Ltbeta humanized genome, vector, non-human mammal, and methods of construction and use

Abstract

The application relates to the field of genetic engineering, and discloses an LTbeta humanized genome, a vector, a non-human mammal, a construction method and application. The LTbeta humanized genome comprises a humanized LTbeta gene and an endogenous LTbeta promoter of a non-human mammal; the humanized LTbeta gene is effectively connected with the endogenous LTbeta promoter of the non-human mammal. The vector comprises the LTbeta humanized genome. By introducing the vector into a non-human mammal cell, a non-human mammal model can be constructed. The non-human mammal model has important application in the fields of drug evaluation and disease research, can more accurately simulate physiological and pathological processes in the human body, and provides a more reliable experimental object and a more effective research means for related research.

Description

Technical Field

[0001] This application relates to the field of genetic engineering, and more specifically, to an LTβ humanized genome, vector, non-human mammal, construction method, and uses. Background Technology

[0002] LTβ (lymphotoxin β) is a type II membrane protein of the tumor necrosis factor (TNF) superfamily. It can form a heterotrimer (mainly LTα1β2) with LTα (lymphotoxin α) and anchor on the surface of lymphocytes, serving as the main ligand for the LTβ receptor (LTβR). The LTβ / LTβR signaling pathway plays a crucial role in the immune system: studies have shown that LTβ is one of the factors that induce inflammatory responses and is essential for the normal development and evolution of lymphoid organs. For example, LTβ activation can promote the formation and differentiation of mucosal and peripheral lymphoid tissues, while the absence of LTβ signaling leads to severe developmental defects in immune organs.

[0003] Classic gene knockout mouse models have demonstrated the importance of LTβ function. LTβ knockout mice lack secondary lymphoid structures such as Peyer's patches, peripheral lymph nodes, splenic germinal centers, and follicular dendritic cells. Compared to LTα-deficient mice, LTβ-deficient mice retain some structures in cervical and mesangial lymph nodes, but exhibit significant overall immune structural abnormalities. Simultaneously, LTβ receptor (LTβR)-deficient mice also show the absence of all peripheral lymph nodes and Peyer's patches, accompanied by disorganization of spleen and thymus structures, and defects in the production of various immune cells (such as dendritic cells, NK cells, and NKT cells) and IgA. These findings suggest that LTβ / LTβR signaling plays a crucial role in the development of secondary lymphoid organs, lymphocyte differentiation, and immune homeostasis.

[0004] In experimental studies, researchers also used transgenic mice to explore the function of LTβ. Sustained activation of T cells expressing LTα and LTβ led to excessive thymic atrophy and abnormal T cell development; however, overexpression of LTβ alone did not induce the same pathological changes. This indicates that LTα is crucial for certain LTβ-mediated effects and also reveals the differences in physiological functions between LTβ and other members of the TNF family. On the other hand, abnormal LTβ expression has also been observed in inflammation and disease models: clinical studies have found that in tissues of chronic inflammatory diseases such as inflammatory bowel disease, sarcoidosis, and tuberculosis, CD4+ expression is significantly elevated. + LTβ expression was significantly elevated in various cell types, including T cells and plasma cells. These results suggest that LTβ is not only crucial for normal immune development but may also play an important role in inflammatory diseases and infections, and could even become a novel therapeutic target.

[0005] Traditionally, mouse models are the most commonly used animal models for studying LTβ, including gene knockout (KO) and transgenic models. KO models are used to study gene deletion effects, while transgenic models (such as overexpression or reporter gene knock-in) are used to study overexpression or gene regulation. In recent years, with the development of genetic engineering technology, humanized mouse models have gradually become an important tool for studying human immune genes. Common strategies for constructing humanized models include: (1) introducing human cDNA sequences into mouse endogenous loci to knock out mouse genes and simultaneously introduce human genes; (2) performing point mutations or exon substitutions in the mouse genome to replace part of the mouse gene sequence with a highly homologous artificially synthesized sequence; (3) inserting human genes into safe loci (such as ROSA26) to ensure stable expression, and tissue-specific promoters or inducing elements can be added as needed; (4) using BAC plasmids to randomly integrate large-sized human genome fragments, which can preserve complete introns and regulatory elements.

[0006] Each of the above methods has its advantages and disadvantages: endogenous site knock-in can improve model stability, but the insertion length is limited and usually only contains cDNA sequences; BAC random insertion can carry larger fragments and retain human regulatory structures, but the integration site is uncontrollable and may lead to position effects. Overall, existing humanization strategies provide multiple avenues for studying human gene function, but designing the optimal scheme for a specific target gene (such as LTβ) requires comprehensive consideration of expression regulation and construction difficulty. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing mouse LTβ models (such as gene knockout) in accurately reproducing the gene expression levels and regulatory mechanisms of human LTβ, making them unsuitable for evaluating targeted drugs or vaccines against human LTβ. Furthermore, traditional transgenic or knock-in methods have limited integration capabilities for large genomic sequences (including promoters, UTRs, and multiple exons / introns), resulting in complex and inefficient construction. Existing models lack reliable strategies to stably express complete LTβ functional fragments under human immune conditions, leading to discrepancies between research results and human pathophysiological environments. Therefore, this invention provides a precise and efficient humanized expression system to improve the accuracy of human LTβ-related disease simulation and the clinical relevance of immune mechanism research.

[0008] To achieve the above-mentioned objectives, this application adopts the following technical solution: Firstly, this application provides an LTβ humanized genome, including Humanized LTβ gene; and Endogenous LTβ promoters in non-human mammals; The humanized LTβ gene is effectively linked to the endogenous LTβ promoter in non-human mammals.

[0009] Furthermore, the humanized LTβ gene contains a segment encoding an amino acid sequence that has at least 95% identity with the human LTβ amino acid sequence; The human LTβ amino acid sequence has the sequence shown in SEQ ID NO:2.

[0010] Furthermore, the non-human mammal is a mouse.

[0011] Secondly, this application provides a vector containing the aforementioned LTβ humanized genome.

[0012] Thirdly, this application provides a non-human mammal containing the aforementioned LTβ humanized genome, or the aforementioned vector.

[0013] Furthermore, the non-human mammal is a mouse.

[0014] Fourthly, this application provides a method for constructing the aforementioned non-human mammal, comprising: (1) Design and construct a humanized LTβ gene targeting vector; (2) The vector obtained in step (1) is introduced into ES cells of non-human mammals and screened and identified. (3) Inject the positive ES cell clones identified in step (2) into blastocysts, transplant them into pseudopregnant mice and obtain chimeric offspring; obtain germline F1 through breeding, and perform genotyping, transcription / protein expression and functional verification on the offspring to confirm the expression and function retention of humanized LTβ in vivo, and obtain non-human mammals that stably inherit the humanized LTβ gene.

[0015] Fifthly, this application provides the use of the LTβ humanized genome, or the vector, or the non-human mammal, or the method of constructing the non-human mammal in simulating the development and function of human secondary lymphoid organs. The application referred to is the application mediated by LTβ.

[0016] Sixthly, this application provides the use of the LTβ humanized genome, or the vector, or the non-human mammal, or the method for constructing the non-human mammal in drug screening and toxicity testing.

[0017] In a seventh aspect, this application provides the use of the LTβ humanized genome, or the vector, or the non-human mammal, or the method of constructing the non-human mammal, in the development of therapeutic targets.

[0018] In summary, this application has the following beneficial effects: 1. This application utilizes gene editing technology to replace the mouse LTβ gene with a human LTβ gene, enabling the simulation of the human immune system in mice. This humanized mouse model can provide more clinically relevant results when studying human diseases and screening drugs. For example, in cancer immunotherapy research, using humanized LTβ mice can better simulate the human tumor microenvironment, aiding in the screening of novel drugs targeting LTβ.

[0019] 2. The transgenic mouse model constructed using the humanized LTβ gene in this application can better mimic the development and function of human secondary lymphoid organs in these mice. This humanized model can not only more accurately reproduce the human immune response process, but also help researchers analyze the disease mechanisms related to LTβ, especially in areas closely related to the immune system such as cancer and autoimmune diseases, thus promoting the development of disease treatment strategies.

[0020] 3. By using humanized LTβ mouse models, researchers can conduct drug screening and toxicity testing in a context closer to the human immune system. These models can provide more accurate toxicity and efficacy assessments for novel drugs, thereby improving the success rate of drug development and reducing the risk of failure in clinical trials. Humanized LTβ mouse models provide an ideal platform to elucidate its role in tumor immune regulation. By expressing the same LTβ membrane protein as in humans in mice, researchers can better analyze its role in the cancer microenvironment. This not only helps in understanding the mechanisms of LTβ in tumor progression but also aids in developing anti-tumor immunotherapeutic strategies targeting LTβ.

[0021] 4. The LTβ humanized mouse model provides a tool that more closely resembles the human physiological environment for studying LTβ-related diseases. By simulating a disease state similar to that in humans in humanized mice, researchers can more accurately evaluate the therapeutic effects of novel drugs. Particularly in the treatment of autoimmune and inflammatory diseases, the LTβ humanized model can help develop more targeted drugs, reduce side effects, and improve treatment efficacy.

[0022] 5. Using the LTβ humanized mouse model, researchers can more directly study key mechanisms in the human immune system, thereby improving the accuracy of experimental results. Because these models better reflect the characteristics of the human immune system, research results can be translated into clinical applications more quickly and effectively. This will significantly improve experimental efficiency, reduce drug development time and costs, and thus accelerate the development of novel immunotherapies. Attached Figure Description

[0023] Figure 1 Schematic diagram of humanized LTβ cDNA vector. Detailed Implementation

[0024] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0025] Example 1: Construction of a humanized LTβ gene targeting vector The upstream genomic fragment of mouse LTβ gene exon 1 was used as the left homologous arm (LA), and the downstream genomic fragment was used as the right homologous arm (RA). Both were cloned into the multiple cloning site of the backbone plasmid.

[0026] Insert the following components sequentially between the left and right axial arms: A DNA fragment containing the human LTβ start codon ATG and the complete coding sequence (as shown in SEQ ID NO: 4); A selection cassette (HygroRcassette) containing the PGK promoter-driven Hygromycin B resistance gene and a polyA tail, with Lox2272 sites on each side of the cassette; A selection cassette (DTA cassette) containing the diphtheria toxin A chain gene and its universal promoter; The cat gene encoding chloramphenicol acetyltransferase; The coding sequence and exon structure of human LTβ.

[0027] The obtained recombinant targeting vector was then sequenced to verify its correct orientation and sequence accuracy.

[0028] Specifically, refer to Figure 1 : Homologous arms (LA / RA): These left and right homologous arms originate from the target genome sequence and are used to integrate the insert fragment into the target locus via homologous recombination. Generally, the longer the homologous arm, the higher the homologous recombination efficiency. In the construction process, LA and RA correspond to the upstream and downstream sequences of the target gene, respectively, and the sequence sandwiched between them is the inserted human coding region and the selection cassette.

[0029] The positive selection cassette (HygroR cassette) contains a PGK promoter-driven Hygromycin B resistance gene and a polyA tail, used to screen for successfully integrated clones in mammalian cells (such as embryonic stem cells). After vector transfection, cells expressing PGK-HygroR survive under puromycin (or hygromycin) selection, enriching homologous integration events. The cassette is designed with Lox2272 sites at both ends to be cleaved during subsequent expression of Cre recombinase, leaving only a single Lox2272 sequence trace.

[0030] The Lox2272 site is a mutant lox site sequence recognized by the Cre recombinase and is not interoperable with the wild-type LoxP sequence. In the diagram, the two Lox2272 sites are located on either side of the HygroR resistance cassette and are aligned in the same direction. In the presence of Cre, the homotype Lox2272 sequence can mediate site-specific excision of the cassette, thereby removing the selection cassette. Because Lox2272 only pairs with itself and does not interchange with regular LoxP, this avoids cross-interaction with other Cre / LoxP systems.

[0031] Negative selection cassette (DTA cassette): Contains the diphtheria toxin A chain gene and its universal promoter (such as the RNA polymerase II promoter) to eliminate random insertion events. When a vector randomly integrates into a non-target site in the genome, DTA expression leads to cell death of the vector-carrying cell, thereby significantly increasing the proportion of positive clones with homologous recombination. Literature reports that adding a DTA negative selection cassette to positive and negative selection vectors can improve targeting efficiency with limited transient cytotoxicity.

[0032] Bacterial selection marker (cat): The cat gene, encoding chloramphenicol acetyltransferase, is used for plasmid selection and amplification in *E. coli*. During plasmid cloning, chloramphenicol resistance is used to select bacterial colonies containing the vector. In practice, chloramphenicol selection vectors are frequently used for humanized targeting vectors or BAC manipulation.

[0033] Chimeric human LTβ coding region (chimeric hLTB CDS): The plasmid illustration shows the coding sequence and exon structure of human LTβ. This indicates that the vector inserts the human LTβ coding region into the target locus in a chimeric manner. The "5' part of exon1" in the figure suggests that the inserted fragment contains a portion of the first human exon to ensure correct assembly and expression of the human sequence.

[0034] The vector design is based on pBeloBAC11, a BAC / low-copy backbone, which is modified and integrated with bacterial selection markers, Lox2272 site, negative selection cassette and other modules to form a targeted vector.

[0035] Example 2: Transfection of ES cells and screening and identification of positive clones Electroporation transfection: Take approximately 1×10 8 One ES cell and 100 μg of linearized plasmid (Example 1). The linearized plasmid was transfected into the ES cells by electroporation at 260 V and 500 μF.

[0036] Drug screening: 48 hours after electroporation, 200 μg / ml G418 was added to the ES cell culture medium for positive screening.

[0037] Cloning and amplification: Select surviving ES cell clones, transfer them to 96-well plates for amplification, and make 96-well plate replicas.

[0038] Genotyping: Plates containing gelatin-amplified ES cell clones were genotyped using PCR and sequencing analysis.

[0039] Primers and expected PCR size are as follows: Example 3: Construction of humanized LTβ mice (1) Design and construct a humanized LTβ gene targeting vector; (2) The vector obtained in step (1) is introduced into ES cells of non-human mammals and screened and identified. (3) Inject the positive ES cell clones identified in step (2) into blastocysts, transplant them into pseudopregnant mice and obtain chimeric offspring; obtain germline F1 through breeding, and perform genotyping, transcription / protein expression and functional verification on the offspring to confirm the expression and function retention of humanized LTβ in vivo, and obtain non-human mammals that stably inherit the humanized LTβ gene.

[0040] The humanized LTβ mouse model constructed using the above scheme exhibited good expression specificity and stability: human LTβ mRNA and protein expression were detected only in individuals carrying the human gene, while wild-type mice did not express human LTβ. Because the target gene is located within the mouse's own gene locus, this model has high genetic stability: the constructed alleles can be stably inherited by offspring, the expression pattern is highly reproducible, and the insertion site effect that may occur in random integration models is avoided.

[0041] Example 4: Application of humanized LTβ mice in drug evaluation This model offers significant advantages in human disease simulation and drug screening. It is well known that the LTβ / LTβR signaling pathway plays a crucial role in various diseases: for example, LTβ expression is elevated in inflammatory and autoimmune diseases, and studies have suggested it as a potential therapeutic target for various infectious or inflammatory diseases; tumor-related studies have also found that tumor-derived LTβ promotes bone metastasis of cancer cells and participates in bone metastasis colonization and osteoclastosis. Using this model, the efficacy of candidate inhibitors or antibody drugs targeting human LTβ can be directly evaluated in vivo, significantly improving clinical relevance. Furthermore, this model simulates the human LTβ gene regulatory environment, allowing for in-depth research into LTβ-mediated immune mechanisms, such as lymphoid organogenesis, IgA secretion regulation, and responses to specific pathogen infections. Compared to conventional mouse models, this invention overcomes the limitations of interspecies sequence differences, achieving in vivo reconstruction of humanized signals, providing a more reliable experimental platform for studying the biological function of human LTβ and targeted therapy.

[0042] Authentic simulation of human LTβ expression: The constructed humanized genome ensures that human LTβ is expressed only in mice carrying human alleles, and the expression tissues are consistent with human physiology.

[0043] Stable and reliable expression: Humanized alleles are obtained by knocking in endogenous sites, which can be stably inherited and maintain high expression consistency in offspring, making them more reproducible than randomly inserted transgenic models.

[0044] Significant advantages: The LTβ signaling pathway is a therapeutic target for various diseases, including autoimmune diseases, infections, and cancer. This model allows researchers to evaluate the efficacy of drugs targeting human LTβ in vivo, such as examining the effects of anti-LTβ monoclonal antibodies on inflammatory lesions or tumor metastases, thus improving the effectiveness of drug screening.

[0045] Breakthrough: Compared with existing mouse models, this human-derived model is the first to stably express the complete human LTβ gene (including key regulatory elements) in mice, providing a more physiologically accurate model system for immune mechanism research and is expected to promote precision medicine research based on human LTβ signaling.

[0046] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

[0047] Sequence List: Mouse amino acid sequence (SEQ ID NO:1) MGTRGLQGLGGRPQGRGCLLLAVAGATSLVTLLLAVPITVLAVLALVPQDQGRRVEKIIGSGAQAQKRLDDSKPSCILPSPSSLSETPDPRLHPQRSNASRNLASTSQGPVAQSSREASAWMTILSPAADSTPDPGVQQLPKGEPETDLNPELPAAHLIGAWMSGQGLSWEASQEEAFLRSGAQFSPTHGLALPQDGVYYLYCHVGYRGRTPPAGRSRARSLTLRSALYRAGGAYGRGSPELLLEGAETVTPVVDPIGYGSLWYTSVGFGGLAQLRSGERVYVNISHPDMVDYRRGKTFFGAVMVG Human amino acid sequence (SEQ ID NO:2) MGTRGLQGLGGRPQGRGCLLLAVAGATSLVTLLLAVPITVLAVLALVPQDQGGLVTETADPGAQAQQGLGFQKLPEEEPETDLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG Mouse gene sequence (SEQ ID NO:3) ATGGGGACACGGGGACTGCAGGGCCTGGGTGGGAGACCCCAGGGGAGGGGCTGCCTCTTGCTGGCTGTGGCAGGAGCTACTTCCCTGGTGACCCTGTTGTTGGCAGTGCCTATCACTGTCCTGGCTGTGCTGGCCTTGGTGCCGCAGGATCAGGGACGTCGGGTTGAGAAGATCATTGGCTCAGGAGCACAGGCTCAGAAAAGACTGGATGACAGCAAACCGTCGTGCATCTTGCCCTCACCCTCTAGCCTCTCAGAGACTCCTGACCCCCGTCTGCATCCTCAGAGATCCAATGCTTCCAGGAATCTAGCCTCCACATCCCAGGGCCCTGTTGCGCAGTCCTCTCGGGAGGCATCTGCATGGATGACCATCCTGTCTCCAGCTGCGGATTCTACACCAGATCCAGGGGTTCAACAGCTGCCAAAGGGGGAACCAGAAACTGACCTCAACCCTGAGCTCCCTGCTGCCCACCTCATAGGCGCTTGGATGAGTGGGCAAGGGCTCAGCTGGGAGGCGAGCCAAGAAGAAGCGTTTCTGAGGAGCGGCGCGCAGTTCTCCCCCACCCACGGGCTGGCGCTGCCACAGGACGGCGTCTATTACCTCTACTGCCACGTCGGGTACAGGGGCAGGACGCCCCCTGCCGGCCGAAGCCGTGCTCGCTCGCTCACGCTGCGCAGCGCCCTGTACCGCGCGGGGGGCGCCTACGGGCGAGGTTCCCCCGAGTTGCTGCTGGAGGGCGCGGAGACAGTCACACCTGTTGTGGACCCCATCGGGTACGGGTCGTTATGGTACACGAGCGTGGGGTTCGGCGGCCTGGCGCAGCTCCGGAGCGGCGAGAGGGTCTACGTTAACATCAGTCACCCCGACATGGTGGACTACAGGAGAGGGAAGACCTTCTTCGGGGCGGTGATGGTGGGGTGA Human gene sequence (SEQ ID NO:4) ATGGGGGCACTGGGGCTGGAGGGCAGGGGTGGGAGGCTCCAGGGGAGGGGTTCCCTCCTGCTAGCTGTGGCAGGAGCCACTTCTCTGGTGACCTTGTTGCTGGCGGTGCCTATCACTGTCCTGGCTGTGCTGGCCTTAGTGCCCCAGGATCAGGGAGGACTGGTAACGGAGACGGCCGACCCCGGGGCACAGGCCCAGCAAGGACTGGGGTTTCAGAAGCTGCCAGAGGAGGAGCCAGAAACAGATCTCAGCCCCGGGCTCCCAGCTGCCCACCTCATAGGCGCTCCGCTGAAGGGGCAGGGGCTAGGCTGGGAGACGACGAAGGAACAGGCGTTTCTGACGAGCGGGACGCAGTTCTCGGACGCCGAGGGGCTGGCGCTCCCGCAGGACGGCCTCTATTACCTCTACTGTCTCGTCGGCTACCGGGGCCGGGCGCCCCCTGGCGGCGGGGACCCCCAGGGCCGCTCGGTCACGCTGCGCAGCTCTCTGTACCGGGCGGGGGGCGCCTACGGGCCGGGCACTCCCGAGCTGCTGCTCGAGGGCGCCGAGACGGTGACTCCAGTGCTGGACCCGGCCAGGAGACAAGGGTACGGGCCTCTCTGGTACACGAGCGTGGGGTTCGGCGGCCTGGTGCAGCTCCGGAGGGGCGAGAGGGTGTACGTCAACATCAGTCACCCCGATATGGTGGACTTCGCGAGAGGGAAGACCTTCTTTGGGGCCGTGATGGTGGGGTGA Primer 1 sequence(SEQ ID NO:5) TGCCTGGAGAGGCAAAGACAGACC Primer 2 sequence(SEQ ID NO:6) TGCGTTGGGTCCACTCAGTAGATGC Primer 2 sequence(SEQ ID NO:7) CCTATCACTGTCCTGGCTGTGCTGG

Claims

1. The LTβ humanized genome, characterized by, include Humanized LTβ gene; and Endogenous LTβ promoters in non-human mammals; The humanized LTβ gene is effectively linked to the endogenous LTβ promoter in non-human mammals.

2. The LTβ humanized genome according to claim 1, characterized in that, The humanized LTβ gene contains a segment encoding an amino acid sequence that has at least 95% identity with the human LTβ amino acid sequence. The human LTβ amino acid sequence has the sequence shown in SEQ ID NO:

2.

3. The LTβ humanized genome according to claim 1, characterized in that, The non-human mammal in question is the mouse.

4. A carrier, characterized in that, It includes the LTβ humanized genome as described in any one of claims 1-3.

5. The use of the LTβ humanized genome according to any one of claims 1-3, or the vector according to claim 4, in the development and function of simulated human secondary lymphoid organs; The application referred to is the application mediated by LTβ.

6. Use of the LTβ humanized genome according to any one of claims 1-3, or the vector according to claim 4, in drug screening and toxicity testing.

7. Use of the LTβ humanized genome according to any one of claims 1-3, or the vector according to claim 4, in the development of therapeutic targets.

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