weeks of age. At 16 weeks of age, the mice were euthanized, embedded in a compound at the optimal cutting temperature, or fixed in 10% formalin, and their kidneys were collected for histological examination. Furthermore, 0.5 μg / g of LCWE emulsified with CFA or PBS 2 μg / g was administered three times a week for the first two weeks, and once a week for the following two weeks, to 2-month-old mice housed under barrier conditions. + / + The mice were injected intraperitoneally. The observation period was extended until the mice reached 8 months of age.

[0120] Urine, terminal serum, spleen (SP), small intestine (SI), inguinal lymph nodes (ILN), mesenteric lymph nodes (MLN), Peyer's patches (PP), femur, and small intestinal mucus diluted with 3 mL of PBS were collected for further analysis.

[0121] Polymerase chain reaction (PCR) and real-time quantification (RT-qPCR) The IGHA1 gene was identified from mouse tail or auricle DNA using the Mouse Direct PCR Kit (B40015, Bimake). RNA was extracted from mouse tissue based on TRIzol (15596026, Invitrogen) or RNAsimple Total RNA Extraction Kit (DP419, TIANGEN), and then reverse transcribed into cDNA using the GoScript® reverse transcription system (A2801, Promega). The resulting cDNA and primers were conjugated with PowerUp® SYBR® Green Master Mix (A25742, Invitrogen), and RT-qPCR analysis was performed on a LightCycler® 480 instrument (Roche). GAPDH was used as the internal reference gene. The primers are shown in Table 1 below. [Table 1]

[0122] Serum enzyme immunosorbent assay (ELISA) and Western blotting The levels of mouse IgA, IgG, IgM, human IgA1, human IgA1-mouse IgG complex, and CD89-conjugated polymer IgA (pIgA) complex in serum were measured by ELISA. Plates were coated overnight with goat F(ab')2 anti-mouse Ig (1012-01, Southern Biotech), goat anti-human IgA F(ab')2 fragment (109-006-011, JacksonImmuno), or recombinant human CD89 protein (10414-H08H, SinoBiological). The plates were blocked at room temperature (RT) for 2 hours in 0.1% Tween-containing phosphate-buffered saline (PBST) containing 1% bovine serum albumin (BSA), after which diluted serum and standard solutions were added and incubated at room temperature for 1 hour. Subsequently, mouse IgA, IgG, IgM, and human IgA1 were separately detected using horseradish peroxidase (HRP)-labeled goat anti-mouse IgA, IgG, and IgM antibodies (1040-05, 1030-05, and 1020-05, Southern Biotech), HRP-labeled mouse anti-human IgA1 antibody (9130-05, Southern Biotech), HRP-labeled goat anti-human IgA α-chain antibody (ab98558, Abcam), or HRP-labeled goat anti-mouse IgG antibody (1030-05, Southern Biotech). To detect LCWE-specific hIgA1 and Gd-IgA1, plates were coated with 2.5 μg / mL of LCWE. For detection, HRP-labeled mouse anti-human IgA1 (9130-05, Southern Biotech), rat monoclonal anti-human Gd-IgA1 antibody (10777, Immuno-Biological Laboratories), and HRP-labeled polyclonal rabbit anti-rat IgG (RS030226, Immunoway Biotechnology) were used.

[0123] All of the above ELISA tests were detected using a tetramethylbenzidine liquid substrate system. Optical density was measured at 450 nm, with wavelength correction performed 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.

[0124] Using a non-reducing loading buffer, serum (0.02 μl / sample) was loaded onto a gradient SDS-PAGE machine (8012011, BioSci, Shenzhen, Guangdong, China), electrophoresis was performed at 150 V for 2 hours, and the samples were transferred to a 0.45 μm PVDF membrane. For N-acetylgalactosamine residue (GalNAc) detection, 2 μg of FPLC-purified hIgA was desialylated with neuraminidase (GK80040, Agilent Technologies) and used as one sample. After blocking with 5% skim milk, the membrane was incubated with HRP-labeled goat anti-mouse IgA (1040-05, Southern Biotech) or HRP-labeled goat anti-human IgA α-chain (ab98558, Abcam) at room temperature for 1 hour to detect IgA. The membrane was detected by enhanced chemiluminescence.

[0125] urine analysis Urinary albumin was measured by ELISA. Plates were coated overnight with goat anti-mouse albumin antibody (A90-134A, Bethyl Laboratories). The plates were blocked at room temperature for 2 hours with 1% BSA-containing PBST, then diluted urine and standard solution were added and incubated at room temperature for 1 hour. Subsequently, horseradish peroxidase (HRP)-labeled anti-mouse albumin antibody (A90-134P, Bethyl Laboratories) was used as the detection antibody. Optical density was measured as described above. Urinary creatinine levels were measured using a creatinine assay kit (DICT-500, BioAssay Systems, Hayward, California, USA). Renal function was evaluated using the urinary albumin / creatinine ratio (ACR, mg / g).

[0126] Flow cytometry assay SP, PP, MLN, and ILN were polished, and bone marrow (BM) was washed with PBS containing 2% fetal bovine serum (FBS) until single cells remained. The suspension was then filtered through a 100 μm cell strainer. Red blood cells from the spleen, bone marrow, and whole blood were lysed with RBC lysis buffer (420301, Biolegend), and the cells were washed with PBS containing 2% FBS. The small intestine was incised longitudinally, cut into several pieces, and washed with RPMI at 200 rpm for 30 minutes. The washed tissue was then digested at 37°C with 20 ml of RPMI containing collagenase IV (V900893, Sigma), dispase II (04942078001M, Roche), and DNase I (SLBF7798V, Sigma) for 35 minutes. The digested tissue was then filtered through a 100 μm cell strainer and washed at least twice with PBS containing 2% FBS. Lymphocytes from digested intestinal tissue were separated by gradient centrifugation using Histopaque-1083 (10831, Sigma).

[0127] Single-cell suspensions were blocked with TruStain FcX (101320, BioLegend) and stained with APC / Cy7-labeled B220 (RA3-6B2, BioLegend), FITC-labeled anti-mouse IgA (1040-30, Southern Biotech), and AF647-labeled anti-human IgA1 (9130-31, Southern Biotech). After three washes, the cells were resuspended in PBS containing 2% FBS and subjected to data acquisition using a flow cytometer (Verse, BD). + The expression rates of mouse IgA or human IgA1 were analyzed in the cell gate.

[0128] Preparation of LCWE As previously mentioned, we obtained cell wall extracts of Lactobacillus casei (ATCC 11578) (Wan F, Wang H, Wang M, et al., J Pathol 2022; 257(3):262-273). Briefly, we collected the bacteria during the exponential growth phase in MRS broth (BD). The bacteria were incubated overnight with 4% sodium dodecyl sulfate (SDS). To remove residual SDS, the cell wall fragments were washed at least eight times. The treated cell wall fragments were sonicated for 2 hours while cooling in a dry ice / ethanol bath. After sonication, the cell wall fragments were centrifuged at 4°C and 38,000 rpm for 1 hour, and the supernatant was collected. The total rhamnose content in the cell walls was measured using the colorimetric phenol-sulfuric acid extraction technique as described above (Lehman TJ, Walker SM, Mahnovski V, et al., Arthritis Rheum 1985; 28:652-659). LCWE used in the IgA renal deposition mouse model was emulsified with CFA as previously published (Wan F, Wang H, Wang M, et al., J Pathol 2022; 257:262-273).

[0129] Tissue fixation, staining, and histopathology Fresh kidney tissue was embedded in a compound at the optimal cutting temperature, and frozen sections were prepared with a thickness of 2 μm. The frozen sections were stained overnight with both Dylight 488-labeled anti-human IgA (ab98553, Abcam), AF488-labeled anti-mouse IgA (1040-30, Southern Biotech), rat anti-mouse CD31 (553370, BD Pharmingen), and Alexa Fluor 555-labeled anti-rat IgG (ab150166, Abcam), as well as rabbit anti-C3 / C3b antibody (ab200999, Abcam) and cy3-labeled anti-rabbit IgG (A0516, Beyotime Biotechnology). After mounting with DAPI Fluoromount-G (0100-20, Southern Biotech), the sections were imaged using a confocal microscope (LSM780, Zeiss).

[0130] The kidneys, small intestine, and spleen were harvested, fixed in 10% formalin solution, and embedded in paraffin blocks. All sections used for histopathology and immunohistochemistry were 2.5 μm thick. Following standard protocols, the kidneys were stained with hematoxylin-eosin, periodate-Schiff, or Masson stain. After antigen recovery, the small intestine and spleen, which contained Peyer's patches, were stained with HRP-labeled anti-human IgA or HRP-labeled anti-mouse IgA. After nuclear counterstaining with hematoxylin as a standard procedure for the DAB chromogen system, the sections were sealed with neutral balsam. For staining of Gd-IgA1 in the small intestine and kidney, dewaxed paraffin sections were antigen-recovered for 30 minutes at room temperature using 0.05% bacterial protease subtilisin A (P5380, Sigma-Aldrich). Nonspecific binding was inhibited using a phosphate-buffered saline blocking solution containing 3% bovine serum albumin. Sections were incubated with rat monoclonal anti-human Gd-IgA1 antibody (10777, Immuno-Biological Laboratories) at 37°C for 1 hour, and then incubated with Alexa Fluor 555-labeled goat anti-rat IgG (ab150166, Abcam) at 37°C for 30 minutes. After staining with Dylight 488-labeled anti-human IgA (ab98553, Abcam) and DAPI Fluoromount-G (0100-20, Southern Biotech), images were obtained using a confocal microscope (LSM780, Zeiss).

[0131] Semi-quantitative immunofluorescence of the kidney was evaluated by measuring the fluorescence intensity in the glomerular mesangial region. The mean fluorescence intensity of the small intestine was evaluated by the ratio of positive integrated density to villous area.

[0132] Electron microscopy Kidney tissue from treated mice was collected in multiple small pieces (approximately 1 mm). 3) was sliced, fixed in a phosphate buffer containing 2.5% glutaraldehyde, post-fixed with 1% osmium tetroxide, and then embedded in Epon 812 resin. Ultrathin sections were stained with uranyl acetate and lead citrate and observed using a transmission electron microscope (JEM-1230; JEOL, Tokyo, Japan).

[0133] B-cell receptor repertory sequencing (BCR seq) Approximately 1 cm of the terminal ileum tissue with PP was washed three times with PBS and then stored in RNA (AM7020, Invitrogen). RNA extraction, library preparation, and data analysis for high-throughput sequencing were all performed by Seqhealth Technology Co., Ltd. (Wuhan, China). Total RNA was extracted from the samples using TRIzol (15596018, Invitrogen), and DNA digestion was performed with DNaseI (EN0521, Thermo Scientific). Nanodrop(™) One C The quality of the RNA was measured by measuring A260 / A280 using a spectrophotometer (ND-ONEC-W, Thermo Scientific). The integrity of the RNA was confirmed by 1.5% agarose gel electrophoresis. Finally, appropriate RNA was quantified using a Qubit(™) RNA HS assay kit (Q32855, Invitrogen) with a Qubit(™) 3 fluorometer (Q33216, Invitrogen).

[0134] Following the manufacturer's instructions, BCR sequencing libraries were prepared using approximately 2 μg of total RNA per sample with the Illumina® 150 KC-Digital® Stranded BCR-seq Library Prep Kit (DT0815-02, Seqhealth Technology). This kit eliminates duplication bias in the PCR and sequencing processes by labeling pre-amplified cDNA molecules with eight random base unit molecular identifiers (UMIs). Library products corresponding to 250–500 bp were enriched, quantified, and finally sequenced on NovaSeq (Illumina®). First, the raw sequencing data was filtered using SOAPnuke (version 1.6.0), and then clean reads were initially clustered according to the UMI sequences, grouping reads with the same UMI sequence into the same cluster. Deduplication-removed consensus sequences were used for BCR-seq analysis. Using MiXCR software (version 3.0.3), these sequences were mapped to the international ImMunoGeneTics (IMGT) database to obtain rearrangements of the V, D, and J fragments and CDR3 sequences. Genes showing significantly different expression were screened using P ≤ 0.05 or double change > 2. BCR diversity was determined by the diversity of the complementarity-determining region (CDR). The CDR3 sequence with the greatest variation in the functional epitope of the antibody is called an identical clone if it consists of the same VDJ gene. Subsequently, Shannon's entropy and Simpson's index were introduced to confirm BCR diversity from the number of non-duplicate clones.

number

[0135] Quantitative analysis of O-glycopeptides in the IgA1 hinge region (HR) by LC-MS analysis. Terminal serum was diluted to 1 ml with phosphate-based saline (PBS) and subjected to hIgA1 purification. In an FPLC system, the IgA1 fraction was purified using an HP affinity chromatography column (17-0716-01, Cytiva) activated with HiTrap NHS conjugated to the F(ab')2 fragment of a goat anti-human IgA antibody (109-006-011, JacksonImmuno). The column was washed with PBS (pH=7.3), and the fraction was eluted with glycine (pH=2.7). The eluate was immediately neutralized with Tris buffer (pH=9.0), collected, and concentrated by ultrafiltration using a 30 kDa regenerated cellulose membrane (UFC8030, Amicon® Ultra-4, Milipore).

[0136] Purified serum hIgA1 was loaded onto a gradient SDS-PAGE (8012011, BioSci, Shenzhen, Guangdong, China) using a non-reducing loading buffer, and electrophoresis was performed at 150V for 1.5-2 hours. Subsequently, the gel was stained with Coomassie protein stain (ab119211, Abcam), and the gel containing hIgA1 monomers and dimers was cleaved for O-glycosylation analysis.

[0137] The bands were cut into smaller fragments (1 mM × 1 mM) and washed with 50 mM ammonium bicarbonate at 37°C. The gel fragments were shrunk three times with 100% acetonitrile (ACN) and rehydrated with 10 mM DTT in 50 mM AmBic, 55 mM iodoacetamide in 50 mM AmBic, or a small amount (10 μL) of 50 mM AmBic, respectively. This small amount of solvent was then digested with trypsin at 37°C for 18 hours, followed by PNGase F treatment at 37°C for 4 hours. The glycosylated peptides were further concentrated on a commercially available HILIC column, dried, and redissolved in 10 μL of 0.1% formic acid before analysis.

[0138] The peptides were first separated using a Thermo Scientific EASY-nLC™ 1200 nano LC system (Thermo Fisher, San Jose) in trap-elute mode, and then discharged into a Thermo Scientific Orbitrap Eclipse mass spectrometer (Thermo Fisher, San Jose) equipped with FAIMS Pro (Thermo Fisher, San Jose). Solvent A was 0.1% formic acid-containing water, and solvent B was 0.1% formic acid-containing 80% ACN. After loading the peptides into a trap column (Thermo Scientific Acclaim PepMap 100 C18, 75 μm × 2 cm, 3 μm, 100 Å) at a flow rate of 10 μL / min for 3 minutes, all peptides were further eluted into an analytical column (Thermo Scientific Acclaim PepMap RSLC, 75 μm × 25 cm, 1.9 μm, 100 Å) at a flow rate of 250 nL / min using gradients of 8%–30% solvent B for 127 minutes and 30%–90% solvent B for 16 minutes. Subsequently, the column was washed for 14 minutes.

[0139] In positive ion mode, full-scan mass spectra were recorded with a resolution of 60000 (200 m / z) in the scan range of 350–2000 m / z. The AGC target was set to 4e5 and the maximum injection time to 50 ms. For dd-MS2 scans, a maximum setting of 1 second was used, followed by 30 seconds of dynamic exclusion. Data were acquired at -45V and -65V using FAMIS Pro. Using calibrated charge-dependent ETD parameters and 27% NCE HCD co-activation, EthcD spectra were obtained with a resolution of 30000 (200 m / z), an AGC target of 4e5, and a maximum injection time of 400 ms.

[0140] Byonic (trademark) (version 3.8.13) and Byologic RUsing software (version 3.8-11-x64, Protein Metrics Inc.), fragmentation data of glycopeptides was extracted from raw data files with a mass tolerance of ±10 ppm for precursor ions and ±20 ppm for fragment ions. The Protein Metrics 78 O-linked glycan library was specified as the O-glycan modification, and intact O-glycopeptides containing the HR peptide HYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR and O-glycans were analyzed. The false detection rate (FDR) was set to 1%. 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 expressed as the AUC ratio to the total glycopeptide content. The average composition of HR O-glycans for each sample was then calculated.

[0141] statistical analysis Continuous normally distributed variables are expressed as mean ± standard deviation (SD). Non-normally distributed variables are expressed as median and interquartile interval (IQR). Differences between two or more normally distributed data sets were compared using t-tests or analysis of variance (ANOVA) and minimum significance (LSD) post-hoc multiple comparisons, and linear trend tests were performed as a supplement.

[0142] All statistical methods and graphs were created using IBM SPSS Statistics 26 software (SPSS Corporation, Chicago, Illinois, USA) and GraphPad Prism 9 software (GraphPad Software Corporation, San Diego, California). For two-tailed tests, a P < 0.05 value was considered statistically significant.

[0143] [Example 1] IGH1 + / + Mouse development In this study, the inventors investigated human immunoglobulin heavy chain constant α1 knock-in (IGHA1 + / + We created mice and investigated the effects of different levels of pathogen exposure on hIgA1 expression and glycosylation. + / +The mice were obtained from GemPharmatech Co., Ltd. (Nanjing, China), and the targeting method is shown in Figure 1A. In short, this IGHA1 + / + Mice were generated using a CRISPR / Cas9 system (Cong L, Ran FA, Cox D, et al., Science (New York, NY) 2013; 339(6121):819-823) with Cas9 mRNA, sgRNA (sgRNA sequence shown in Table 2 below), and donor cells, which were simultaneously injected into C57BL / 6J zygotes by microinjection. In other words, the human IGHA1 genome fragment (its nucleic acid sequence is shown in SEQ ID NO: 33) replaced the mouse Igha locus. The amino acid sequence of the human IgA1 heavy chain constant region expressed in the humanized mice is shown in SEQ ID NO: 34. Next, the embryos were transplanted into pseudopregnant female mice to generate C57BL / 6J F0 mice. These mice were screened by PCR analysis using specific primers (primer sequences are shown in Table 3 below). This assay also determined the IGHA1 genotype and confirmed that the humanized IGHA1 allele was transmitted through the germline. Next, F0 mice were backcrossed with C57BL / 6J mice for two generations to obtain homozygosity in which the endogenous mouse Igha gene was replaced with the human IGHA1 gene. [Table 2] [Table 3]

[0144] Typical WT, IGHA1 + / - and IGHA1 + / + PCR analysis of tail DNA extracted from mice confirmed that the mouse Igha gene was successfully replaced by the human IGHA1 gene (Figure 6A). The expression of hIgA1 and mouse IgA mRNA in leukocytes of each group showed that human IgA1 replaced IGHA1. + / - and IGHA1 + / + We demonstrated that it can be properly translated in mice (Figure 6B). IGHA1 + / +The circulating levels of human IgA1 protein in mice were comparable to those of mouse IgA protein in the WT control group (Figure 6C). As expected, IgA1 + / + No mouse IgA was detected in the mice. Western blot analysis of serum samples from each group confirmed that chimeric IgA1 was expressed as complete immunoglobulin (Figure 1B).

[0145] Most IgA is secreted from IgA plasma cells in mucosal-associated lymphoid tissue. To evaluate the normal expression of the human IGHA1 gene in immune organs and mucosal organs, WT, IGHA1 + / - and IGHA1 + / + mRNA expression of mouse IgA and human IgA1 was examined in the mouse spleen (Figure 6D) and small intestine (Figure 6E). + / + In line with the absence of mouse IgA in mouse blood cells, IGHA1 + / - and IGHA1 + / + Normal production of chimeric human IgA1 was observed in mice. Flow cytometry analysis of multiple organs (represented as B220+ cells) revealed that the human IGHA1 gene is IGHA1 + / - and IGHA1 + / + It was demonstrated that IGHA1 was expressed in the spleen, small intestine, bone marrow, and lymph nodes of mice, but not in wild-type mice (Figure 6F-I). These results suggest that IGHA1 + / - and IGHA1 + / + It was found that the IgA expression system in mice is identical to that of wild-type mice.

[0146] Immunohistochemical analysis of tissues regarding the presence of mouse IgA and human IgA revealed that, compared to WT mice, IGHA1 + / - and IGHA1 + / + Similar localization patterns of antibody-secreting cells were found in mouse lymphoid and mucosal tissues. Plasma cells were located around rare Peyer's patches (Figure 1C), in the lamina propria of the intestinal mucosa, and intestinal crypts (Figure 1D). Overall, the human IGHA1 gene was fully expressed, and as a result, IGHA1 + / +Mouse IgA was replaced in the mice.

[0147] IGHA1 + / + To elucidate the ability of mice to generate diverse immunoglobulins through variable and diverse binding gene (VDJ) rearrangements, somatic hypermutation (SHM), and class switch recombination (CSR), the inventors performed B cell receptor (BCR) sequencing of terminal ileum tissue containing Peyer's patches. The inventors used specific primers to amplify immunoglobulin subtypes (hIgA1, IgA, IgG, IgM, IgD, IgE) and found that IGHA1 + / + We found that the mice exhibited a similar immunoglobulin subtype composition to WT mice (Figure 1E). IGHA1 + / + The dominant subtypes in the terminal ileum tissue of mice and wild-type mice were hIgA1 (>90%) and IgA, respectively. The inventors defined identical VDJ genes as clones and evaluated BCR diversity using Shannon's entropy and Simpson's indices, which measure the number of non-duplication clones. Higher values ​​of Shannon's entropy and Simpson's indices were associated with greater BCR diversity in the samples (Feutrill, A. and M. Roughan, Entropy (Basel), 2021. 23(8)). IGHA1 + / + The diversity distribution of hIgA1 in mice and the diversity distribution of mouse IgA in wild-type mice were similar, although the absolute number of hIgA1 clones was slightly increased. The frequency of use of the V and J genes in hIgA1 was also similar to that of mouse IgA (Figure 1F, Figure 7A). Furthermore, the complementarity-determining region 3 (CDR3) sequence, which typically consists of 8-20 amino acids and showed the highest diversity of functional epitopes, exhibited a uniform normal distribution in both hIgA1 and IgA (Figure 1G). IGHA1 + / +In mice, BCR clusters were increased compared to WT mice, but cluster analysis of immunoglobulin heavy chains (IGH) showed no significant difference, as shown in the Venn diagram (Figure 7B) illustrating 3055 common IGH sequences. The distribution of the first 100 IGH clusters was uniform, and no significant difference was observed between the two systems (Figure 7C). Overall, IGH1 + / + To address the complexities of mucosal immunity, the mice successfully underwent class switching and affinity maturation, synthesizing a diverse range of immunoglobulins similar to wild-type mice.

[0148] [Example 2] LCWE-induced IGH1 + / + Mouse development and characterization Pathogen exposure promotes hIgA1 production in multiple organs. IgA is the most abundant antibody isotype produced in humans, and is mainly found in mucosal regions, but is also present in the blood (Perse, M. and Z. Veceric-Haler, The Role of IgA in the Pathogenesis of IgA Nephropathy. Int J Mol Sci, 2019. 20(24)). In the human body, IgA can exist in three forms: mainly monomers (mIgA), multimers (mainly dimers; pIgA, dIgA), and IgA-containing complexes. IGHA1 raised in GF, SPF, and CV. + / + All mice synthesized three forms of hIgA1 (Figures 2A-B, Figure 8A). As pathogen exposure levels increased, IGHA1 + / + Serum levels of mIgA (approximately 160kD) and pIgA, particularly dIgA (approximately 340kD), increased in mice. Serum Gd-IgA1 levels also changed depending on the rearing conditions (Figure 2B). IGHA1 mice reared under CV conditions. + / + The increased production of Gd-IgA1 in mice revealed that Gd-IgA1 production is affected by infection.

[0149] To clarify the cause of the elevated serum hIgA1 levels, the inventors measured the mRNA expression levels of IGHA1 in lymphocytes derived from SP, PP, MLN, and ILN (Figure 2C). RT-qPCR results showed that IGHA1 mRNA expression increased in proportion to exposure in both systemic and mucosal sites that produce hIgA1 in mice raised under GF, SPF, and CV conditions. Interestingly, the inventors found that in systemic sites such as SP and ILN, SPF-raised mice showed IGHA1 expression levels similar to GF, while in mucosal sites such as PP and MLN, they showed expression levels equivalent to CV. This is likely because, under SPF conditions, IGHA1 + / + Although the mice established symbiotic bacteria in their mucous membranes, there were not enough pathogens to stimulate the abundant production of systemic hIgA1.

[0150] In humans and mice, approximately 80% of all plasma cells in the body reside 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. Front Immunol, 2019. 10:p. 1891). In the SI mucosal lamina propria of GF mice, almost no hIgA1-positive cells were detected. The inventors of this invention have developed IGHA1 + / + When symbiotic bacteria colonize mice, SI hIgA + A significant increase in the mean fluorescence intensity (MFI) of plasma cells was observed (Figures 2D-E). These results indicate that the elevation of serum hIgA1 levels is caused by multiple immune tissues, and that both systemic and mucosal sites are involved in this process.

[0151] LCWE antigen-induced IGHA1 + / + In mice, levels of complexes containing serum Gd-IgA1 and hIgA1 were elevated. LCWE, used in the IgA renal deposition mouse model, was emulsified with complete Freund's adjuvant (CFA) as previously published (Wan F, Wang H, Wang M, et al., J Pathol 2022; 257:262-273). Two-month-old IgA1 mice were reared under barrier conditions. + / + Mice were intraperitoneally injected with LCWE (emulsified with CFA) or PBS eight times and observed until they reached eight months of age (Figure 3A, Figure 9A). Gd-IgA1 was identified as an early factor in the pathogenesis of IgA nephropathy (Wyatt, RJ and BA Julian, IgA nephropathy. N Engl J Med, 2013. 368(25):p. 2402-14; Lai, KN, et al., IgA nephropathy. Nat Rev Dis Primers, 2016. 2:p. 16001; Novak, J., et al., IgA Nephropathy. Semin Nephrol, 2018. 38(5): p. 461-476). Furthermore, circulating IgA-containing immune complexes are known to be the most common and pathogenic form of renal 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, JW, et al., Perspectives on how mucosalimmuneresponses and gut microbiome shape IgA nephropathy and future therapies. Theranostics, 2020. 10(25):p. 11462-11478). After LCWE antigen stimulation, IGHA1 + / + Mice produced LCWE-specific hIgA1 and Gd-IgA1 (Figure 3B). LCWE-induced IGH1 + / + (In this application, "IGHA + / +In mice (also known as "LCWE"), the absolute value of serum Gd-IgA1 and its relative ratio to hIgA1 were significantly increased (Figures 3C-D). Furthermore, both the hIgA1-mouse IgG complex and the CD89-bound pIgA complex reached higher levels after stimulation, even at 8 months of age (Figure 3E). Serum IgM increased with age, and IgG peaked immediately after stimulation (Figure 9B).

[0152] PBS and LCWE-induced IGHA1 + / + Serum hIgA1 was purified from mice, and the hIgA1 protein in the gel was stained with an anti-hIgA1 antibody for identification (Figure 3F). Subsequently, the hIgA1 gel band was excised and subjected to LC-MS analysis of hIgA1 HR O-glycopeptide (Figure 10). Compared with the PBS group, LCWE-induced IGHA1 + / + The Gal / GalNAc ratio in mouse IgA1 HR decreased, but the number of GalNAc residues remained unchanged (Figure 3G-H).

[0153] Overall, under strong mucosal stimulation, both Gd-IgA1 and hIgA1 immune complexes were successively elevated, simulating the first to third hits of the "multi-hit" hypothesis.

[0154] The TD and TI pathways played a role in strengthening mucosal-derived Gd-IgA1. The terminal ileum is a site of PP concentration and functions as a major site for antigen sampling and immune induction in gut-associated lymphoid tissue (GALT) 50. LCWE-induced IGHA1 + / + In mouse small intestinal mucus, the concentrations of hIgA1 and Gd-IgA1 were high (Figure 4A). Gd-IgA1 in the SI mucosal lamina propria +To understand the process of plasma cell enhancement (Figure 4B), mRNA levels in the terminal ileum were analyzed. Both T cell-dependent (TD) and T cell-independent (TI) immune responses regulated naive B cells that undergo class switch recombination and produce IgA1 in the intestinal tract (Tezuka, H. and T. Ohteki, Regulation of IgA Production by Intestinal Dendritic Cells and Related Cells. Front Immunol, 2019. 10:p. 1891). TGFβ was affected by both the TD and TI pathways, and the inventors found that transcription levels increased 1000-fold in the LCWE-induced group compared to the other three groups (Figure 4E). Furthermore, IgA1 class switching occurred directly via the TI pathway in response to BAFF and APRIL secreted from Toll-like receptor ligand-activated dendritic cells (DCs) in the PP and 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). LCWE stimulation increased BAFF and APRIL mRNA levels in terminal ileal tissue (Figure 4C-D). Transcriptional levels of induced nitric oxide (NO) synthase (iNOS) also increased (Figure 4F). iNOS is expressed in dendritic cells (DCs) and macrophages, mediating massive NO production, which in turn induces TGFβ receptors on B cells in the TD pathway and BAFF and APRIL expression on dendritic cells 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).In other words, transcriptional levels of inflammatory cytokines increased in both the TD and TI pathways, thereby stimulating IGHA1 with pathogens. + / + The mice were able to produce more Gd-IgA1 secretory plasma cells in their intestinal tract.

[0155] Gd-IgA1 caused severe complement activation and pathological damage in the kidneys. The gradient is consistent with the elevated levels of serum hIgA1 and hIgA1-containing complexes, and the inventors have found that LCWE-induced IGHA1 + / + We observed deposition of hIgA1, particularly Gd-IgA1, in the glomerular mesangial region of mice. These mice showed continuous and stable human IgA1 (positive rate: 100%) renal mesangial deposition and C3 co-deposition (positive rate: 80%) up to 8 months of age (Figure 5A). Compared to the PBS group, LCWE-induced IGHA1 + / + Mice showed suppressed increases in semi-quantitative scores for C3 and hIgA1 (Figure 5B-C). Histologically, significant increases in mesangial dilation, cell hyperplasia, and tubulointerstitial fibrosis were observed. Electron microscopy revealed LCWE-induced IGHA1 + / + Typical electron-dense mesangial deposits were observed 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 + / +In mice, no inflammation of the cardiovascular system or aorta was observed (Figure 11A). In LCWE-induced wild-type mice, compared to previously 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 sustained and stable IgA deposition model. J Pathol, 2022. 257(3):p. 262-273), pathological Gd-IgA1 induced LCWE-induced IgA1 + / + In mice, complement C3 activation, mesangial dilation, and vascular endothelial cell proliferation were more severe (see Table 4 below). However, no significant differences were observed in clinical indicators such as serum creatinine, urea nitrogen, and urinary albumin / creatinine ratio after stimulation (Figure 11B). [Table 4]

[0156] Consideration Insights from IgAN models provide valuable information on various aspects of the pathogenesis of IgAN and can contribute to the development of IgAN-specific drugs (Suzuki, H. and Y. Suzuki, Mine Models of Human IgA Nephropathy. Semin Nephrol, 2018. 38(5):p. 513-520). Mucosal polymer IgA and Gd-IgA1 are important in the pathogenesis of IgAN (Schena, FP and SN Cox, Biomarkers and Precision Medicine in IgA Nephropathy. Semin Nephrol, 2018. 38(5):p. 521-530). The novel IgAN mouse models described herein can further deepen our understanding of this disease. The inventors have developed an IgAN mouse model expressing chimeric hIgA1 having an intact O-glycosylated hinge region. + / +We successfully created a mouse model. Upon exposure to pathogens and strong antigenic stimulation, mucosal hypersecretion type HR hypogalactosylated hIgA1 showed significant renal deposition and was more effective in inducing complement activation and inflammatory injury.

[0157] IGHA1 created in this application + / + The mouse model successfully expressed a chimeric hIgA1 that possesses both the human IgA1 heavy chain constant region and the mouse variable region, but lacks mouse IgA. During the immune response, B lymphocyte progenitor cells reassemble the V(D)J gene fragment to generate the antigen-binding region of the BCR (Cooper, MD and MN Alder, The evolution of adaptive immune systems. Cell, 2006. 124(4):p. 815-22), and via SHM and CSR, produce high-affinity antibodies with biological effector function (Tezuka, H. and T. Ohteki, Regulation of IgA Production by Intestinal Dendritic Cells and Related Cells. Front Immunol, 2019. 10:p. 1891). Depending on the BCR sequence, IgA1 + / + The immunoglobulin gene structures of mice and wild-type mice are identical. Compared to α1KI mice (Duchez, S., et al., Premature replacement of mu with alphaimmunoglobulin chains impairs lymphopoiesis and mucosal homing but proves plasma cell maturation. Proc Natl Acad Sci USA, 2010. 107(7):p. 3064-9), which lacked mouse IgM but had mouse IgA, IGHA1 + / +Mice were able to better defend against pathogens and mimic human mucosal immunity. In the human body, 75-90% of serum IgA is monomeric, 10-15% is in the form of multimers, and 1% is bound to circulating immune complexes (Perse, M. and Z. Veceric-Haler, The Role of IgA in the Pathogenesis of IgA Nephropathy. Int J Mol Sci, 2019. 20(24)). The third hit in the IgAN "multi-hit" hypothesis is the formation of Gd-IgA1-containing immune complexes, which can form autoaggregates by complexing with anti-Gd-IgA1 antibodies (IgG or IgA) or soluble CD89 (Selvaskandan, H., J. Barratt, and CK Cheung, Immunological drivers of IgA nephropathy: Exploring the mucosa-kidney link. Int J Immunogenet, 2022. 49(1):p. 8-21). Levels of poly-IgA immune complexes were significantly higher in IgAN patients compared to healthy controls (Zhang, X., et al., Poly-IgA Complexes and Disease Severity in IgA Nephropathy. Clin J Am Soc Nephrol, 2021. 16(11):p. 1652-1664). Although the mice did not express CD89, the inventors used a recombinant CD89 affinity probe as an ex vivo tool and IGHA1 + / +We were able to measure polyhIgA1 levels in mouse serum. With increased exposure to the pathogen, not only the levels of the hIgA1-IgG complex but also the CD89-bound polyhIgA1 complex significantly increased. Furthermore, it has been reported that elevated Gd-IgA1 serum concentrations were observed in up to 90% of IgAN patients from various 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 inventors of this invention have developed IGHA1 + / + We confirmed that the hIgA1 hinge region in mice is O-glycosylated. Furthermore, under LCWE stimulation, qualitatively, HR showed a decrease in Gal modification. In terms of quantity, serum Gd-IgA1 levels increased fivefold compared to the control group. We are the first in the world to report a mouse model with elevated Gd-IgA1 levels.

[0158] IGHA1 + / +The function and effects of chimeric IgA1 in mice are related to the symbiotic bacterial flora and inflammatory responses (Monteiro, RC, Role of IgA and IgA fc receptors inflammation. J Clin Immunol, 2010. 30(1):p. 1-9; Wilmore, JR, et al., Commensal Microbes Induce Serum IgA Responses that Protect against Polymicrobial Sepsis. Cell Host Microbe, 2018. 23(3):p. 302-311.e3). New evidence has confirmed the crucial role of mucosal immunity in the pathogenesis of IgAN (Gesualdo, L., V. Di Leo, and R. Coppo, The mucosalimmune system and IgA nephropathy. Semin Immunopathol, 2021. 43(5):p. 657-668).Current experimental findings from IgAN-like mouse models support the idea that mucosal polymer IgA, induced by mucosal exposure to food and microbial antigens, can induce IgAN-like damage in the glomeruli (McCarthy, DD, 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, RC 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, PPs, and other MALTs (GALTs, T cells, B cells, etc.) are present and interact with captured antigens (Megha, KB and PV Mohanan, Role of immunoglobulin and antibody in disease management. Int J Biol Macromol, 2021. 169:p. 28-38). IGHA1 in mice raised under CV conditions compared to mice raised under SPF conditions. + / + Human IgA1 expression in mice was enhanced at these immune induction sites. However, LCWE-induced IgA1 + / + The lamina propria of mouse SI mucosa contains hIgA + and Gd-IgA1 + Numerous plasma cells are present, which indicate germinal center (GC) independent or LCWE-induced IgA +This reflects the involvement of short GCs in B cell production (Seifert, M. and R. Kuppers, Human Memory B Cells. Leukemia, 2016. 30(12):p. 2283-2292). We elucidated that mRNA levels in the terminal ileum affect mucosal hIgA1 production not only through the TD pathway but also through the TI pathway. Therefore, LCWE-induced IGHA1 + / + Mice are very suitable for studying the mucosal renal axis in IgA nephropathy.

[0159] LCWE-induced mice exhibited pathological features including IgA1 and C3 deposition. Observations up to 8 months of age revealed histological lesions such as IgAN-like mesangial hyperplasia, endovascular cell proliferation, segmental sclerosis, interstitial inflammation, and tubular atrophy, with crescent formation observed in some mice. Compared to previously reported LCWE-induced wild-type mice (Wan, F., et al., Sustained release of Lactobacillus casei cell wall extract can induce a sustained and stable IgA deposition model. J Pathol, 2022. 257(3):p. 262-273), the complement activation ability of pathological Gd-IgA1 was higher in LCWE-induced IgA1. + / + The mice exhibited more pronounced complement C3 activation, mesangial dilation, and intratubular cell proliferation compared to WT-LCWE mice and PBS-LCWE mice.

[0160] In summary, the inventors have established the world's first mouse model with elevated serum Gd-IgA1 levels. This animal model is expected to be used not only to elucidate the mechanisms of IgA nephropathy, which are influenced by both genetic and environmental factors, but also to contribute to the exploration of new therapeutic strategies and the acceleration of new drug development. Our research group has established the IGHA1 + / +The in vivo effects of chimeric fusion of IgA protease and Fc were investigated using mice (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. J Am Soc Nephrol, 2022. 33(5):p. 918-935). Therefore, this model has considerable potential in promoting IgAN drug development and facilitating clinical interpretation.

[0161] [Example 3] Immune stimulation by different immunostimulants At baseline, 2-month-old IGHA1 + / + Mice were administered total Freund's adjuvant (CFA, catalog number F5881) by intraperitoneal injection once every two weeks for a total of six doses. The dose was 2 μg / g (mouse body weight) per dose. Samples were collected multiple times from mice aged 5 to 7.5 months.

[0162] These results showed that the levels of human IgA1, mouse IgG, mouse IgM, and human IgA1-mouse IgG complex in the serum of 5-month-old mice were all significantly elevated (Figure 12).

[0163] 7.5-month-old IGHA1 + / + In frozen kidney sections of mice (No. 6-14, a total of 9 mice), the positive rates for human IgA, C3, IgM, and IgG, measured by immunofluorescence staining, were 100%, 88.9%, 100%, and 33.3%, respectively (Figure 13).

[0164] In the PAS pathological staining assay of the kidney, an increase in matrix was observed in the glomerular mesangial region of one 4-month-old mouse (No. 1). In 5-month-old mice (Nos. 2-3), an increase in matrix and cells, a significant increase in endothelial cells, and segmental glomerulosclerosis were observed in the glomerular mesangial region (Figure 14). In addition, mild hyperplasia was observed in the glomerular mesangial region of 6-month-old mice (Nos. 4-5), and the lesions disappeared compared to the 5-month-old mice. In 7.5-month-old mice (Nos. 6-14), the lesions entered the chronic phase, and proliferative lesions were observed in the glomerular mesangial region of all 9 mice. For example, in mouse No. 6, an increase in matrix and cells, as well as segmental glomerulosclerosis, was observed in the glomerular mesangial region (Figure 15).

[0165] When serum creatinine (SCR) and urinary albumin / creatinine ratio (ACR) levels were measured, no statistically significant difference was observed between 5-month-old (6-needle) mice and 7.5-month-old mice. However, the ACR values ​​of mice exhibiting relatively severe renal pathological phenotypes (e.g., mouse No. 6) were significantly higher than those of other test mice and control mice (Figure 16).

[0166] In summary, stimulation with CFA alone resulted in elevated serum IgA and complex levels in IGHA1 humanized mice, with IgA deposition reaching 100% in the glomerular mesangial region of 7.5-month-old mice. Renal pathological changes could be classified into acute and chronic phases. During administration (5 months of age), the matrix and cells in the glomerular mesangial region increased, with a significant increase in endothelial cells. After discontinuation of administration (6 months of age), the lesions disappeared. Subsequently, the renal pathological changes entered the chronic phase, with increased matrix and cells in the glomerular mesangial region of 7.5-month-old mice, and glomerular segmental sclerosis was observed. In mice with relatively severe disease, the urinary albumin / creatinine ratio (ACR) was significantly increased.

[0167] In the examples, other types of immunostimulants were also tested, including saponins, aluminum hydroxide (Al(OH)3), lipopolysaccharides (LPS), incomplete Freund's adjuvant (IFA), CFA, and LCWE emulsified with CFA (LCWE-CFA). All of these immunostimulants are commercially available, for example, saponins (Sigma, catalog number: SAE0073), aluminum hydroxide (Thermo, catalog number: 77161), lipopolysaccharides (Sigma, catalog number: L2630), incomplete Freund's adjuvant (Sigma, catalog number: F5506), and CFA (Sigma, catalog number: F5881). The administration protocol is described in Table 5 below. For each group, at baseline, 2-month-old IGHA1 + / + Each test sample was injected intraperitoneally into mice, and 2-month-old mice raised under barrier conditions were subjected to IGHA1 + / + Mice were given intraperitoneal injections three times a week for the first two weeks, and once a week for the following two weeks. The observation period was extended until the mice reached eight months of age. [Table 5]

[0168] Serum human IgA levels in each group before and after stimulation are shown in Figures 22A and 22B. As shown in Figures 22A and 22B, serum human IgA levels significantly and continuously increased in the LCWE-CFA group and were maintained at relatively high levels. Serum human IgA levels also significantly increased in the Al(OH)3, CFA, and IFA groups, but decreased in the later stages.

[0169] Figures 23A and 23B show the serum human IgA-mouse IgG complex levels in each group before and after stimulation. As shown in Figures 23A and 23B, the serum human IgA-mouse IgG complex levels in the Al(OH)3, IFA, CFA, and LCWE-CFA groups increased significantly, and the serum human IgA-mouse IgG complex in the LCWE-CFA group reached the highest level.

[0170] The urinary albumin / creatinine ratio (ACR) for each group before and after stimulation is shown in Figures 24A and 24B. As shown in Figures 24A and 24B, no significant differences were observed in the urinary ACR of each group.

[0171] Figure 25 shows the human IgA (hIgA) and C3 deposition levels in each group, as measured by immunofluorescence and PAS pathological staining assays of the kidney, as well as PAS staining. As shown in Figure 25, the LCWE-CFA group had a higher hIgA positivity rate and deposition intensity than all other groups, with the CFA group ranking second. In the Al(OH)3 group, the C3 deposition positivity rate and deposition intensity were higher than all other groups, with the LCWE-CFA group ranking second. Furthermore, the Al(OH)3, CFA, and LCWE-CFA groups showed an increase in substrate, expansion of the glomerular mesangial region, and a significant increase in endothelial cells, and crescent formation in some mice.

[0172] The semi-quantitative scores of immunofluorescence images and microscopic images of mice in each group are shown in Table 6 and Figure 26 below. As shown in Figure 26, no significant difference was observed in the intensity of mouse IgM in each group (Figure 26A). The intensity of human IgA (hIgA) in the LCWE-CFA group was higher than in all other groups, with the CFA group ranking second (Figure 26B). The C3 deposition intensity in the Al(OH)3 group was higher than in all other groups, with the LCWE-CFA group ranking second (Figure 26C). [Table 6]

[0173] [Example 4] Immune stimulation via different administration routes (intraperitoneal and intranasal) At baseline, LCWE emulsified with PBS or CFA (intranasal LCWE group or intraperitoneal LCWE group) was administered to 2-month-old IGHA1 + / + Mice were administered either intranasally (in) or intraperitoneally (ip) for a period of one month, with administration three times a week for the first two weeks and once a week for the following two weeks. These samples were collected from seven-month-old mice.

[0174] As shown in Figure 17, the results showed that, compared to the PBS group, both human IgA1 and mouse IgG levels in the serum of 4-month-old mice were significantly increased in the intraperitoneal LCWE group. In the intranasal LCWE group, human IgA1 levels tended to increase, but the difference was not significant compared to the PBS group. In the intraperitoneal LCWE group, the levels of human IgA1, mouse IgG, mouse IgM, and human IgA1-mouse IgG complex in the serum of 7-month-old mice were all significantly increased. For example, the human IgA1 level in the intraperitoneal LCWE group was twice that of the human IgA1 level in the PBS group. However, no significant differences were observed between the intranasal LCWE group and the PBS group for these biomarkers (Figure 17).

[0175] Immunofluorescence analysis of frozen kidney sections from 7-month-old mice revealed that the human IgA1 positivity rate was 75% in the intranasal LCWE group, lower than the 100% in the intraperitoneal LCWE group. The positivity rate was 12.5% ​​in the intranasal LCWE group and 40% in the intraperitoneal LCWE group (Figure 18).

[0176] In a PAS pathological staining assay of kidneys from 7-month-old mice, no significant pathological changes were observed in the intranasal LCWE group, whereas an increase in substrate and cells in the glomerular mesangial region was observed in the intraperitoneal LCWE group (Figure 19).

[0177] In summary, a comparison between the intranasal LCWE group and the intraperitoneal LCWE group revealed that serum IgA levels increased in the intranasal LCWE group at the end of the intranasal administration period (4 months of age), and the IgA deposition rate in the glomerular mesangial region of 7-month-old mice was 75%, lower than the 100% in the intraperitoneal LCWE group. The positivity rate and intensity of IgA and C3 were also lower in the intraperitoneal LCWE group than in the intraperitoneal LCWE group. Furthermore, no pathological changes in the kidneys were observed in the intraperitoneal LCWE group.

[0178] While embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, and substitutions can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and equivalents.

Claims

1. A method for creating genetically modified non-human animals, (i) The step of modifying the genome of a non-human animal to incorporate a nucleotide containing a heterologous gene encoding the human IgA heavy chain constant region or a fragment thereof into the endogenous Igha gene locus of the non-human animal, (ii) A method comprising the step of treating the non-human animal prepared in step (i) with a formulation containing an immunostimulant.

2. The method according to claim 1, wherein step (i) includes introducing nucleotides containing the heterogene into pluripotent cells of a non-human animal to obtain non-human animal pluripotent cells containing the heterogene, and producing a non-human animal using the non-human animal pluripotent cells containing the heterogene.

3. The method according to any one of claims 1 to 2, wherein the nucleotide containing the heterologous gene is introduced into the pluripotent cells of the non-human animal by the CRISPR / Cas system.

4. The method according to any one of claims 1 to 3, wherein the CRISPR / Cas system comprises a Cas9 protein and a guide RNA that targets a guide RNA target sequence within the endogenous Igha locus.

5. The method according to any one of claims 1 to 4, wherein the pluripotent cells are embryonic stem (ES) cells.

6. The method according to any one of claims 1 to 5, wherein part or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha gene locus is deleted and replaced with the nucleotides containing the heterologous gene.

7. The method according to any one of claims 1 to 6, wherein the non-human animal does not express an endogenous non-human IgA heavy chain constant region.

8. The method according to any one of claims 1 to 7, wherein the nucleotide containing the heterologous gene is operably linked to an endogenous regulatory element of the non-human animal (e.g., a promoter, enhancer, silencer, etc.).

9. The method according to any one of claims 1 to 8, wherein the nucleotide containing the heterologous gene is a genomic fragment of the human IGHA1 gene.

10. The method according to any one of claims 1 to 9, wherein the heterologous gene comprises both a human IGHA1 coding sequence and a non-coding sequence.

11. The method according to any one of claims 1 to 10, wherein the non-human animal produces a chimeric IgA1 in which the heavy chain comprises a non-human variable region and a human constant region or a fragment thereof.

12. The method according to any one of claims 1 to 11, wherein the human IgA1 heavy chain constant region of the non-human animal includes an O-glycosylated hinge region.

13. The method according to any one of claims 1 to 12, wherein the heterologous gene is a human IGHA1 gene encoding a human IgA1 heavy chain constant region or a fragment thereof.

14. The method according to any one of claims 1 to 13, wherein the preparation comprises an immunostimulant and an antigen.

15. The method according to any one of claims 1 to 14, wherein the preparation comprises an immunostimulant.

16. The method according to any one of claims 1 to 15, wherein the immunostimulant is selected from the group consisting of aluminum adjuvants, emulsion adjuvants, biological adjuvants, and saponins.

17. The aforementioned aluminum adjuvant is aluminum hydroxide (Al(OH) ３ ), selected from the group consisting of aluminum phosphate, aluminum hydroxyphosphate, and amorphous aluminum hydroxyphosphate sulfate (AAHS), optionally, The emulsion adjuvant is an oil-in-water emulsion adjuvant or a water-in-oil emulsion adjuvant, and optionally, The method according to any one of claims 1 to 16, wherein the biological adjuvant is selected from the group consisting of lipopolysaccharides or derivatives thereof, Toll-like receptor (TLR) agonists (e.g., TLR4 ligand, TLR7 ligand, TLR8 ligand, and TLR9 ligand), immunostimulatory oligonucleotides (e.g., DNA or dsRNA containing CpG), cytokines (e.g., IL-2, IL-7, IL-12, IL-15, and IL-23) or variants thereof, interferons (e.g., IFN-α or IFN-γ), colony-stimulating factors (e.g., M-CSF and GM-CSF), tumor necrosis factor, endotoxins, and lipids (e.g., lipid A or analogs thereof).

18. The method according to any one of claims 1 to 17, wherein the aluminum adjuvant is aluminum hydroxide, optionally the emulsion adjuvant is selected from the group consisting of complete Freund's adjuvant (CFA), incomplete Freund's adjuvant, MF59, and AS03, and optionally the biological adjuvant is lipopolysaccharide or a derivative thereof.

19. The method according to any one of claims 1 to 18, wherein the antigen comprises a rhamnose source.

20. The method according to any one of claims 1 to 19, wherein the rhamnose source is Lactobacillus casei cell wall extract (LCWE).

21. The method according to any one of claims 1 to 20, wherein in step (ii), the non-human animal prepared in step (i) is treated with a formulation comprising LCWE emulsified with CFA.

22. The method according to any one of claims 1 to 21, wherein in step (ii), the formulation is injected intraperitoneally into the non-human animal.

23. The method according to any one of claims 1 to 22, wherein the preparation is administered by intraperitoneal injection to the non-human animal in a continuously low dose.

24. The method according to any one of claims 1 to 23, wherein the preparation is administered to the non-human animal by intraperitoneal injection in a dose of less than 1 μg of LCWE and / or less than 5 μL of CFA per gram of the non-human animal.

25. The method according to any one of claims 1 to 24, wherein the formulation is administered to the non-human animal for at least three months.

26. The method according to any one of claims 1 to 25, wherein the formulation is administered to the non-human animal prepared in step (i) three times a week for the first two weeks and once a week for the following two weeks for one month, or the formulation is administered to the non-human animal prepared in step (i) once every two weeks for three months.

27. The method according to any one of claims 1 to 26, wherein the non-human animal is a rodent, a mammal, or a non-human primate.

28. The method according to any one of claims 1 to 27, wherein the rodent is a mouse or a rat.

29. A non-human animal produced by the method described in any one of claims 1 to 28.

30. A non-human animal whose genome contains a genetically modified endogenous Igha locus comprising a heterologous gene encoding the human IgA heavy chain constant region or a fragment thereof, wherein the non-human animal exhibits clinical signs of an IgA deposition-related disease (e.g., IgA nephropathy).

31. The non-human animal according to claim 29 or 30, wherein the non-human animal shows elevated levels of galactose-deficient IgA protein.

32. The non-human animal according to any one of claims 29 to 31, wherein the non-human animal has shown elevated levels of galactose-deficient IgA protein in its serum or intestinal mucus.

33. The non-human animal according to claim 1, wherein part or all of the endogenous non-human animal Igha gene at the endogenous non-human animal Igha gene locus is deleted and replaced with a nucleotide containing the heterologous gene.

34. The non-human animal according to any one of claims 29 to 33, wherein the non-human animal does not express an endogenous non-human IgA heavy chain constant region.

35. The non-human animal according to any one of claims 29 to 34, wherein the nucleotide containing the heterologous gene is operably linked to an endogenous regulatory element of the non-human animal (e.g., a promoter, enhancer, silencer, etc.).

36. The non-human animal according to any one of claims 29 to 35, wherein the nucleotide containing the heterologous gene is a genomic fragment of the human IGHA1 gene.

37. The non-human animal according to any one of claims 29 to 36, wherein the heterologous gene comprises both a human IGHA1 coding sequence and a non-coding sequence.

38. The non-human animal according to any one of claims 29 to 37, wherein the non-human animal produces chimeric IgA1 in which the heavy chain comprises a non-human variable region and a human constant region or a fragment thereof.

39. The non-human animal according to any one of claims 29 to 38, wherein the human IgA heavy chain constant region of the non-human animal includes an O-glycosylated hinge region.

40. The non-human animal according to any one of claims 29 to 39, wherein the heterologous gene is a human IGHA1 gene encoding the human IgA1 heavy chain constant region or a fragment thereof.

41. The non-human animal according to any one of claims 29 to 40, wherein the non-human animal is a rodent, a mammal, or a non-human primate.

42. The non-human animal according to any one of claims 29 to 41, wherein the rodent is a mouse or a rat.

43. Use of a non-human animal according to any one of claims 29 to 42 as an animal model for IgA deposition-related diseases.

44. The use according to any one of claims 1 to 43, wherein the IgA deposition-related disease is selected from the group consisting of IgA nephropathy, herpetiform dermatitis, Henoch-Schönlein purpura (also known as IgA vasculitis), Kawasaki disease, Henoch-Schönlein nephritis, renal impairment due to IgA vasculitis, IgA rheumatoid factor-positive rheumatoid arthritis, IgA-mediated anti-GBM disease, or IgA-mediated ANCA-associated vasculitis.

45. The use according to claim 43 or 44, wherein the IgA deposition-related disease is IgA nephropathy, IgA vasculitis, or Kawasaki disease.

46. A method for evaluating the efficacy of a candidate drug for treating or preventing an IgA deposition-related disease, comprising: preparing a non-human animal as described in any one of claims 29 to 42; 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 control non-human animal that has not been administered the candidate drug.

47. A method for identifying candidate drugs for treating or preventing IgA deposition-related diseases, comprising: preparing a non-human animal according to any one of claims 29 to 42; 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 control non-human animal that has not been administered the candidate drug.

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